Canine atopic dermatitis(CAD)is a prevalent genetically susceptible infammatory and pruritic allergic skin condition afecting not only the health of dogs but also the quality of life of their owners.Interleukin-31(IL-...Canine atopic dermatitis(CAD)is a prevalent genetically susceptible infammatory and pruritic allergic skin condition afecting not only the health of dogs but also the quality of life of their owners.Interleukin-31(IL-31)and interleukin-31 receptor alpha(IL-31RA)are essential for the development of pruritus in primates and mice.Hence,it is expected that inhibiting IL-31RA will be an efective approach to alleviate pruritus.The purpose of the study was to produce anti-canine IL-31RA polyclonal antibodies(anti-IL-31RA pAbs)and evaluate their efcacy in inhibiting house dust mite(HDM)-evoked pruritic responses.Dogs were immunized with antigens formed by IL-31RA recombinant short peptides coupled to BSA to produce anti-IL-31RA pAbs.The CAD model was developed by using HDM allergen stimulation,and the efects of IL-31RA pAbs on the reduction of pruritus in CAD model dogs were examined.The Canine Atopic Dermatitis Extent and Severity Index(CADESI)-4 and pruritus Visual Analog Scale(pVAS)were utilized to evaluate pruritic responses,and skin tissue samples were collected from the inguinal area for pathological assessment of skin infammatory cell infltration.The results showed that anti-IL-31RA pAbs with high titers(1:128,000)and specifcity were efectively produced.In the CAD model group,the severity of skin damage,pruritus score,infammatory cell infltration and level of infammatory factors were considerably elevated.Anti-IL-31RA pAbs relieved pruritic behavior and dermatitis in dogs compared to placebo-treated dogs.In conclusion,anti-IL-31RA pAbs efectively suppressed CAD in vivo and are anticipated to be an efective novel treatment for pruritic skin disorders such as CAD.展开更多
Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of...Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3<sup>rd</sup> immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4<sup>th</sup> immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.展开更多
[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene...[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.展开更多
[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and to prepare its polyclonal antibody. [Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants ...[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and to prepare its polyclonal antibody. [Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained and confirmed by Western-blot analysis. [Result] The polyclonal anti- body was successfully prepared and could efficiently detect the expressed antigen. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein in rice.展开更多
Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophor...Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.展开更多
Izumol is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumol was fractionated...Izumol is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumol was fractionated into 6 fragments （F0-F5） which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 （DE3） and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumol, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumol ascetic polyclonal antibody with intraperitoneal injection of S 180 cells. Subsequently, the anti-GST-Izumol polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumol fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumol proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumol in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumol to the egg surface.展开更多
A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Tw...A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.展开更多
BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most ...BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using Protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant protein, which served as an antigen. Rat LRRN3 protein was primarily expressed in cerebral cortex neurons, hippocampal dentate gyrus granule cell layer neurons, and cerebellar Purkinje cells.展开更多
A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as th...A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.展开更多
To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given in...To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin （ASIg） or sheep nonimmune serum immunoglobulin （NSIg）. At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lurrg weight, dressing percentage, and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate, cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH, meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.展开更多
Three polychlorinated biphenyls （PCBs） congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized. The three result...Three polychlorinated biphenyls （PCBs） congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized. The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens. Three of the resultant polyclonal antibodies （Pabs） were obtained. The antiserum exhibited relatively high antibody titres （1：32-64） in double agar diffusion.展开更多
Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C...Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.展开更多
Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism a...Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body’s homeostatic mechanisms, we for the first time, report successful prokary- otic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.展开更多
Testis-specific protease 50 （TSP50） is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising targ...Testis-specific protease 50 （TSP50） is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSPS0 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector PeDNA3.1. Rabbit anti-TSPS0 polyclonal antibodies were prepared by means of intramuscular injection of peDNA3.1-TSPS0 into the rabbits. Titem of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSPS0 fusion protein as an antigen. In addition, we examined the expression of TSPS0 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.展开更多
A polyclonal antibody against the currently concerned estrogenic bisphenol compounds was produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used to synthesize the complete antigen in which...A polyclonal antibody against the currently concerned estrogenic bisphenol compounds was produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used to synthesize the complete antigen in which the characteristic bisphenol structure was exposed to the largest extent. The produced polyclonal antibody showed high specificity and affinity for bisphenol A.展开更多
The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies ag...The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21（DE3）plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay （ELISA）, Western blotting and immunohistochemical staining （IHC）. The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marrnota himalayan—— for the research of infectious diseases of hepatitis viruses and liver cancer treatment.展开更多
Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays a...Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.展开更多
Polyclonal antibodies collected from the blood of animals and humans experimentally immunised or spontaneously immunised respectively can be injected into patients to protect them against pathogens, toxins, tumours et...Polyclonal antibodies collected from the blood of animals and humans experimentally immunised or spontaneously immunised respectively can be injected into patients to protect them against pathogens, toxins, tumours etc. This approach is severely limited by the availability of human polyclonal antibodies of interest. Moreover, polyclonal antibodies from animals are recognised as antigens by patients and are thus rapidly rejected and inactivated. To circumvent this problem, animals (essentially rabbits, chicken, pigs and cows) are being genetically engineered. Their immunoglobulin genes are being inactivated and the corresponding human immunoglobulin genes are being transferred to them. These animals will be immunized and it is expected that large amounts of pure human polyclonal antibodies will be extracted from their blood to be administered to patients. The possible acceptability problem of this approach is under a case study of the European Union Pegasus project.展开更多
Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain an...Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.展开更多
Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled...Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.展开更多
基金the National Natural Science Foundation of China(Grant No.32072938)。
文摘Canine atopic dermatitis(CAD)is a prevalent genetically susceptible infammatory and pruritic allergic skin condition afecting not only the health of dogs but also the quality of life of their owners.Interleukin-31(IL-31)and interleukin-31 receptor alpha(IL-31RA)are essential for the development of pruritus in primates and mice.Hence,it is expected that inhibiting IL-31RA will be an efective approach to alleviate pruritus.The purpose of the study was to produce anti-canine IL-31RA polyclonal antibodies(anti-IL-31RA pAbs)and evaluate their efcacy in inhibiting house dust mite(HDM)-evoked pruritic responses.Dogs were immunized with antigens formed by IL-31RA recombinant short peptides coupled to BSA to produce anti-IL-31RA pAbs.The CAD model was developed by using HDM allergen stimulation,and the efects of IL-31RA pAbs on the reduction of pruritus in CAD model dogs were examined.The Canine Atopic Dermatitis Extent and Severity Index(CADESI)-4 and pruritus Visual Analog Scale(pVAS)were utilized to evaluate pruritic responses,and skin tissue samples were collected from the inguinal area for pathological assessment of skin infammatory cell infltration.The results showed that anti-IL-31RA pAbs with high titers(1:128,000)and specifcity were efectively produced.In the CAD model group,the severity of skin damage,pruritus score,infammatory cell infltration and level of infammatory factors were considerably elevated.Anti-IL-31RA pAbs relieved pruritic behavior and dermatitis in dogs compared to placebo-treated dogs.In conclusion,anti-IL-31RA pAbs efectively suppressed CAD in vivo and are anticipated to be an efective novel treatment for pruritic skin disorders such as CAD.
