AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HB...AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.展开更多
To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellu...To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.展开更多
Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmet...Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3' terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4 -ADNF-9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the pBV220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.展开更多
This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The...This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection(P<0.01 vs. control group). RT-PCR showed that the level of HBV mRNA decreased by 80.2%(t=–99.22, P<0.01), the genomic HBV DNA by 92.8%(t=–73.06, P<0.01), and the supernatant of HBV DNA copy number by 89.8%(t=–47.13, P<0.01) in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.展开更多
Myostatin(MSTN)is a negative regulator of skeletal muscle growth,in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells,we constructed co-expression v...Myostatin(MSTN)is a negative regulator of skeletal muscle growth,in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells,we constructed co-expression vector pcDNA3.1-ProMSTNshRNA,transfected it into muscle satellite cells by Liposome 2000,and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h.The expressions of P21 and CDK2 were detected by Western blot and real-time PCR.The results showed that the cell vitality of experimental groups significantly increased than that of the negative control,and cells in S phase also increased significantly(P<0.05).After knocked down MSTN gene,P21 expression decreased(P<0.05),but CDK2 gene expression increased(P<0.05).These results indicated that MSTN gene expression was associated with P21 and CDK2,the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited,which provided a theoretical basis for the study on transgenic cattle.展开更多
Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ templa...Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220,and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3'terminal of the signal and leader peptides of NT4,and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.展开更多
The Aspergillus niger XynB gene and core promoter region of porcine RELMβgene were cloned into pcDNA3.1(-),and an intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP carrying green fluorescence and Myc ...The Aspergillus niger XynB gene and core promoter region of porcine RELMβgene were cloned into pcDNA3.1(-),and an intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP carrying green fluorescence and Myc double tags was constructed.The vector was transfected into human colon cancer cells(HT29)and human liver cancer cells(Bel7402)using liposomes.Fluorescence microscopy revealed that the vector could specifically express green fluorescent protein(GFP)in HT29 cells.RT-PCR and Western Blot were performed on the HT29 cells transfected with the expression vector,and the results showed that the XynB gene was normally transcribed in HT29 cells,and the target protein expression was detected in the cells.展开更多
Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by...Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase展开更多
A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will b...A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.展开更多
Introduction:Multiple myeloma (MM) is a geriatric disease with onset at an average age of approximately 61 years.With the aging of the population,the incidence rate of MM is climbing.In the United States,the annual in...Introduction:Multiple myeloma (MM) is a geriatric disease with onset at an average age of approximately 61 years.With the aging of the population,the incidence rate of MM is climbing.In the United States,the annual incidence rate of MM is 2-5/100,000.Multiple myeloma accounts for approximately 1% of all tumor eases and slightly 】10% of cases with hematologic malignancy.Although an展开更多
Introduction:Multiple myeloma(MM)is a geriatric dis-ease with onset at an average age of approximately 61years.With the aging of the population,the incidence rate ofMM is climbing.In the United States,the annual incid...Introduction:Multiple myeloma(MM)is a geriatric dis-ease with onset at an average age of approximately 61years.With the aging of the population,the incidence rate ofMM is climbing.In the United States,the annual incidence展开更多
Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmi...Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using展开更多
Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin(PLF N) . A pair of primers was designed with Oligo6.0 and used to amplify PLF N gene. It was in accordance with the cha...Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin(PLF N) . A pair of primers was designed with Oligo6.0 and used to amplify PLF N gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR;the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLF N was created and identified by using plasmid extraction,PCR,restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393,Lactobacillus plantarum KLDS 1.0344,Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS1.0413 by electroporation,and produced the recombinant strains of pPG612-PLF N /L. casei,pPG612-PLF N /L. plantarum,pPG612-PLF N /L. paracasei and pPG612-PLF N /L. pentosus,respectively. The results indicated that PLF N gene had inserted into the expression vectors and achieved multiple Lactobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobacillus.展开更多
High yield,high quality,stable yield,adaptability to growth period,and modern mechanization are the basic requirements for crops in the 21st century.Soybean oleic acid is a natural unsaturated fatty acid with strong a...High yield,high quality,stable yield,adaptability to growth period,and modern mechanization are the basic requirements for crops in the 21st century.Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability.Known as a safe fatty acid,it has the ability to successfully prevent cardiovascular and cerebrovascular disorders.Improving the fatty acid composition of soybean seeds,can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans,but also have important significance in human health,and provide the possibility for the development of soybean oil as a new energy source.Hence,the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean.In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis,a 662 bp fragment was acquired by specific PCR amplification,and the pMD18T cloning vector was linked by the use of a seamless cloning technique.Bioinformatics analysis of the signal peptide prediction,subcellular localization,protein hydrophobicity,transmembrane region analysis,a phosphorylation site,protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out.The plasmid of the gene editing vector is pBK041.The overexpression vector was transformed from pCAMBIA3301 as the base vector,and overexpression vector were designed.Positive plants were obtained by genetic transformation by the pollen tube channel method.Fluorescence quantitative PCR was performed on the T2 generation plants to detect the relative expression levels in different tissues.Southern Blot was used to detect the presence of hybridization signal.Screening genes BAR,35S,and NOS in plants were identified by conventional PCR.10 seeds with high and low oleic acid content were chosen for quantitative PCR identification,and finally,the concentration and morphology of soybean fatty acids were identified by nearfar infrared spectroscopy.On 10 seeds with an upper and lower oleic acid content,a quantitative fluorescence analysis was done.In Southern blot hybridization,the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy.Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots,stems,leaves,and seeds was 1.70,1.67,3.83,and 4.41,respectively.Positive expression seeds had a 4.77%increase in oleic acid content.The level of oleic acid in the altered seeds was reduced by 4.13%when compared to CK,and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean.There has not been a discussion of gene cloning or functional verification.The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid,which lays a foundation for the study of soybean with high oleic acid.展开更多
基金Supported by Grants from National Natural Science Foundation of China, No. 30901344
文摘AIM: To generate recombinant adenoviral vector con-taining calreticulin (CRT)-hepatitis B surface antigen (HBsAg) fusion gene for developing a safe, effective and HBsAg-specific therapeutic vaccine.METHODS: CRT and HBsAg gene were fused using polymerase chain reaction (PCR), endonuclease diges-tion and ligation methods. The fusion gene was cloned into pENTR/D-TOPO transfer vector after the base pairs of DNA (CACC) sequence was added to the 5′ end. Adenoviral expression vector containing CRT-HBsAg fusion gene was constructed by homologous recombinan-tion. The human embryo kidney (HEK) 293A cells were transfected with linearized DNA plasmid of the recombi-nant adenoviral vector to package and amplify recombi-nant adenovirus. The recombinant adenovirus titer was characterized using the end-dilution assay. The expres-sion of the CRT/HBsAg fusion protein in Ad-CRT/HBsAg infected 293A cells was detected by Western blotting.RESULTS: The CRT-HBsAg fusion gene was char-acterized by PCR and sequencing and its length and sequence were confirmed to be accurate. The CRT-HB-sAg fusion gene recombinant pENTR/D-TOPO transfer vector was constructed. The recombinant adenoviral vector, Ad-CRT/HBsAg, was generated successfully. The titer of Ad-CRT/HBsAg was characterized as 3.9 × 1011 pfu/mL. The CRT-HBsAg fusion protein was ex-pressed by HEK 293A cells correctly. CONCLUSION: CRT/HBsAg fusion gene recombinant replication-defective adenovirus expression vector is constructed successfully and this study has provided an experimental basis for further studies of Hepatitis B vi-rus gene therapy.
