High efficient expression of human endostatin in E.coli and its antiangiogensis activity / 中国病理生理杂志

AIM: To examine the expression of human endostatin in E.coli , produce its fusion protein antibody and observe its biological activity. METHODS: Endostatin gene was amplified by polymerase chain reaction,recombined with plasmid vector pGEX-2T and induced expression with IPTG.The protein activity was tested by endothelial cell proliferation inhibitory assay.Inclusion body crudely purified was used to generate polyclonal antibody to detect its expression at mouse's liver and kidney etc. RESULTS: The protein expressed was 20 kD after digestion by thrombin,it appeared the anti-angiogenesis activity and Western blotting indicated the expression of endostatin in liver and kidney of mouse. CONCLUSION: The successful expression of human endostatin and the preparation of polycolonal antibody indicated its potential application in anti-angiogenesis therapy and diagnosis tumors.

Construction of an eukaryotic expression vector encoding human granzyme B and it's expression in Hep2 cells / 中国病理生理杂志

AIM: To construct pVAX1-GrB. METHODS: Lymphocytes from human laryngeal carcinoma tissue were separated from tumor tissue. The fragment of granzyme B (GrB) was amplified by RT-PCR and was recombined to the downstream of T7 promoter in the vector pVAX1. The construction was transfected into Hep2 cells with lipofectamine 2000. The expression of protein was identified by indirect immunofluorescent antibody assay. RESULTS: It has been proved that the sequence of the RT-PCR product was totally consistent with the data of GenBank by DNA sequencing analysis. The GrB cDNA fragment was cloned into the vector of pVAX1 in the right direction and the open reading fragment of GrB was maintained. The target protein was detected in the transfected Hep2 cells. CONCLUSION: The pVAX1-GrB plasmid was successfully constructed and expressed. [

Signal peptide sequence of human interleukin-2 influenced hEndostatin gene expression and protein secretion in HepG2 cells / 中国病理生理杂志

AIM: The role of human interleukin-2(IL-2) signal peptide sequence in the effect of human Endostatin (hEndostatin) expression and secretion was investigated in HeG2 cells. METHODS: RT-PCR and Western-blotting were conduct to observe mRNA level difference of hEndostatin gene, its protein expression and secretion level difference between with hIL-2 signal peptide sequence and without it. RESULTS: mRNA level of hEndostatin gene in HepG2 (pBlast-hIL2-hEndo) cells was higher than that in HepG2(pBlast-hEndo)( P