文摘Human African trypanosomiasis (HAT), commonly known as sleeping sickness is one of the neglected tropical diseases (NTDs), which is fatal if left untreated. Its diagnosis is a challenge since the signs and symptoms of the primary phase are not specific, the existing diagnostic methods have low sensitivity and specificity, and the available drugs have some toxicity. New, robust, and cost-effective techniques are needed for the early identification of parasites. This study aimed to assess the sensitivity and specificity of two different types of polyclonal antibodies against T. b. gambiense using antigen detection ELISA. Polyclonal antibodies against the expressed proteins Tbg I2 and Tbg I17 were produced using New Zealand white rabbits. The antibody titer measured was greater than 32 g/L after the 3<sup>rd</sup> immunization for the expressed protein Tbg I2. For the expressed protein Tbg I17, the antibody titer measured was greater than 32 g/L after the 4<sup>th</sup> immunization. The sensitivity and specificity of the Tbg I2 polyclonal antibody confirmed with Polymerase Chain Reaction (PCR) as gold standard were respectively 89.5% and 80.6%, while for the Tbg I17 polyclonal antibody, the sensitivity and specificity were respectively 92.1% and 88.9%. The area under the curve for the Tbg I2 polyclonal antibody was 0.90 ± 0.032, while for the Tbg I17 polyclonal antibody, the area under the curve was 0.92 ± 0.0. The Tbg I17 polyclonal antibody produced in New Zealand white rabbits has good sensitivity and good specificity;it can be successfully used in the diagnosis of HAT.
文摘[Objective] This study aimed to clone the E. coil NrfA gene and construct the pET-28a （＋）-NrfA prokaryotic expression vector for preparation of polyclonal anti- body against E. coil NrfA. [Method] E. coil NrfA gene was cloned from the E. coli genome DNA by PCR and inserted into the vector pET-28a（＋） to construct prokary- otic expression vector pET-28a （＋）-NrfA. E. coil NrfA protein was expressed by IPTG induction and purified. Polyclonal antibody against NrfA protein was prepared by im- munizing rabbit with routine method. The specificity and titer of polyclonal antibody was confirmed by ELISA and Western Blotting. [Result] The constructed prokaryotic expression vector pET-28a（＋）-NrfA was induced by IPTG, the recombinant NrfA pro- tein could be expressed effectively. The titer of rabbit anti-NrfA polyclonal antibody obtained by immunization and purification was about 1：204 900. Western Blotting anal- ysis indicated that the obtained polyclonal antibody against E. coil NrfA protein had high titer and high specificity. [Conclusion] E. coil NrfA gene was cloned and the prokaryotic expression vector pET-28a （＋）-NrfA was constructed successfully, poly- clonal antibody with high titer and high specificity was prepared, which laid the foun- dation for the study of NrfA in different strains of bacteria.
基金Supported by Higher School Science and Technology Innovation Project of Guangdong Province (No.2012KJCX0021)National Natural Science Foundation of China (No.30700052)~~
文摘[Objective] This study aimed to conduct prokaryotic expression of rice SDG711 C-terminal and to prepare its polyclonal antibody. [Method] C-terminal of rice SDG711 containing relatively intensive antigen determinants was selected for prokaryotic expression, prokaryotic expression vector pET28a-711C was constructed and the recombinant plasmid was transformed into Escherichia coli BL21 （DE3） competent cells. The recombinant fusion protein was induced by IPTG and purified to immunize a New Zealand white rabbit as the antigen, and polyclonal antibody was obtained and confirmed by Western-blot analysis. [Result] The polyclonal anti- body was successfully prepared and could efficiently detect the expressed antigen. [Conclusion] This study laid the foundation for further investigating the functions of SDG711 protein in rice.
文摘Serum immunoglobulin from the mandarin fish, or the so called Chinese perch, Siniperca chuatsi (Basilewsky), was successfully purified using affinity chromatography. Heavy and light chains were detected on electrophoresis gel, with molecular weights being estimated at 72 and 29 kDa, respectively. The tetrameric IgM of S. chuatsi was calculated to be 808 kDa. The rabbit polyclonal antisera against the purifed immunoglobulin were developed and tested by Western blot analysis. The antisera reacted strongly with the heavy chains of S. chuatsi immunoglobulin. Humoral immune responses of the mandarin fish can then be examined using the developed polyclonal antibody.
