To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites ...To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.展开更多
Porcine epidemic diarrhea (PED), a devastating enteric disease in pigs, is caused by PEDvirus (PEDV)(1)Reduced severity of clinical diseases was reported to associate with neutralizing antibody titers in colostrum. Ho...Porcine epidemic diarrhea (PED), a devastating enteric disease in pigs, is caused by PEDvirus (PEDV)(1)Reduced severity of clinical diseases was reported to associate with neutralizing antibody titers in colostrum. However, viral neutralization assay(VN) is laborious and not suitable for routine diagnosis. Spike protein plays an important role in stimulating neutralizing antibody that might be suitable for PEDV diagnosis.展开更多
Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production ...Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production of antibodies and the consequent transfer of these to the piglets via colostrum to prevent diarrhea during the neonate period and after weaning. The objective of this study was to evaluate the immune response of the sows immunized with recombinant ETEC proteins (F4, F5, F6, F18 and F41). The immune response of the sows immunized with the recombinant proteins was compared with a commercial vaccine containing ETEC bacterins. The study was performed on a commercial farm and included nine pregnant sows divided into three groups: G1 was vaccinated with recombinant proteins (n = 3);G2 was vaccinated with the commercial vaccine (n = 3);and G3 was vaccinated with sterile buffered saline (PBS) (n = 3). All the sows were fed a balanced diet without antibiotics and water ad libitum. The recombinant fimbriae stimulated the specific humoral immune response of the immunized sows. There was a statistically significant increase in the levels of antibodies to the fimbriae F4 (K88), F5 (K99), F6 (987P) and F18 in the sows vaccinated with the recombinant proteins compared with the control group. The colostrum IgG titers for all fimbriae in all the immunized sows were significantly increased compared to the control group. Additionally, all the piglets exhibited significantly increased antibody levels relative to all fimbriae when compared with those in the unimmunized control group, demonstrating successful antibody transfer via colostrum of the sows to the piglets.展开更多
Trichoderma is an ascomycete fungal genus widely distributed in the soils.Several species were selected,engineered and utilized for protein production for decades.The high extracellular secretion capability and eukary...Trichoderma is an ascomycete fungal genus widely distributed in the soils.Several species were selected,engineered and utilized for protein production for decades.The high extracellular secretion capability and eukaryotic post-translational modification machinery make Trichoderma spp.particularly interesting hosts.In this review,we summarized the recombinant proteins produced in Trichoderma since 2014,concerning their origins,hosts,promoters,terminators,signal peptides,yields and commonly used media.Meanwhile,strategies and merging trends in protein production and strain engineering are classified and summarized regarding codon optimization,promoter utilization,transcription factor regulation,post-translational modification and proteolytic degradation inhibition.With state-of-art biotechnologies and more available expression platforms,Trichoderma spp.could be more successful hosts to produce recombinant proteins as desired,i.e.better enzyme formula for efficient cellulose degradation or functional protein with high purity and yield.展开更多
It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But ...It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.展开更多
Objective In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis(LB) and screened out the appropriate antigens to support the production of a Chines...Objective In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis(LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA(enzyme-linked immunosorbent assay) kit for LB. Methods Six recombinant antigens, Fla B.g, Osp C B.a, Osp C B.g, P39 B.g, P83 B.g, and Vls E B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic(ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. Results Two Ig G(Vls E and Osp C B.g) and two Ig M(Osp C B.g and Osp C B.a) antigens were left by the logistic regression model screened. Vls E had the highest specificity for syphilis samples in the Ig G test(87.7%, P<0.05). Osp C B.g had the highest diagnostic value in the Ig M test(AUC=0.871). Interactive effects between Osp C B.a and Fla B.g could reduce the specificity of the ELISA. Conclusion Three recombinant antigens, Osp C B.g, Osp C B.a, and Vls E B.a, were useful for ELISAs of LB. Additionally, the interaction between Osp C B.a and Fla B.g should be examined in future research.展开更多