文摘To study the therapeutic effects of herpes simplex virus thymidine kinase gene transferred by the EBV based expression vector on experimental hepatocellular carcinoma, pDR2 TK gene was delivered into human hepatocellular carcinoma cell line SMMC 7721 by using liposome mediated transfection technique,and then gene expression was detected by RT PCR, and the killing effects were examined through MTT method. In the nude mice hepatoma model,the antitumor effects of pDR2 TK /GCV system was evaluated in terms of tumor growth. MTT results showed that the pDR2 TK /GCV had cytotoxic effect and about 70 % SMMC 7721 cells were killed when GCV was at 1000 μmol/L. In vivo experiment showed that the tumor size in nude mice with transferred pDR2 TK gene was significantly smaller than that in control group . On the 10th day the tumor in 3 mice (60 %) disappeared completely after GCV treatment. It is concluded that the pDR2 TK/GCV system has marked killing effects on the experimental hepatocellular carcinoma.
文摘Objective To construct a prokaryotic expression vector bearing fusion gene NT4-ADNF-9 for future studies on genetic therapies for sensorineural deafness. Methods Double strand ADNF-9 cDNA was synthesized using asymmetrical primer/ templates and ligated to the 3' terminal of signal and leader peptides of neurotrophin 4 (NT4). The fusion gene NT4 -ADNF-9, was subcloned into prokaryotic expression vector pBV220, and named pBV220/ NT4-ADNF-9. DNA sequence of the fusion gene was analyzed. The fusion protein was isolated by SDS-PAGE and its bioactivity was evaluated using primary culture of day 8 chicken embryonic DRGcells. Results The correct sequence of fusion gene NT4-ADNF-9 was successfully subcloned into the pBV220 vector. The expressed ADNF-9 protein showed its effects in promoting cell survival and neurite growth. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 was constructed successfully and the expressed fusion protein demonstrated satisfactory bioactivity.
基金supported by grants from the Independent Innovation Research Fund of Huazhong University of Science and Technology(No.2016YXMS200)Natural Science Foundation of the Science and Technology Department of Hubei Province(No.ZRMS2017000406)
文摘This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection(P<0.01 vs. control group). RT-PCR showed that the level of HBV mRNA decreased by 80.2%(t=–99.22, P<0.01), the genomic HBV DNA by 92.8%(t=–73.06, P<0.01), and the supernatant of HBV DNA copy number by 89.8%(t=–47.13, P<0.01) in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.
基金Supported by the Major Special Projects of New Product Training of Transgenic Organisms(zx080072008-2008)
文摘Myostatin(MSTN)is a negative regulator of skeletal muscle growth,in order to study the effect of inhibition MSTN expression on the proliferation of bovine skeletal muscle satellite cells,we constructed co-expression vector pcDNA3.1-ProMSTNshRNA,transfected it into muscle satellite cells by Liposome 2000,and detected cell proliferation changes by CCK-8 method and flow cytometry after 48 h.The expressions of P21 and CDK2 were detected by Western blot and real-time PCR.The results showed that the cell vitality of experimental groups significantly increased than that of the negative control,and cells in S phase also increased significantly(P<0.05).After knocked down MSTN gene,P21 expression decreased(P<0.05),but CDK2 gene expression increased(P<0.05).These results indicated that MSTN gene expression was associated with P21 and CDK2,the proliferation of skeletal muscle satellite cells could be promoted while MSTN was inhibited,which provided a theoretical basis for the study on transgenic cattle.
基金supported by the National Natural Science Foundation of China (No.30471877)
文摘Objective To construct the prokaryotic expression vector bearing fusion gene NT4-ADNF-9 and lay foundation for further study on genetic therapy of neurosensory deafness. Methods By means of asymmetrical primer/ template,double stranded cDNA of activity dependent neurotrophic factor-9 (ADNF-9) was obtained,which included restriction enzymes sites on the two extremities. ADNF-9 cDNA was ligated to the signal and leader peptides of neurotrophin 4 (NT4),and the fusion gene was named NT4-ADNF-9. Then it was subcloned into prokaryotic expression vector pBV220,and called pBV220/ NT4-ADNF-9. Results Evidences of DNA sequence analysis and restriction enzymes digestion showed that we recombined ADNF-9 cDNA to the 3'terminal of the signal and leader peptides of NT4,and the fusion gene was subcloned into pBV220 successfully. Bioactivity of the products was proved that it could support the cell survival and neurite growth in the primary cultures of dorsal root ganglia (DRG) of embryonic day-8 chicken neurons as compared to the control. Conclusion Prokaryotic expression vector pBV220/NT4-ADNF-9 can be constructed successfully and the bioactivity is satisfactory.