基金supported by the Natural Science Foun-dation of Inner Mongolia Autonomous Region, China(20080404MS0505)the National Training Foun-dation for Talents of Basic Sciences, China (J0730648)
文摘Izumol is a novel member of the immunoglobulin superfamily locating on sperm, and is indispensable for sperm-egg fusion. According to its immunoglobulin-like domain in the extracellular region, Izumol was fractionated into 6 fragments （F0-F5） which were ligated with pGEX-4T1 to construct the prokaryotic expression vectors pGEX-Fn. The recombinant plasmids were transformed into Escherichia coli BL21 （DE3） and the GST-Fn fusion proteins were expressed successfully by induction with IPTG. GST-F0, a recombinant fusion protein of GST with the full length of extracellular region of mature cashmere goat Izumol, was purified by polyacrylamide gel slicing method and was used as an antigen to immunize the Kunming mouse to generate anti-GST-Izumol ascetic polyclonal antibody with intraperitoneal injection of S 180 cells. Subsequently, the anti-GST-Izumol polyclonal antibody was purified with miscellaneous antigen by glutaraldehyde cross-linking method. Western blotting analysis showed that the purified ascetic polyclonal antibody had high affinity to all 6 GST-Izumol fragment fusion proteins. Immunohistochemical analysis with this antibody displayed that the cashmere goat Izumol proteins were at the equatorial segment of sperm head surface. These results indicate that this polyclonal antibody has high specificity and lays the foundations for further study on the expression pattern of Izumol in cashmere goat testis and binding abilities of each extra-membrane fragment of Izumol to the egg surface.
基金This work was financially supported by National Natural Science Foundation of China （20447003）.
文摘A competitive enzyme-linked immunosorbent assay （ELISA） suitable for the determination of the urea herbicide isoproturon, 3-（4-isopropylphenyl）-l,l-dimethylurea, in food and environmental samples was developed. Two haptens named 1-（3- carboxypropyl）-3-（4-isopropylphenyl）-1-methylurea （hapten 4C） and 1-（5-carboxypentyl）-3-（4-isopropylphenyl）-1- methylurea （hapten 6C） were synthesized. The haptens were coupled to bovine serum albumin （BSA） and ovalbumin （OVA）, respectively, using the N-hydroxysuccinimide reaction. The hapten 6C-BSA conjugate was used as the immunogen, with which a high-titer anti-isoproturon polyclonal antibody （pAb） was successfully obtained by immunization of New Zealand white rabbits. The hapten 4C-OVA conjugate was used as coating antigen and a method of the indirect competitive ELISA for isoproturon was established. The haptens were confirmed with TLC, IR, and 1H NMR. The conjugation molar ratios of hapten 4C to OVA and hapten 6C to BSA were 36：1 and 46：1, respectively, as calculated by a UV spectrophotometry. The highest titer of the anti-isoproturon sera determined by a non-competitive indirect ELISA procedure was 1.6 × 10^5. The optimal concentrations of the coating antigen and the dilution of the anti-isoproturon sera used in the ELISA were 0.1 mg·L^-1 and 1.0 × 10^5, respectively. The concentration of isoproturon that inhibits 50% of antibody-antigen binding （IC50） was 0.07 mg·mL^-1. The cross-reactivities of six urea herbicides including chlorbromuron, fluometuron, monolinuron were lower than 0.1%. Isoproturon is a small molecule without immune activity and active functional group for attaching to carrier protein. To produce an antibody against isoproturon with high titer and high specificity is the most important step in the development of an immunochemical method for the determination of isoproturon in food and environmental samples. The two haptens synthesized in this study have carboxyl groups and accommodate different lengths of spacer arms, and the phenyl and isopropyl groups are fully exposed. An anti-isoproturon polyclonal antibody with high titer and high specificity was successfully obtained by immunization of rabbits with the conjugate of the hapten attached to the protein carrier.