The engineering genetic technology has developed Bt maize events which contain recombinant protein that will be safe for the consumer. The aflatoxins are contaminants present in maize capable of producing cancer and d...The engineering genetic technology has developed Bt maize events which contain recombinant protein that will be safe for the consumer. The aflatoxins are contaminants present in maize capable of producing cancer and decreasing the immune response in human, additionally contained polyphenols compounds considered non nutritive. The objective of this study was to identify the presence of recombinant protein in hybrid and local varieties of corn and evaluate the content of aflatoxins and tannins. 25 samples of white grain maize for human consumption were collected, 12 were for hybrid maize and 13 local varieties, from the states of Hidalgo, Mexico and Morelos. Samples were analyzed for Cry1Ab/Cry1Ac, using lateral flow strip method, crude protein and lipids by standard methods. Aflatoxins were assessed by comercial Elisa kit and tannins by spectroscopy method. The data were grouped in a completely random model and an analysis of variance was performed. The results indicated that 44.5% of hybrid corn was positive by Bt-Cry1Ab/1Ac proteins, containing 9.02% ± 2.5 lipids and 11.33% ± 2.2 crude protein, 189 ± 0.92 mg/g of tannins and 6.36 ± 3.3 μg·g-1 aflatoxins. The local maize samples (55.5%) were negative to Bt-Cry1Ab/ Cry1Ac, which protein content was of 8.68% ± 0.90, 6.14% lipids ± 2.3, 273 ± 0.40 mg/100g tannin and 7.15 ± 3.3 μg·g-1 of aflatoxins. In conclusion, we observed an improvement of nutrient composition in hybrid maize with Bt proteins, and decrease in tannins content comparing with some local varieties without Bt proteins. The effectiveness of Bt maize expressing the Cry1Ab/ Cry1Ac in reducing aflatoxin contamination was not observed, therefore, additive affects of aflatoxins contamination in maize Bt-Cry need to be further investigated in cancer disease development.展开更多
Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing E...Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.展开更多
A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins form microbial cells. The process was narned...A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins form microbial cells. The process was narned as simultanecus cell disruption and aqueous two-phase extraction (ADATE). Advantages, such as high cell disruption efficiency, biochemical activities proservation of proteins, cell debris elimination, and preliminary puriffcation of the target protein were being clairmed. When this technique was employed for isolating recombinant Tumor Necrois Factor (TNF) from E.coli, overall protein codcentration and TNF activity were found to have been increased. More than 85% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficinet was greater than 3 and the TNF puriflcation fsctop was greater than 6. It is zhown that less separation steps were being utilized in the new techniqne, meaning a reduction in separation time and less process extractors required.展开更多
To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira b...To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.展开更多
Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have b...Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have been proposed to identify essential proteins. Unfortunately, most methods based on network topology only consider the interactions between a protein and its neighboring proteins, and not the interactions with its higher-order distance proteins. In this paper, we propose the DSEP algorithm in which we integrated network topology properties and subcellular localization information in protein–protein interaction(PPI) networks based on four-order distances, and then used random walks to identify the essential proteins. We also propose a method to calculate the finite-order distance of the network, which can greatly reduce the time complexity of our algorithm. We conducted a comprehensive comparison of the DSEP algorithm with 11 existing classical algorithms to identify essential proteins with multiple evaluation methods. The results show that DSEP is superior to these 11 methods.展开更多
Globally,the prevalence of anxiety and depression has reached epidemic proportions.Food-derived protein hydrolysates and peptides delivered through dietary supplementation can avoid the negative risks associated with ...Globally,the prevalence of anxiety and depression has reached epidemic proportions.Food-derived protein hydrolysates and peptides delivered through dietary supplementation can avoid the negative risks associated with traditional pharmaceuticals while delivering superior anxiolytic and antidepressant effects.This review summarizes current research on food-derived anxiolytic and antidepressant protein hydrolysates and peptides,and subsequently analyses their physicochemical characteristics and elaborates on their mechanisms.The aim of this work is to contribute to the in-depth study and provide a theoretical foundation for the development of related products to better serve patients with anxiety and depression.展开更多
Cowpea (Vigna unguiculata L. Walp) is a multi-purpose legume with high quality protein for human consumption and livestock. The objective of this work was to develop near-infrared spectroscopy (NIRS) prediction models...Cowpea (Vigna unguiculata L. Walp) is a multi-purpose legume with high quality protein for human consumption and livestock. The objective of this work was to develop near-infrared spectroscopy (NIRS) prediction models to estimate protein content in cowpea. A total of 116 cowpea breeding lines with a wide range of protein contents (19.28 % to 32.04%) were selected to build the model using whole seed and ground seed samples. Partial least-squares discriminant analysis (PLS-DA) regression technique with different pre-treatments (derivatives, standard normal variate, and multiplicative scatter correction) were carried out to develop the protein prediction model. Results showed: 1) spectral plots of both the whole seed and ground seed showed higher spectral scatter at higher wavelengths (>1450 nm), 2) data pre-processing affects prediction accuracy for bot whole seed and ground seed samples, 3) prediction using ground seed samples (0.64 R<sup>2</sup> 0.85) is better than the whole seed (0.33 R<sup>2</sup> 0.78), and 4) the data pre-processing second derivative with standard normal variate has the best prediction (R<sup>2</sup>_whole seed = 0.78, R<sup>2</sup>_ground seed = 0.85). The results will be of interest in cowpea breeding programs aimed at improving total seed protein content.展开更多