基金Northern Jiangsu Science and Technology Special Project(XZ-SZ201921)the Technology Program of Gaoyou City(GY201914)。
文摘The Aspergillus niger XynB gene and core promoter region of porcine RELMβgene were cloned into pcDNA3.1(-),and an intestine-specific expression vector pcDNA3.1-RELMβ-XynB-Myc-GFP carrying green fluorescence and Myc double tags was constructed.The vector was transfected into human colon cancer cells(HT29)and human liver cancer cells(Bel7402)using liposomes.Fluorescence microscopy revealed that the vector could specifically express green fluorescent protein(GFP)in HT29 cells.RT-PCR and Western Blot were performed on the HT29 cells transfected with the expression vector,and the results showed that the XynB gene was normally transcribed in HT29 cells,and the target protein expression was detected in the cells.
文摘Objective To construct green fluorescent protein (GFP)-labeled pSELECT-GFP zeohBMP2 eukaryotic expression vector.Methods The encoding fragment of hBMP2 gene was obtained from a recombinant plasmid pcDNA3.1/CT-hBMP2 by using polymerase
文摘A number of eukaryotic expression vectors have been developed for use as DNA vaccines. They showed varying abilities to initiate immune responses;however, there is little data to indicate which of these vectors will be the most useful and practical for DNA vaccines in different species. This report examines the use of five expression vectors with different promoters and Kozak sequence to express the same hemagglutinin (HA) protein of an H6N2 avian influenza virus for DNA vaccination in chickens. Although intramuscular vaccination with seven DNA constructs elicited no or limited measurable H6 HA antibody responses in Hy-Line chickens, variable reduction in virus shedding for either oropharyngeal or cloacal swabs post-virus challenge were observed. This indicated that all DNA constructs generated some levels of protective immunity against homologous virus challenge. Interestingly, lower dose (50 or 100 μg) of plasmid DNAs consistently induced better immune response than higher dose (300 or 500 μg). In the transfection experiments there appeared to be a hierarchy in the in vitro expression efficiency in the order of pCAG-optiHAk/ pCAG-HAk > pCI-HAk > VR-HA > pCI-HA > pCI-neo-HA > pVAX-HA. Since the level of in vitro expression correlates with the level of immune response in vivo, in vitro expression levels of the DNA constructs can be used as an indicator for pre-selection of plasmid vaccines prior to in vivo assessment. Moreover, our results suggested that the Kozak sequence could be used as an effective tool for DNA vaccine design.