基金the National Natural Science Foundation of China,No.30600224,30700438,30600636No.39 Grant by China Postdoctoral Science Foundation,No.20060390886
文摘BACKGROUND： Studies have shown that the Repeat superfamily, could be related to neural LRRN3, a member of the Neuron Leucine-Rich development, differentiation, information transmission, and other functions, but most studies have focused on nucleic acid levels and few have reported on LRRN3 protein levels. OBJECTIVE： To prepare rabbit anti-rat LRRN3 polyclonal antibody and to observe protein tissue expression profiles. DESIGN, TIME AND SEI-rlNG： In vitro, molecular, biological experiments were performed from October 2007 to April 2009 in Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. MATERIALS： Immunization antigen, namely rat MaI-LRRN3C-His recombinant protein, was provided by the Laboratory of Neurobiology at Xiangya School of Medicine, Central South University. METHODS： Rat Mal-LRRN3C-His recombinant protein was used to immunize male, New Zealand rabbits, and rabbit anti-rat LRRN3 polyclonal antibody was prepared. MAIN OUTCOME MEASURES： Antibody purification was conducted using Protein A affinity chromatography, and the LRRN3 anti-serum titer was identified using enzyme-linked immunosorbent assay. Immunohistochemical techniques and Western blot preliminary tests were used to determine LRRN3 protein expression profiles in adult rats. RESULTS： A highly purified rabbit anti-rat LRRN3 polyclonal antibody was obtained. Western Blot results from rat brain total protein revealed a band at 79 kD, which was consistent with the size of LRRN3. Immunohistochemistry results showed that protein was mainly expressed in the central nervous system, and no significant positive signals were observed in other tissues. Positive cells included neurons of cerebral cortex and hippocampal dentate gyrus granule cell layer, and cerebellar Purkinje cells. There was no positive expression in glial cells. CONCLUSION： Rabbit anti-rat LRRN3 polyclonal antibody was successfully prepared at a high purity from the prokaryotic-expressed MaI-LRRN3C-His recombinant protein, which served as an antigen. Rat LRRN3 protein was primarily expressed in cerebral cortex neurons, hippocampal dentate gyrus granule cell layer neurons, and cerebellar Purkinje cells.
文摘A polyclonal antibody-based antigen-capture ELISA （AC-ELISA） has been developed for detection of Canine parvovirus （CPV） antigens in faecal samples of dogs. The assay uses rabbit anti-CPV polyclonal antibody as the capture antibody, guinea pig anti-CPV polyclonal antibody as tracing antibody and anti-guinea pig HRPO conjugate as the detection system. The optimum dilution of the capture antibody and the tracing antibody capable of detecting the CPV-2 antigens was found to be 1：1 600 and 1：400, respectively, in the check-board titration. In this study, a total of 152 samples （129 faecal samples and 23 cell culture supernatant） were tested both by AC-ELISA and by polymerase chain reaction （PCR）. Of the samples tested, 69 and 78 samples were found positive by AC-ELISA and PCR, respectively. The AC-ELISA had relative sensitivity, relative specificity and accuracy of 88.4%, 100.0% and 91.4% respectively. The analytical sensitivity of AC-ELISA was estimated to be 102.8 TCID50/mL whereas PCR sensitivity was 100.8 TCIDs0/mL. The AC-ELISA is a simple, quick and reliable method for screening large numbers of faecal samples of dogs suspected of CPV infection.
基金the grants from the Key Project of Natural Science Foundation of Yunnan Province, China （2000C005Z） the National NaturzA Science Foundation of China （30260079）.