Plant height influences plant architecture,lodging resistance,and yield performance.It is modulated by gibberellic acid(GA)metabolism and signaling.DELLA proteins,acting as central repressors of GA signaling,integrate...Plant height influences plant architecture,lodging resistance,and yield performance.It is modulated by gibberellic acid(GA)metabolism and signaling.DELLA proteins,acting as central repressors of GA signaling,integrate various environmental and hormonal signals to regulate plant growth and development in Arabidopsis.We examined the role of two DELLA proteins,GmRGAa and GmRGAb,in soybean plant height control.Knockout of these proteins led to longer internodes and increased plant height,primarily by increasing cell elongation.GmRGAs functioned under different light conditions,including red,blue,and far-red light,to repress plant height.Interaction studies revealed that GmRGAs interacted with the blue light receptor GmCRY1b.Consistent with this,GmCRY1b partially regulated plant height via GmRGAs.Additionally,DELLA proteins were found to stabilize the protein GmSTF1/2,a key positive regulator of photomorphogenesis.This stabilization led to increased transcription of GmGA2ox-7b and subsequent reduction in plant height.This study enhances our understanding of DELLA-mediated plant height control,offering Gmrgaab mutants for soybean structure and yield optimization.展开更多
Background: Preeclampsia is reported to complicate 2% - 8% of pregnancies globally and is an important cause of maternal and perinatal morbidity and mortality. The aetiology and pathogenesis are still poorly understoo...Background: Preeclampsia is reported to complicate 2% - 8% of pregnancies globally and is an important cause of maternal and perinatal morbidity and mortality. The aetiology and pathogenesis are still poorly understood and substantial improvement has not been made in the prediction, prevention and treatment of the disease. Objective: To compare the frequency of activated protein C resistance (APC-R) in patients with pre-eclampsia to that of normotensive pregnant women and to determine the correlation between activated protein ratio (APC-ratio) and the severity of pre-eclampsia. Methodology: A cross-sectional study was carried out in 100 pre-eclamptic patients and 100 normotensive pregnant controls. The APC-ratio was determined using the modified activated partial thromboplastin time. Study participants with APC-ratio of less than 2.0 were defined as having APC-R. Data was analyzed using SPSS version 22.0. Results: Mean APC-ratio was significantly lower in pre-eclamptics (2.89 ± 1.70) compared to normotensive pregnant women (3.57 ± 1.06) (p = 0.0008) and the levels were also higher in mild (2.95 ± 1.15) compared to severe pre-eclamptics (2.62 ± 1.14). The frequency of APC-R was 26% among women with pre-eclampsia compared to 4% among normotensive controls (p = 0.000). Among 100 pre-eclamptic women 7 (21.2%) out of 33 with mild pre–eclampsia had APC-R, while 19 (28.4%) out of 67 with severe pre-eclampsia had APC-R. APC-ratio had a significant negative correlation with mean arterial blood pressure (r = −0.324;p = 0.000) and proteinuria (r = −0.379;p = 0.000) among study participants. Conclusion: The frequency of activated protein c resistance is significantly higher in pre-eclamptics compared to normotensive pregnant women and this is more pronounced in those with severe pre-eclampsia compared with those with mild disease. APC-R may therefore be used as a marker of severity in the disease.展开更多
Primary Budd-Chiari syndrome (BCS) is a spontaneously fatal disease characterized by an obstruction of the hepatic venous outflow tract due to thrombosis or a primary disease of the venous wall. The primary form of BC...Primary Budd-Chiari syndrome (BCS) is a spontaneously fatal disease characterized by an obstruction of the hepatic venous outflow tract due to thrombosis or a primary disease of the venous wall. The primary form of BCS is extremely rare. This is a disease mainly affecting young adults of both sexes. Clinical manifestations are variable;they can be asymptomatic, acute, or subacute but mostly chronic. Several causes have been identified, such as myeloproliferative syndrome, antiphospholipid syndrome, paroxysmal nocturnal hemoglobinuria, and inherited thrombotic disorders. Data on primary BCS in Sub-Saharan Africa is rare as most publications available are case reports. In these reports, the causes are unknown with poor prognosis in most cases often leading to patient death. We herein present a case report of a male patient diagnosed with a primary BCS at Yaoundé General Hospital (Cameroon) caused by a Protein C deficiency who presented with ascites decompensating liver cirrhosis. Treatment was based on anticoagulants, diuretics and laxatives administration. Two years after the diagnosis, the patient is alive with clinical and paraclinical improvement.展开更多
The unfolded protein response(UPR)is a cellular stress response pathway activated when the endoplasmic reticulum,a crucial organelle for protein folding and modification,encounters an accumulation of unfolded or misfo...The unfolded protein response(UPR)is a cellular stress response pathway activated when the endoplasmic reticulum,a crucial organelle for protein folding and modification,encounters an accumulation of unfolded or misfolded proteins.The UPR aims to restore endoplasmic reticulum homeostasis by enhancing protein folding capacity,reducing protein biosynthesis,and promoting protein degradation.It also plays a pivotal role in coordinating signaling cascades to determine cell fate and function in response to endoplasmic reticulum stress.Recent research has highlighted the significance of the UPR not only in maintaining endoplasmic reticulum homeostasis but also in influencing various physiological processes in the nervous system.Here,we provide an overview of recent findings that underscore the UPR’s involvement in preserving the function and viability of neuronal and myelinating cells under physiological conditions,and highlight the critical role of the UPR in brain development,memory storage,retinal cone development,myelination,and maintenance of myelin thickness.展开更多
Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis ...Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.展开更多
文摘To clone human arresten gene and investigate biological activity of the recombinant protein.Methods Human arresten gene was obtained from the plasmid pGEMArr and subcloned into the BamHⅠ and Pst Ⅰ restriction sites of prokaryotic expression vector pRSET containing T7 promoter.The recombinant plasmid pRSETAN was subsequently transformed into the strain E.coli BL21(DE3),and the target gene was expressed under induction of IPTG.The expressed protein was extracted,purified by Ni 2+ chelation affinity chromatography and refoled.The effect of the recombinant protein on proliferation of human umbilical vein endothelial cells (HUVECs) was also analyzed with the MTT assay.Results Endonuclease digesting and DNA sequencing confirmed that the arresten gene was correctly inserted into the expression vector.The recombinant protein was hightly expressed in the form of inclusion body in the host bacteria after induction.SDS-PAGE analysis revealed that the recombinant protein with a molecular weight of 26×103 amounted to 27% of the total bacterial proteins.The purity of the expected protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could reach over 96% through affinity chromatography.After renaturation,the recombinant protein could significantly suppress proliferation of human umbilical vein endothelia cells(HUVECs) induced by vascular endothelial growth factor(VEGF).Conclusion Human arresten gene was successfully cloned into the expression vector pRSET and expressed at high level in Escherichia coli.Purified and refolded arresten protein could effectively inhibit proliferation of vascular endothelia cells.2 refs.
文摘Porcine epidemic diarrhea (PED), a devastating enteric disease in pigs, is caused by PEDvirus (PEDV)(1)Reduced severity of clinical diseases was reported to associate with neutralizing antibody titers in colostrum. However, viral neutralization assay(VN) is laborious and not suitable for routine diagnosis. Spike protein plays an important role in stimulating neutralizing antibody that might be suitable for PEDV diagnosis.
文摘Enteric disorders in pigs are related to the fimbriae F4 (K88), F5 (K99), F6 (987P), F41 and F18 of enterotoxigenic Escherichia coli (ETEC). Immunization of sows with adhesins is important to stimulate the production of antibodies and the consequent transfer of these to the piglets via colostrum to prevent diarrhea during the neonate period and after weaning. The objective of this study was to evaluate the immune response of the sows immunized with recombinant ETEC proteins (F4, F5, F6, F18 and F41). The immune response of the sows immunized with the recombinant proteins was compared with a commercial vaccine containing ETEC bacterins. The study was performed on a commercial farm and included nine pregnant sows divided into three groups: G1 was vaccinated with recombinant proteins (n = 3);G2 was vaccinated with the commercial vaccine (n = 3);and G3 was vaccinated with sterile buffered saline (PBS) (n = 3). All the sows were fed a balanced diet without antibiotics and water ad libitum. The recombinant fimbriae stimulated the specific humoral immune response of the immunized sows. There was a statistically significant increase in the levels of antibodies to the fimbriae F4 (K88), F5 (K99), F6 (987P) and F18 in the sows vaccinated with the recombinant proteins compared with the control group. The colostrum IgG titers for all fimbriae in all the immunized sows were significantly increased compared to the control group. Additionally, all the piglets exhibited significantly increased antibody levels relative to all fimbriae when compared with those in the unimmunized control group, demonstrating successful antibody transfer via colostrum of the sows to the piglets.
基金This work was supported by the National Key R&D Program of China(No.2018YFA0902200)the National Natural Science Foundation of China(Nos.21838001,21525625,31961133018 and 81803409)the Fundamental Research Funds for the Central Universities(Nos.buctrc201810 and XK1802-8).
文摘Trichoderma is an ascomycete fungal genus widely distributed in the soils.Several species were selected,engineered and utilized for protein production for decades.The high extracellular secretion capability and eukaryotic post-translational modification machinery make Trichoderma spp.particularly interesting hosts.In this review,we summarized the recombinant proteins produced in Trichoderma since 2014,concerning their origins,hosts,promoters,terminators,signal peptides,yields and commonly used media.Meanwhile,strategies and merging trends in protein production and strain engineering are classified and summarized regarding codon optimization,promoter utilization,transcription factor regulation,post-translational modification and proteolytic degradation inhibition.With state-of-art biotechnologies and more available expression platforms,Trichoderma spp.could be more successful hosts to produce recombinant proteins as desired,i.e.better enzyme formula for efficient cellulose degradation or functional protein with high purity and yield.