文摘Introduction:Multiple myeloma (MM) is a geriatric disease with onset at an average age of approximately 61 years.With the aging of the population,the incidence rate of MM is climbing.In the United States,the annual incidence rate of MM is 2-5/100,000.Multiple myeloma accounts for approximately 1% of all tumor eases and slightly 】10% of cases with hematologic malignancy.Although an
文摘Introduction:Multiple myeloma(MM)is a geriatric dis-ease with onset at an average age of approximately 61years.With the aging of the population,the incidence rate ofMM is climbing.In the United States,the annual incidence
文摘Objective To construct p IRES2-ZsG reen1/FⅨexpression vector,using the pcDNA/FⅨplasmid containing FⅨcDNA as template,and expressing in HEK-293cells.Methods The total ORF of FⅨgene was amlified from pcDNA/FⅨplasmid,then the amplified fragment was clonded into the p IRES2-ZsG reen1 vector using
基金Supported by National Natural Science Foundation of China (30871809,31072057)Principal Fund of Northeast Agricultural University
文摘Lactobacillus was selected as a bacterial carrier for expression of N-lobe of porcine lactoferrin(PLF N) . A pair of primers was designed with Oligo6.0 and used to amplify PLF N gene. It was in accordance with the characters of translational fusions from gene and expression vector plasmid. A 1 077 bp fragment of the gene from PLF was cloned from mammary gland tissue of the lactating sow on the third day by RT-PCR;the gene was connected with the vector plasmid pPG612.1 and transformed into the host strain JM109. The recombinant expression vector plasmid pPG612-PLF N was created and identified by using plasmid extraction,PCR,restriction enzyme digestion and sequence analysis. The recombinant plasmid was transformed into Lactobacillus casei ATCC393,Lactobacillus plantarum KLDS 1.0344,Lactobacillus paracasei KLDS 1.0652 and Lactobacillus pentosus KLDS1.0413 by electroporation,and produced the recombinant strains of pPG612-PLF N /L. casei,pPG612-PLF N /L. plantarum,pPG612-PLF N /L. paracasei and pPG612-PLF N /L. pentosus,respectively. The results indicated that PLF N gene had inserted into the expression vectors and achieved multiple Lactobacillus expression systems. It electes the base for the expression and production of recombinant porcine lactoferrin in Lactobacillus.
基金funded by the National Major Special Project for Breeding New Varieties of Genetically Modified Organisms(2016ZX08004-004)National Natural Science Foundation of China(31771817).
文摘High yield,high quality,stable yield,adaptability to growth period,and modern mechanization are the basic requirements for crops in the 21st century.Soybean oleic acid is a natural unsaturated fatty acid with strong antioxidant properties and stability.Known as a safe fatty acid,it has the ability to successfully prevent cardiovascular and cerebrovascular disorders.Improving the fatty acid composition of soybean seeds,can not only speed up the breeding process of high-quality high-oil and high-oleic soybeans,but also have important significance in human health,and provide the possibility for the development of soybean oil as a new energy source.Hence,the aim of this study was to analyze the high oleic acid elated gene GmSAM22 in soybean.In this research the soybean oleic acid-related gene GmSAM22 was screened out by Genome-wide association analysis,a 662 bp fragment was acquired by specific PCR amplification,and the pMD18T cloning vector was linked by the use of a seamless cloning technique.Bioinformatics analysis of the signal peptide prediction,subcellular localization,protein hydrophobicity,transmembrane region analysis,a phosphorylation site,protein secondary and tertiary structure and protein interaction analysis of the protein encoded by the SAM22 gene was carried out.The plasmid of the gene editing vector is pBK041.The overexpression vector was transformed from pCAMBIA3301 as the base vector,and overexpression vector were designed.Positive plants were obtained by genetic transformation by the pollen tube channel method.Fluorescence quantitative PCR was performed on the T2 generation plants to detect the relative expression levels in different tissues.Southern Blot was used to detect the presence of hybridization signal.Screening genes BAR,35S,and NOS in plants were identified by conventional PCR.10 seeds with high and low oleic acid content were chosen for quantitative PCR identification,and finally,the concentration and morphology of soybean fatty acids were identified by nearfar infrared spectroscopy.On 10 seeds with an upper and lower oleic acid content,a quantitative fluorescence analysis was done.In Southern blot hybridization,the SAM22 gene was integrated into the recipient soybean plant in hands of a sole copy.Fluorescence quantitative PCR appeared that the average relative expression of the SAM22 gene in roots,stems,leaves,and seeds was 1.70,1.67,3.83,and 4.41,respectively.Positive expression seeds had a 4.77%increase in oleic acid content.The level of oleic acid in the altered seeds was reduced by 4.13%when compared to CK,and it was discovered that the GmSAM22 gene could be a regulatory and secondary gene that promotes the conversion of stearic acid to oleic acid in soybean.There has not been a discussion of gene cloning or functional verification.The cloning and genetic transformation of the soybean SAM22 gene can effectively increase the content of oleic acid,which lays a foundation for the study of soybean with high oleic acid.
