Prokaryotic expression and purification of the cDNA of recombinant human cytokeratin 9 / 西安交通大学学报(医学版)

Objective To clone and fuse the cDNA of human cytokeratin 9 in prokaryotic expression system,and purify and identify the fusion protein.Methods The cDNA fragment of human cytokeratin 9 was amplified from human keratinocyte (HaCaT) total RNA with specific primers.The PCR products were cloned into vector pET-28a,then the fusion protein of his-CK9 was induced by IPTG.The expressed fusion protein of his-CK9 was purified by nickel ion affinity chromatography and identified by SDS-PAGE and Western blot.Results The sequencing proved that the recombinant vector of the cDNA of CK9 was correct.The fusion protein of his-CK9 was induced to be expressed in E.coli.The fusion protein of his-CK9 was highly purified and his-CK9 showed specific binding to the commercialized antibodies of CK9.Conclusion The recombinant vector of pET-28a-CK9 has been successfully constructed,and the fusion protein of his-CK9 has been successfully expressed and purified.

Preparation of a polyclonal antibody against human LYZL4 and its expression in the testis / 中华男科学杂志

Objective@#To prepare a polyclonal antibody against human lysozyme-like protein 4 (LYZL4) expressed in the prokaryotic system and identify the distribution of LYZL4 in the testis.@*METHODS@#The full-length cDNA of LYZL4 was cloned into the pET32a plasmid and the expression of the recombinant LYZL4 (rLYZL4) was induced by IPTG. The rLYZL4 was purified by Ni-NTA and chitin affinity chromatography respectively and its bactericidal activity was observed by bilayer agar plate diffusion assay. The purified rLYZL4 was used as an immunogen to generate the polyclonal antibody, followed by examination of the antibody titer by ELISA and its specificity by Western blot. The distribution of LYZL4 in human tissue, sperm and seminal plasma was identified and its subcellular localization in the testis was determined by immunohistochemistry.@*RESULTS@#rLYZL4 was expressed efficiently in the prokaryotic system and exhibited no bacteriolytic activity against M. lysodeikticus and E. coli. The anti-rLYZL4 polyclonal antibody could bind the recombinant protein with a high sensitivity and specificity. LYZL4 was identified in the testis, epididymis and sperm protein extracts and localized in the acrosomal region of round and elongating spermatids.@*CONCLUSIONS@#An anti-rLYZL4 polyclonal antibody was successfully prepared using the prokaryotic expression system. LYZL4 was detected in the acrosomal region of round and elongating spermatids, suggesting an association with the structure and function of the acrosome.

Expression of norovirus G Ⅱ.4 GZ121 strain P protein and analysis of its binding activity with HBGAs receptor / 中华微生物学和免疫学杂志

Objective To construct a prokaryotic expression system of norovirus (NoⅤ) G Ⅱ-4 strain P protein (P particle and P dimer) and to explore its binding activity and patterns with HBGAs receptor.Methods P domain sequence of GZ121 NoⅤ ORF2 gene was cloned and its phylogenic tree was constructed to identify the gene cluster.The pGEX-4T-1-based expression plasmids were constructed respectively by inserting P domain gene fragments with hinge and P-CDCRGDCFC without hinge,and then transformed into BL21 to express fusion proteins,which was induced with 0.6 mmol/L IPTG at 22℃ overnight.P proteins were purified by thrombin cutting and characterized by FPLC.The binding patterns of NoⅤ P protein to HBGAs antigens were analyzed by EIA.Results P region gene of GZ121 belonged to genotype G Ⅱ.4/2004 cluster.SDS-PAGE analysis showed the relative molecular weight of P particle and P dimer was about 36×103,which was consistent with other reports.The peak appeared at 830×103 confirmed the formation of P particle by FPLC.The expression of P protein was further confirmed by Western blot.The EIA results showed that GZ121 P protein could bind to saliva of A-group,B-group and O-group secretors,but not to nonsecretor.The binding affinity of P particle was 80-100 times higher than that of P dimer.Compared with VA387 P particle,it showed stronger binding affinity to O-group,but weaker to A-group.Conclusion The NoⅤ GⅡ-4 GZ121 P proteins including P particle and P dimer were successfully expressed and HBGAs receptor binding assays were established.This pave the way for further studies on the evolution dynamics of NoⅤ G Ⅱ.4 strains and the development of NoⅤ vaccines.