基金supported in part by the Chinese National Basic Research Program(973)2009CB118900Chinese National Science Foundation Project30771451Boyce Thompson Institute Project BTI-QAU1-23-2007
文摘It is well known that Tn5B1-4 (commercially known as the High Five) cell line is highly susceptible to baculovirus and provides superior production of recombinant proteins when compared to other insect cell lines.But the characteristics of the cell line do not always remain stable and may change upon continuous passage.Recently an alphanodavirus,named Tn5 Cell Line Virus (or TNCL Virus),was identified in High Five cells in particular.Therefore,we established a new cell line,QB-Tn9-4s,from Trichoplusia ni,which was determined to be free of TNCL virus by RT-PCR analysis.In this paper,we describe the development of a novel cell clone,QB-CL-B,from a low passage QB-Tn9-4s cell line and report its susceptibility to AcMNPV,and the level of recombinant protein production.This cell clone was similar to its parental cells QB-Tn9-4s and Tn5B1-4 cells in morphology and growth rate;although it also showed approximately the same responses to AcMNPV infection and production of occlusion bodies,there were higher levels of recombinant protein production in comparison to QB-Tn9-4s (parental cells) and High5 cells.
基金supported by the National Science and Technology Major Project[No.2013ZX10004-001]
文摘Objective In this study, we evaluated the diagnostic efficiency of six recombinant proteins for the serodiagnosis of Lyme borreliosis(LB) and screened out the appropriate antigens to support the production of a Chinese clinical ELISA(enzyme-linked immunosorbent assay) kit for LB. Methods Six recombinant antigens, Fla B.g, Osp C B.a, Osp C B.g, P39 B.g, P83 B.g, and Vls E B.a, were used for ELISA to detect serum antibodies in LB, syphilis, and healthy controls. The ELISA results were used to generate receiver operating characteristic(ROC) curves, and the sensitivity and specificity of each protein was evaluated. All recombinant proteins were evaluated and screened by using logistic regression models. Results Two Ig G(Vls E and Osp C B.g) and two Ig M(Osp C B.g and Osp C B.a) antigens were left by the logistic regression model screened. Vls E had the highest specificity for syphilis samples in the Ig G test(87.7%, P<0.05). Osp C B.g had the highest diagnostic value in the Ig M test(AUC=0.871). Interactive effects between Osp C B.a and Fla B.g could reduce the specificity of the ELISA. Conclusion Three recombinant antigens, Osp C B.g, Osp C B.a, and Vls E B.a, were useful for ELISAs of LB. Additionally, the interaction between Osp C B.a and Fla B.g should be examined in future research.
文摘The engineering genetic technology has developed Bt maize events which contain recombinant protein that will be safe for the consumer. The aflatoxins are contaminants present in maize capable of producing cancer and decreasing the immune response in human, additionally contained polyphenols compounds considered non nutritive. The objective of this study was to identify the presence of recombinant protein in hybrid and local varieties of corn and evaluate the content of aflatoxins and tannins. 25 samples of white grain maize for human consumption were collected, 12 were for hybrid maize and 13 local varieties, from the states of Hidalgo, Mexico and Morelos. Samples were analyzed for Cry1Ab/Cry1Ac, using lateral flow strip method, crude protein and lipids by standard methods. Aflatoxins were assessed by comercial Elisa kit and tannins by spectroscopy method. The data were grouped in a completely random model and an analysis of variance was performed. The results indicated that 44.5% of hybrid corn was positive by Bt-Cry1Ab/1Ac proteins, containing 9.02% ± 2.5 lipids and 11.33% ± 2.2 crude protein, 189 ± 0.92 mg/g of tannins and 6.36 ± 3.3 μg·g-1 aflatoxins. The local maize samples (55.5%) were negative to Bt-Cry1Ab/ Cry1Ac, which protein content was of 8.68% ± 0.90, 6.14% lipids ± 2.3, 273 ± 0.40 mg/100g tannin and 7.15 ± 3.3 μg·g-1 of aflatoxins. In conclusion, we observed an improvement of nutrient composition in hybrid maize with Bt proteins, and decrease in tannins content comparing with some local varieties without Bt proteins. The effectiveness of Bt maize expressing the Cry1Ab/ Cry1Ac in reducing aflatoxin contamination was not observed, therefore, additive affects of aflatoxins contamination in maize Bt-Cry need to be further investigated in cancer disease development.