文摘To detect the effects of the polyclonal antibodies raised in sheep against porcine adipocyte plasma membranes on the porcine carcass composition and meat quality, 30 pigs assigned into 6 treatment groups were given intraperitoneal injections of sheep antipig adipocyte plasma membrane immunoglobulin （ASIg） or sheep nonimmune serum immunoglobulin （NSIg）. At the end of the experiment, the pigs were slaughtered at 90 kg body weight, and carcasses and meat quality were evaluated. The results showed that when pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, 20 mg purified ASIg twice at 15 and 60 kg body weight, or 20 mg purified ASIg at 60 kg body weight, respectively, their lean meat percentage, fat meat percentage, backfat thickness, loin eye area leaf fat weight, caul fat weight, heart weight, liver weight, and kidney weight were significantly affected. However, the kidney weight, lurrg weight, dressing percentage, and spleen weight did not remarkably change. Our results indicated that pigs intraperitoneally immunized with 20 or 30 mg ASIg at 15 kg body weight, and 20 mg ASIg twice at 15 and 60 kg body weight, have significantly different drip loss rate, cooked meat ratio, tenderness, storage loss rate, muscle fiber diameter, moisture content, dry matter content, crude protein content, and crude fat content from the control group that received 20 mg NSIg at 15 kg body weight. However, meat pH, meat color value, meat marbling score, inosinate, and myohemoglobin were not significantly affected. Our results indicated ASIg could not significantly affect the content of most muscular amino acids and intramuscular fatty acids.
基金supported by the National Natural Science Foundation of China(No.20677008)The Research Fund for the Doctoral Program of Higher Education(20060255004)Shanghai Leading Academic Discipline Project(No.B604).
文摘Three polychlorinated biphenyls （PCBs） congeners and their corresponding haptens bearing four carbon length carboxylic groups that can be linked to a protein for raising antibodies were synthesized. The three resultant immunogens were fabricated and used to stimulate immune responses in rabbits to survey the characteristics of the haptens. Three of the resultant polyclonal antibodies （Pabs） were obtained. The antiserum exhibited relatively high antibody titres （1：32-64） in double agar diffusion.
基金Supported by the National Natural Science Foundation of China(Nos.30700827 and 30871301)Jilin Provincial Science & Technology Department of China(Nos.20070719 and 20080731)Northeast Normal University,China(Nos.20070401,NENU-STC07005)
文摘Aquaporins（AQPs） are specific membrane channels for water and other small nonionic molecules.In order to overcome the difficulties to generate the effictive antibody of membrane protein,we selected the cytoplasmic C-terminus of Aquaporin 1（AQP1） as an unique antigen.The long C-terminus of mouse AQP1 was overexpressed in the Glutathione S-tansferase Gene Fusion System.On the basis of the resonable amounts of soluable membrane protein peptides,we prepared the specific antibody.To pursure this object,we constructed pGEX-4T-1/mAQP1（DNA sequence from 700 to 801 bp） recombinant plasmid and transformed it into Escherichia coli BL21 cells.The GST-AQP1 C-terminal hydrophilic peptide fusion protein was induced by IPTG and further purified by Glutathione Sepharose 4B to obtain the right size fusion protein.Then we immunized the New Zealand rabbits to prepare the antiserum.The purified AQP1 antibody showed high sensitivity by ELISA assay and high specificity by Western blot with AQP1 null mice served as negative control.Finally,we also checked the AQP1 localization in the mouse renal tissues in wild type of mice and AQP1 null mice served as negative control.We demonstrated that AQP1 was highly expressed at the descending limb of Henle tube using our purified AQP1 antibody,which was consistent with previous report.The successful design and preparation of AQP1 antibody through GST technique is an example as making antibodies against a specific membrane protein.