Prokaryotic expression and purification of two human mammaglobin gene subtypes / 医学研究生学报

Objective:To clone the cDNA in full-length of human mammaglobin,do prokaryotic expression and purify hMAM protein,as a basis for early diagnosis of breast cancer.Methods :hMAM cDNA was amplified through RT-PCR from breast cancer tissue and breast cancer cell line MD-MB453,and the recombination pQE40-hMAM vector was constructed and expressed in E.Coli.M15 after induction by IPTG.The fusion protein was purified with Ni-NTA-His affinity chromatography. Results: Two subtypes of hMAM cDNA and the hMAM(Isoform) cDNA were found,in which consisted of 270 bp,different from the wildtype hMAM cDNA of 279 bp on nine continuous base pair missing.The fusion protein formed inclusion body in prokaryotic expression system and the renatured protein was purified which purity was about 97%.Conclusion: The recombinant hMAM was successfully purified.

Prokaryotic expression, purification, identification of human cystatin C and preparation of its antiserum / 第三军医大学学报

Objective To construct a prokaryotic expression vector of cystatin C (Cys C), purify Cys C protein produced by the expression system, and prepare its antiserum. Methods Total RNA was isolated from HL-60 cells, and human Cys C gene was amplified with RT-PCR. The cDNA fragment was cloned into pMD18-T vector and which was confirmed by sequencing. The enzyme-digested target fragment was cloned into PET-32(a) expression vector and transfected into E.coli. BL 21(DE3), in which Cys C expression was induced. After the inclusion body protein was purified through Ni2+ affinity chromatography, processed by dialysis, identified by Western blotting, a rabbit was immunized with the fusion protein, and the antiserum was obtained. Results The result of DNA sequence analysis showed that the cloned Cys C gene sequence was completely corresponding to GenBank data. SDS-PAGE and Western blotting showed that the expressed Cys C fusion protein was about 35?103, mainly existing in the inclusion body of E.coli., that could be purified through Ni2+ affinity chromatography. The titer of the antiserum to the purified protein was 1∶8 000 by ELISA, and Western blotting confirmed that the antiserum reacted specifically to the Cys C protein. Conclusion A recombinant Cys C protein and the specific polyclonal antibody have been obtained, which provides a basis for establishment of immunoassays of human Cys C.

Construction,expression and purification of pTAT-HIF-1?-PAS-B in prokaryotic expression system / 第三军医大学学报

Objective To construct a prokaryotic expression vector for a fusion protein, TAT protein transduction domain (PTD) and the PAS-B domain of hypoxia inducible factor 1?(HIF-1?), and then express and purifr the fusion protein. Methods The expression plasmids pTAT-PAS-B, pET-PAS-B and pTAT-EGFP were constructed respectively, and transformed into E. coli. BL21(DE3)pLysS strain to be induced by IPTG. The obtained proteins were analyzed by SDS-PAGE and Western blotting. The fusion protein were purified with Ni-NTA-His affinity chromatography. Results The three recombinant plasmids were constructed successfully. The objective fusion proteins were obtained by optimizing the conditions for expression and purification. Conclusion The successful expression and purification of the fusion protein TAT-HIF-1?PAS-B has laid the foundation for using it to modulate the activity of HIF-1? in vivo.

Purification and characterization of recombinant murine endostatin in E. coli

Endostatin, a carboxyl-terminal fragment of collagen XVIII is known as an anti-angiogenic agent, that specifically inhibits the proliferation of endothelial cell and the growth of several primary tumor. We report here the purification and characterization of the recombinant murine endostatin (rmEndostatin) which was expressed in a prokaryotic expression system. This rmEndostatin has similar physiochemical properties of yeast-produced recombinant endostatin, and it also specifically inhibits the proliferation and migration of bovine capillary endothelial cells stimulated by basic fibroblast growth factor. The biological activity of rmEndostatin was also shown by its anti-angiogenic ability on the chorioallantoic membrane of chick embryo in vivo. In this article, we demonstrate the refolding and purification of rmEndostatin, expressed using E. coli system, to a biologically active and soluble form. In addition, these results confirm the activity of endostatin as a potent anti-angiogenic agent. Copyright 2000 Academic Press.

Construction and identification of prokaryotic expression vector of human cystatin C / 重庆医科大学学报

Objective:To Construct a prokaryotic expression vector of cystatin C(Cys C)to provide a basis of purify Cys C protein produced by the expression system.Methods:Total RNA was isolated from HL-60 cells,and human Cys C gene was amplified with RT-PCR.The cDNA fragment was cloned into pET32a(+)vector,which was confirmed with sequencing and electrophoresis.Results:The result of electrophoresis showed that Cys C had been cloned into pET32a(+)vector,and the result of.DNA sequence analysis also showed the cloned Cys C gene sequence was completely correspondent to GeneBank data.Conclusion:The prokaryotic expression vector of cystatin C(Cys C)was obtained,providing a basis of purification of Cys C protein produced by the expression system.