基金supported by National Key R&D Program of China [2017YFC1200503]Emergency research Program on the prevention and control of Ebola hemorrhagic fever [1061400100275]
文摘Objective In previous studies, we immunized mice with Ebola recombinant protein vaccine and gene vector vaccine. Both stimulated high levels of humoral immunity. In this work, we constructed a pseudovirus containing Ebola membrane proteins to verify whether the two immunization strategies can induce neutralizing antibodies in mice. Methods A pseudovirus containing an Ebola virus membrane protein based on the HIV-1 viral gene sequence was constructed and evaluated using a known neutralizing antibody. The titer of the neutralizing antibody in the sera of mice immunized with the recombinant protein and the gene vector vaccine was examined using a neutralization test. Results Ebola pseudovirus was successfully prepared and applied for neutralizing antibody detection. Immunological experiments showed that recombinant protein GP-Fc and gene vaccine pVR-modGP-Fc had good immunogenicity. The titer of the bound antibody in the serum after 8 weeks of immunization in mice was more than 1:105, and the recombinant protein induced greater humoral immunity. The results of the neutralization test based on the Ebola pseudovirus system demonstrated that both vaccines induced production of protective antibodies, while the gene vaccine induced a higher titer of neutralizing antibodies. Conclusion An Ebola pseudovirus detection system was successfully established and used to evaluate two Ebola vaccines. Both produced good immunogenicity. The findings lay the foundation for the development of new Ebola vaccines and screening for neutralizing monoclonal antibodies.
基金Supported by the National Natural Science Foundation of China(No.295256O9 and 29736180).
文摘A new technique was developed for the integrated processing of cell disruption and aqueous two-phase extraction in a high-speed bead mill to separate intracellular proteins form microbial cells. The process was narned as simultanecus cell disruption and aqueous two-phase extraction (ADATE). Advantages, such as high cell disruption efficiency, biochemical activities proservation of proteins, cell debris elimination, and preliminary puriffcation of the target protein were being clairmed. When this technique was employed for isolating recombinant Tumor Necrois Factor (TNF) from E.coli, overall protein codcentration and TNF activity were found to have been increased. More than 85% of TNF was partitioned into the top phase and all cell debris were in the bottom phase. The partition coefficinet was greater than 3 and the TNF puriflcation fsctop was greater than 6. It is zhown that less separation steps were being utilized in the new techniqne, meaning a reduction in separation time and less process extractors required.
文摘To investigate the existence of the major outer membrane protein (MOMP) gene LipL32 in 15 dominant Chinese strains of 15 serogroups of Leptospira interrogans and 2 international strains of 2 serogroups of Leptospira biflexa, and to clone and construct the expression system as well as to identify the recombinant proteins, genomic DNAs from strains of leptospira were prepared by routine phenol-chloroform method, and the fragments of the LipL32 gene with the whole length from the strains were amplified with high fidelity PCR. The target amplification products were sequenced after T-A cloning, and the expression system for the genes were thereby constructed. Expression of the recombinant proteins was identified by using SDS-PAGE after induction with IPTG at different dosages. Western blot assays with rabbit antiserum against the whole cell of TR/PatocⅠ of Leptospira and immunized serum with rMOMPs were used to determine the immunoreactivity and immunogenicity of the recombinant proteins. Microscopic agglutination test was used to determine the cross- agglutination titres in rabbit sera immunized with rMOMPs, and the cell adherence model of Leptospira was used to examine the blocking effects of rabbit antisera against these rMOMPs. It was found that the LipL32 gene could be found in all the 17 strains of Leptospira mentioned above with two different genotypes, i.e. LipL32/1 and LipL32/2. Amounts of expressions of rMOMP1 and rMOMP2 after IPTG accounted for 40% and 10% of the total bacterial proteins respectively. Both rMOMP1 and rMOMP2 could combine with the rabbit antiserum against leptospiral TR/PatocⅠ, and could induce the production of agglutination antibodies to these 17 strains of Leptospira with 1∶2 to 1∶64 MAT titres. The rabbit anti-rMOMP1 and anti-MOMP2 antibodies at 1∶2 to 1∶16 dilutions could efficiently block adherence of Leptospira. It concludes that all the Leptospira tested in the present study possess LipL32/1 or LipL32/2 genes, and the constructed expression system can express the rMOMP1 and rMOMP2. These recombinant proteins are showed to have good immunogenicity and satisfactory immunoreactivity.
基金Acknowledgment This work was supported by grants from the National Natural Science Foundation （30671563, 30700597） and Key Project of Science and Technology of Gansu Province （0801NKDA034） and by gifts from the Development Planning Project of Science and Technology of Lanzhou （07-2-12, 2008- l- 167）.