文摘Pregnane and Xenobiotic Receptor (PXR; or Steroid and Xenobiotic Receptor, SXR), a new member of the nuclear receptor superfamily, is thought to modulate a network of genes that are involved in xenobiotic metabolism and elimination. To further explore the role of PXR in body’s homeostatic mechanisms, we for the first time, report successful prokary- otic expression and purification of full-length PXR and preparation of polyclonal antibody against the whole protein. The full-length cDNA encoding a 434 amino acids protein was sub-cloned into prokaryotic expression vector, pET-30b and transformed into E. coli BL21(DE3) cells for efficient over expression. The inclusion body fraction, containing the expressed recombinant protein, was purified first by solubilizing in sarcosine extraction buffer and then by affinity column chromatography using Ni-NTA His-Bind matrix. The efficacy of anti-PXR antibody was confirmed by immunocytology, Western blot analysis, EMSA and immunohistochemistry. The antibody obtained was capable of detecting human and mouse PXR with high specificity and sensitivity. Immunofluorescence staining of COS-1 cells transfected with human or mouse PXR showed a clear nuclear localization. Results from immunohistochemistry showed that level of PXR in liver sections is immunologically detectable in the nuclei. Similar to exogenously transfected PXR, Western blot analysis of cell extract from HepG2 and COLO320DM cells revealed a major protein band for endogenous PXR having the expected molecular weight of 50 kDa. Relevance of other immunodetectable bands with reference to PXR isoforms and current testimony are evaluated. Advantages of antibody raised against full-length PXR protein for functional characterization of receptor is discussed and its application for clinical purposes is envisaged.
文摘Testis-specific protease 50 （TSP50） is a testis-specific oncogene, which is abnormally activated in most tested patients with breast cancer. This property makes it an attractive molecular marker and a promising target for the diagnosis and therapy of breast cancer. In order to obtain the protective and specific polyclonal antibodies for further research, TSPS0 cDNA was amplified by RT-PCR from normal human testicular tissue, and inserted into eukaryotic expression vector PeDNA3.1. Rabbit anti-TSPS0 polyclonal antibodies were prepared by means of intramuscular injection of peDNA3.1-TSPS0 into the rabbits. Titem of the anti-sera were measured by ELISA and Western blotting with the E. coli cell lysate containing the induced GST-TSPS0 fusion protein as an antigen. In addition, we examined the expression of TSPS0 in both breast cancer cell line MCF-7 and breast cancer tissue by immunofluorescent and immunohistochemistry analysis.
基金This work was financially supported by the National Natural Science Foundation of China(Grant No.20075001).
文摘A polyclonal antibody against the currently concerned estrogenic bisphenol compounds was produced according to a new scheme. 4,4-Bis (4-hydroxyphenyl) valeric acid was used to synthesize the complete antigen in which the characteristic bisphenol structure was exposed to the largest extent. The produced polyclonal antibody showed high specificity and affinity for bisphenol A.
基金This project was supported by grants from National Natural Sciences Foundation of China (No 30571646)the Na-tional Basic Research Program (Program 973, No 2005CB522901)
文摘The objective of this study is to express the carbohydrate recognition domain （CRD） of the asialoglycoprotein receptor （ASGPR） H1 and H2 subunits of Marmota himalayan in vitro, and develop polyclonal antibodies against the recombinant proteins. RT-PCR was used to amplify ASGPR CRDH1 and CRDH2 from the liver tissue of Marmota himalayan. The products of amplification were subcloned into prokaryotic expression vector pRSET-B, and expressed in E.coli BL21（DE3）plysS. The recombinant proteins were purified using Ni-NTA spin column. The purified proteins were inoculated into BALB/c mice to develop polyclonal antibodies. The sensitivity and specificity of antibodies were evaluated by enzyme-linked immunosorbent assay （ELISA）, Western blotting and immunohistochemical staining （IHC）. The polyclonal antibodies showed high sensitivity and specificity against both denaturated and native ASGPR proteins. We successfully amplified and expressed the ASGPR CRDs of Marmota himalayan. The nucleic sequences of ASGPR CRDH1 and CRDH2 of Marmota himalayan have been submitted to Genbank and the sequence ID are DQ 845465 and DQ845466, respectively. The proteins and antibodies prepared can be used for targeting gene therapy in a new animal model-Marrnota himalayan—— for the research of infectious diseases of hepatitis viruses and liver cancer treatment.
