Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.M...Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.展开更多
[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragmen...[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragments were inserted into plasmid pET28a to obtain recombinant vectors pET28a-S1 and pET28a-S2, respectively. These recombinant vectors were transformed into E. coli BL21, and expression of S1 and S2 fragments were induced by IPTG. The conditions of expression and purification were optimized. [Result] The S1 and S2 fragments were amplified and successfully expressed in E. coli. [Conclusion] This research provides detection antigens for follow-up development of SARS vaccine.展开更多
[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and p...[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and preliminarily identified biologic activity of the purified fusion proteins. [ Method ] ZF and CT cod- ing regions were amplified from MDM2 cDNA library by PCR and separately inserted into prokaryotie expression vector pET28b to construct the recombinant plas- mids. After verification by enzyme digestion, the recombinant plasmids were separately transformed into E. coli DH5c~ competent cells. The expressed recombinant plasmids were purified using Ni-NTA magnetic beads and identified by SDS-PAGE and Western blotting. [ Result] The molecular weight of His-ZF and His-CT fu- sion proteins was 21 and 31 kD, respectively. E Conclusion] Recombinant fusion proteins containing ZF and CT of MDM2 were obtained successfully, which laid the foundation for subsequent protein crystallization and three-dimensional structure analysis.展开更多
The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vect...The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.展开更多
基金Nanjing Science and Technology Plan Project(No.ZX20200009)Jiangsu Province Postgraduate Research and Practice Innovation Program(No.SJCX22-0895)。
文摘Objective:To construct a secretory eukaryotic expression vector of DSG2 fused with the Fc region of the human IgG,to validate its expression in 293T cells,and to purify the secretory protein with biological activity.Methods:The DSG2 extracellular domain fragment gene(DSG2ex),was amplified by PCR,and was inserted into the eukaryotic expression plasmid pCMV3-IgG1 to construct the recombinant eukaryotic expression plasmid-pCMV3-DSG2ex-IgG1.The successfully constructed eukaryotic expression plasmid was transfected into 293T cells to express and secrete DSG2 extracellular domain protein.The targeted protein was purified from the cell culture supernatant by Protein A affinity chromatography and confirmed by Western Blotting and ELISA.Results:The pCMV3-DSG2ex-IgG1 eukaryotic expression plasmid was successfully constructed.The highest protein expression level was obtained with 293T cells after 96 h of transfection.The relative molecular mass of the purified product was between 100 and 130 kDa was estimated by SDS-PAGE,which was consistent with the expectation.The yield of the purified protein reached 0.8 mg/ml with a purity over 90%.The purified DSG2 extracellular domain protein with IgG1 tag was recognized by IgG monoclonal antibodies by Western blotting.Moreover,the ELISA results showed that the prepared DSG2 extracellular domain protein had significant binding activity to human type 55 adenovirus Fiber Knob protein(HAdV-55).Conclusion:A simple and efficient method for eukaryotic expression and purification of human soluble DSG2 extracellular domain protein was successfully established,and biologically active DSG2 extracellular domain protein was purified,which laid the foundation for the later study of its protein function and anti-adenovirus drugs.
基金funded by Natural Science Foundation of Jian-gsu Province (BK2009660)Huaiyin Institute of Technology Foundation (35170706)
文摘[Objective] To obtain pure recombinant S1 and S2 of SARS S protein. [Method] Using asymmetric PCR and ligation with endonuclease, S1 and S2 fragments of SARSV HK strain S gene were synthesized. Then, these two fragments were inserted into plasmid pET28a to obtain recombinant vectors pET28a-S1 and pET28a-S2, respectively. These recombinant vectors were transformed into E. coli BL21, and expression of S1 and S2 fragments were induced by IPTG. The conditions of expression and purification were optimized. [Result] The S1 and S2 fragments were amplified and successfully expressed in E. coli. [Conclusion] This research provides detection antigens for follow-up development of SARS vaccine.
基金Supported by Project of Donghua UniversitySpecial Fund of Basic Research and Operating Expenses for Central Universities and Colleges in China(14D110511)
文摘[ Objective] This study aimed to construct the His-tagged prokaryotic expression vectors harboring zinc finger domain （ZF） and central domain （ acidic domain ＆ zinc finger domain, CT） of MDM2, respectively, and preliminarily identified biologic activity of the purified fusion proteins. [ Method ] ZF and CT cod- ing regions were amplified from MDM2 cDNA library by PCR and separately inserted into prokaryotie expression vector pET28b to construct the recombinant plas- mids. After verification by enzyme digestion, the recombinant plasmids were separately transformed into E. coli DH5c~ competent cells. The expressed recombinant plasmids were purified using Ni-NTA magnetic beads and identified by SDS-PAGE and Western blotting. [ Result] The molecular weight of His-ZF and His-CT fu- sion proteins was 21 and 31 kD, respectively. E Conclusion] Recombinant fusion proteins containing ZF and CT of MDM2 were obtained successfully, which laid the foundation for subsequent protein crystallization and three-dimensional structure analysis.
基金Supported by NSFC-Joint Personnel Training Fund of Henan Province(U1204327)Special Fund for Construction of Provincial Key Laboratory in Henan Province(122300413217)
文摘The paper was to obtain the VP1 protein of FMDV serotype A with high activity. With recombinant plasmid pMD19A-T-vp1 as the tem- plate, vpl gene fragment amplified by PCR was connected into prokaryotic expression vector pET28a to construct recombinant plasmid pET-A-vpl. The E. coli BL21 （DE3） strain containing recombinant plasmid pET-A-vpl were induced by IPTG. SDS-PAGE showed that VP1 protein was ex- pressed in the form of inclusion body, and its molecular weight was about 29 ku. Based on the optimizing IPTG concentration and expression time, the largest expression of VP1 protein was induced by 0.3 mmol/L IPTG for 6 h at 37 ℃. Western-Blot analysis indicated that the expression of VP1 protein could be specifically recognized by positive serum of FMDV serotype A. ELISA test showed that VP1 inclusion body protein had high activity after purification by washing and renaturation by urea concentration gradient dialysis.