基金Project supported by the Gansu Province Industrial Support Plan (Grant No.2023CYZC-25)the Natural Science Foundation of Gansu Province (Grant No.23JRRA770)the National Natural Science Foundation of China (Grant No.62162040)。
文摘Essential proteins are inseparable in cell growth and survival. The study of essential proteins is important for understanding cellular functions and biological mechanisms. Therefore, various computable methods have been proposed to identify essential proteins. Unfortunately, most methods based on network topology only consider the interactions between a protein and its neighboring proteins, and not the interactions with its higher-order distance proteins. In this paper, we propose the DSEP algorithm in which we integrated network topology properties and subcellular localization information in protein–protein interaction(PPI) networks based on four-order distances, and then used random walks to identify the essential proteins. We also propose a method to calculate the finite-order distance of the network, which can greatly reduce the time complexity of our algorithm. We conducted a comprehensive comparison of the DSEP algorithm with 11 existing classical algorithms to identify essential proteins with multiple evaluation methods. The results show that DSEP is superior to these 11 methods.
基金supported by the National Key Research and Development Program of China (2021YFD2100402)the National Natural Science Foundation of China (81903275)the Fund of the Cultivation Project of Double First-Class Disciplines of Food Science and Engineering,Beijing Technology&Business University (BTBUYXTD202203)。
文摘Globally,the prevalence of anxiety and depression has reached epidemic proportions.Food-derived protein hydrolysates and peptides delivered through dietary supplementation can avoid the negative risks associated with traditional pharmaceuticals while delivering superior anxiolytic and antidepressant effects.This review summarizes current research on food-derived anxiolytic and antidepressant protein hydrolysates and peptides,and subsequently analyses their physicochemical characteristics and elaborates on their mechanisms.The aim of this work is to contribute to the in-depth study and provide a theoretical foundation for the development of related products to better serve patients with anxiety and depression.
文摘Cowpea (Vigna unguiculata L. Walp) is a multi-purpose legume with high quality protein for human consumption and livestock. The objective of this work was to develop near-infrared spectroscopy (NIRS) prediction models to estimate protein content in cowpea. A total of 116 cowpea breeding lines with a wide range of protein contents (19.28 % to 32.04%) were selected to build the model using whole seed and ground seed samples. Partial least-squares discriminant analysis (PLS-DA) regression technique with different pre-treatments (derivatives, standard normal variate, and multiplicative scatter correction) were carried out to develop the protein prediction model. Results showed: 1) spectral plots of both the whole seed and ground seed showed higher spectral scatter at higher wavelengths (>1450 nm), 2) data pre-processing affects prediction accuracy for bot whole seed and ground seed samples, 3) prediction using ground seed samples (0.64 R<sup>2</sup> 0.85) is better than the whole seed (0.33 R<sup>2</sup> 0.78), and 4) the data pre-processing second derivative with standard normal variate has the best prediction (R<sup>2</sup>_whole seed = 0.78, R<sup>2</sup>_ground seed = 0.85). The results will be of interest in cowpea breeding programs aimed at improving total seed protein content.
基金supported by the Sci-Tech Innovation 2030(2022ZD0400701-2)Agricultural Science and Technology Innovation Program of CAAS+1 种基金the National Natural Science Foundation of China(31871705)the Central Public-Interest Scientific Institution Basal Research Fund。
文摘Plant height influences plant architecture,lodging resistance,and yield performance.It is modulated by gibberellic acid(GA)metabolism and signaling.DELLA proteins,acting as central repressors of GA signaling,integrate various environmental and hormonal signals to regulate plant growth and development in Arabidopsis.We examined the role of two DELLA proteins,GmRGAa and GmRGAb,in soybean plant height control.Knockout of these proteins led to longer internodes and increased plant height,primarily by increasing cell elongation.GmRGAs functioned under different light conditions,including red,blue,and far-red light,to repress plant height.Interaction studies revealed that GmRGAs interacted with the blue light receptor GmCRY1b.Consistent with this,GmCRY1b partially regulated plant height via GmRGAs.Additionally,DELLA proteins were found to stabilize the protein GmSTF1/2,a key positive regulator of photomorphogenesis.This stabilization led to increased transcription of GmGA2ox-7b and subsequent reduction in plant height.This study enhances our understanding of DELLA-mediated plant height control,offering Gmrgaab mutants for soybean structure and yield optimization.
文摘Background: Preeclampsia is reported to complicate 2% - 8% of pregnancies globally and is an important cause of maternal and perinatal morbidity and mortality. The aetiology and pathogenesis are still poorly understood and substantial improvement has not been made in the prediction, prevention and treatment of the disease. Objective: To compare the frequency of activated protein C resistance (APC-R) in patients with pre-eclampsia to that of normotensive pregnant women and to determine the correlation between activated protein ratio (APC-ratio) and the severity of pre-eclampsia. Methodology: A cross-sectional study was carried out in 100 pre-eclamptic patients and 100 normotensive pregnant controls. The APC-ratio was determined using the modified activated partial thromboplastin time. Study participants with APC-ratio of less than 2.0 were defined as having APC-R. Data was analyzed using SPSS version 22.0. Results: Mean APC-ratio was significantly lower in pre-eclamptics (2.89 ± 1.70) compared to normotensive pregnant women (3.57 ± 1.06) (p = 0.0008) and the levels were also higher in mild (2.95 ± 1.15) compared to severe pre-eclamptics (2.62 ± 1.14). The frequency of APC-R was 26% among women with pre-eclampsia compared to 4% among normotensive controls (p = 0.000). Among 100 pre-eclamptic women 7 (21.2%) out of 33 with mild pre–eclampsia had APC-R, while 19 (28.4%) out of 67 with severe pre-eclampsia had APC-R. APC-ratio had a significant negative correlation with mean arterial blood pressure (r = −0.324;p = 0.000) and proteinuria (r = −0.379;p = 0.000) among study participants. Conclusion: The frequency of activated protein c resistance is significantly higher in pre-eclamptics compared to normotensive pregnant women and this is more pronounced in those with severe pre-eclampsia compared with those with mild disease. APC-R may therefore be used as a marker of severity in the disease.