基金Supported by Youth Fund of Hubei Academy of Agricultural Sciences(2013NKYJJ12)
文摘Japanese encephalitis （JE） is a central nervous system disease caused by Japanese encephalitis virus （JEV）, which can infect human and a variety of animals and cause irreversible nerve damages. NS3 protein plays an important role in the process of JEV polyprotein hydrolysis, which is essential for JEV replication. Therefore, NS3 protein may be used as a potential drug target to treat Japanese encephalitis. In this study, the pET-28a-NS3 plasmid was successfully constructed and expressed in E. coli BL21 （ DE3 ） under IPTG induction. The molecular weight of the expressed recombinant protein was 55 ku, which was consistent with the expected result. The positive serum was prepared by immunizing BALB/c mice with NS3 protein and identified by indirect immunofluorescence （IFA）. The results showed that there was a fluorescence reaction between the prepared positive serum of NS3 protein and cells infected with JEV.
文摘Polyclonal antibodies collected from the blood of animals and humans experimentally immunised or spontaneously immunised respectively can be injected into patients to protect them against pathogens, toxins, tumours etc. This approach is severely limited by the availability of human polyclonal antibodies of interest. Moreover, polyclonal antibodies from animals are recognised as antigens by patients and are thus rapidly rejected and inactivated. To circumvent this problem, animals (essentially rabbits, chicken, pigs and cows) are being genetically engineered. Their immunoglobulin genes are being inactivated and the corresponding human immunoglobulin genes are being transferred to them. These animals will be immunized and it is expected that large amounts of pure human polyclonal antibodies will be extracted from their blood to be administered to patients. The possible acceptability problem of this approach is under a case study of the European Union Pegasus project.
基金The work was supported by grant from The National Natural Sciences Foundation of China (No.30070280)
文摘Objectives The cellular repressor of E1A-activated genes (CREG), a novel gene, was recently found to play a role in inhibiting cell growth and promoting cell differentiation. The purpose of this study was to obtain antibody against CREG protein and to study the expression of CREG protein in human internal thoracic artery cells (HITASY) which express different patterns of differentiation markers after serum withdrawal. Methods The open reading frame of CREG gene sequence was amplified by PCR and cloned into the pGEX-4T-1 vector. Glutathione-S-transferase (GST)-CREG fusion protein was expressed in E. Coli BL21 and purified from inclusion bodies by Sephacryl S-200 chromatography. Rabbits were immunized with the purified GST-CREG protein. Western blot examined with immunohistochemistry staining and the protein expression level was analyzed by Western blot in HITASY cells after serum removal. Results It was confirmed by using endonuclease digesting and DNA sequencing that the PCR product of CREG was correctly inserted into the vector. The GST-CREG protein was purified with gel filtration chromatography. Polyclonal antibody against GST-CREG was obtained from rabbits. CREG protein immunohistochemistry staining displayed a perinuclear distribution in the cytoplasm of HITASY cells. Results from Western blot suggested that comparing with the untreated cells upregulation of CREG polyclonal antibody against CREG was comfirmed. Using this antibody, the changes of CREG protein expression was observed in the process of phenotypic modulation of HITASY cells. These results provide basic understanding on the relationship of CREG gene with the cell phenotypic conversion.
文摘Survivin, a novel member of inhibitor of apoptosis（IAP） protein family, is aberrantly expressed in cancer but undetectable in normal, differentiated adult tissues. The cancer-specific expression of survivin, coupled with its importance in inhibiting cell death and in regulating cell division makes it a useful diagnostic marker of cancer and a potential target for cancer treatment. Survivin cDNA amplified from the total RNA of 293 cells through RT-PCR was cloned into prokaryotic expression vector pRSET-B. The recombinant plasmid pRSET-B-Surv was expressed in E.coli BL21, and the relative molecule mass（Mr） of expressed fusion protein was approximately 21000. The recombinant protein was purified through Ni^2＋ affinity chromatography column and characterized by SDS-PAGE and Western blot. The purified recombinant protein was then injected into rabbits, and antisurvivin polyclonal antibody with a high titer was obtained.