文摘Primary Budd-Chiari syndrome (BCS) is a spontaneously fatal disease characterized by an obstruction of the hepatic venous outflow tract due to thrombosis or a primary disease of the venous wall. The primary form of BCS is extremely rare. This is a disease mainly affecting young adults of both sexes. Clinical manifestations are variable;they can be asymptomatic, acute, or subacute but mostly chronic. Several causes have been identified, such as myeloproliferative syndrome, antiphospholipid syndrome, paroxysmal nocturnal hemoglobinuria, and inherited thrombotic disorders. Data on primary BCS in Sub-Saharan Africa is rare as most publications available are case reports. In these reports, the causes are unknown with poor prognosis in most cases often leading to patient death. We herein present a case report of a male patient diagnosed with a primary BCS at Yaoundé General Hospital (Cameroon) caused by a Protein C deficiency who presented with ascites decompensating liver cirrhosis. Treatment was based on anticoagulants, diuretics and laxatives administration. Two years after the diagnosis, the patient is alive with clinical and paraclinical improvement.
基金supported by grants from the National Institutes of Health,No.NS105689(to WL)the Department of Defense through the Multiple Sclerosis Research Program,No.W81XWH-22-1-0757(to WL).
文摘The unfolded protein response(UPR)is a cellular stress response pathway activated when the endoplasmic reticulum,a crucial organelle for protein folding and modification,encounters an accumulation of unfolded or misfolded proteins.The UPR aims to restore endoplasmic reticulum homeostasis by enhancing protein folding capacity,reducing protein biosynthesis,and promoting protein degradation.It also plays a pivotal role in coordinating signaling cascades to determine cell fate and function in response to endoplasmic reticulum stress.Recent research has highlighted the significance of the UPR not only in maintaining endoplasmic reticulum homeostasis but also in influencing various physiological processes in the nervous system.Here,we provide an overview of recent findings that underscore the UPR’s involvement in preserving the function and viability of neuronal and myelinating cells under physiological conditions,and highlight the critical role of the UPR in brain development,memory storage,retinal cone development,myelination,and maintenance of myelin thickness.
基金supported by the National Natural Science Foundation of China(32001110)Training Program for Cultivating Highlevel Talents by the China Scholarship Council(2021lxjjw01)Open Project of State Key Laboratory of Plateau Ecology and Agriculture,Qinghai University(2021-KF-004)。
文摘Changes in protein abundance and reversible protein phosphorylation(RPP)play important roles in regulating hypometabolism but have never been documented in overwintering frogs at high altitudes.To test the hypothesis that protein abundance and phosphorylation change in response to winter hibernation,we conducted a comprehensive and quantitative proteomic and phosphoproteomic analysis of the liver of the Xizang plateau frog,Nanorana parkeri,living on the Qinghai-Xizang Plateau.In total,5170 proteins and 5695 phosphorylation sites in 1938 proteins were quantified.Based on proteomic analysis,674 differentially expressed proteins(438 up-regulated,236 down-regulated)were screened in hibernating N.parkeri versus summer individuals.Functional enrichment analysis revealed that higher expressed proteins in winter were significantly enriched in immune-related signaling pathways,whereas lower expressed proteins were mainly involved in metabolic processes.A total of 4251 modified sites(4147 up-regulated,104 down-regulated)belonging to 1638 phosphoproteins(1555 up-regulated,83 down-regulated)were significantly changed in the liver.During hibernation,RPP regulated a diverse array of proteins involved in multiple functions,including metabolic enzymatic activity,ion transport,protein turnover,signal transduction,and alternative splicing.These changes contribute to enhancing protection,suppressing energy-consuming processes,and inducing metabolic depression.Moreover,the activities of phosphofructokinase,glutamate dehydrogenase,and ATPase were all significantly lower in winter compared to summer.In conclusion,our results support the hypothesis and demonstrate the importance of RPP as a regulatory mechanism when animals transition into a hypometabolic state.
