Expression and purification of JC virus VP2 fusion protein and preparation of its polyclonal antibody / 中华传染病杂志

Objective To obtain the antigen and antibody of JC virus(JCV)VP2.Methods The JCV VP2 gene were amplified from a cerebrospinal fluid sample by polymerase chain reaction (PCR)and confirmed by sequencing.Then,the gene was cloned into plasmid pET32a(+)to construct recombinant prokaryotic expression vector pET-32a(+)-VP2.The recombinant plasmid was transformed into the competent E.coli BL21.Induced with isopropyl-β-D-1-1 thiogalactopyranoside (IPTG),E.coli BL21 were subsequently crushed by ultrasound.The gene expression in the supernatant was analyzed by Western blot.Thereafter,the expressed protein was purified by isoeleetric point method.The polyclonal antibody against JCV VP2 protein was obtained from the BALB/c mouse immunized with the purified protein.Results The VP2 fusion protein was expressed in the E.coli BL21.The recombinant fusion protein was expressed by IPTG induetion with relative molecular mass of 58.5×10~3.Sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDSPAGE)analysis showed that the expression level was highter after 6-10 h of IPTG induction.The recombinant protein had good antigenicity which was confirmed by BALB/c mice immunized with the protein.Conclusions The successful expression and purification of VP2 fusion protein and the antibody will be valuable for the study on the biological function of VP2 and JCV epidemiologieal investigation.

Expression of t antigen fusion protein of JC virus and preparation of its polyclonal antibody / 中华传染病杂志

Objective To construct prokaryotic expression vector carrying jc virus(JCV)t-antigen gene,express and purify this fusion protein.Methods The JCV t-antigen gene from a cerebrospinal fluid sample was amplified using polymerase chain reaction(PCR)method.After sequencing.the gene was cloned into plasmid pET32a(+)to construct recombinant prokaryotic expression vector pET32a(+)-t.The t-antigen fusion protein was expressed by isopropy-~D-thiogalactoside(IPTG)induction and prepared in large scale,then purified by Ni+affinity column chromatography.The polyclonal antibody was obtained from the BAI.B/C mouse immunity by the purified protein.Results The relative molecular nlass of recombinant protein expressed by pET32a(+)-t was about 41 000.Sodium dodeeylsulfate-polyaerylamide gel electrophoresis(SDS-PAGE)showed that the fusion protein W&S highly expressed after 3.5～20.Oh of IPTG induction.The antigenicity of the purified protein Was well confirmed by Western blot.The anti-mousepolyclonal antibody was obtained successfully from immunized BALB/c mice.Conclusions The prokaryotic expression vector pET32a(+)-t is successful constructed and the fusion protein is expressed and purified.Furthermore,the antibody of JCV small envelop protein t is successfully prepared.This work provides vMuable information for further study on epidemiology and biological function of t antigen.

Prokaryotic expression of HCV NS5ATP1 gene and preparation of polyclonal antibody / 解放军医学杂志

Objective To construct prokaryotic expression vector of hepatitis C virus NS5ATP1 gene, and to induce its expression in E. coli. To purify the fusion protein and obtain its polyclonal antibody from immunized New Zealand rabbits. Methods The NS5ATP1 gene, which was cut from the vector pGBKT7-NS5ATP1, which was self-constructed by the authors, was cloned into plasmid pET32a(+) to construct the pET32a(+)-NS5ATP1 prokaryotic expression vector. It was proved that the recombinant plasmid was constructed correctly by sequencing. And then the expression vector was transformed into the competent E. coli DH5? and BL21. After being induced with IPTG, the NS5ATP1 fusion protein was expressed and analyzed with SDS-PAGE and Western blot. The transformed bacteria were fragmented by ultrasonic and then separated by SDS-PAGE. The fusion protein formed inclusion body. They were then purified and re-natured through Ni+ affinity column chromatography. The purified pET32a(+)-NS5ATP1 fusion protein was used to immunize New Zealand rabbits to gain polyclonal antibody. The specificity and immunizing potence of polyclonal antibody were evaluated by Western blot and ELISA. Results After transferring pET32a(+)-NS5ATP1 plasmid into DH5? and BL21 and induced with IPTG, the NS5ATP1 fusion protein of about 56kD was highly expressed. SDS-PAGE analysis showed that the fusion protein products were mainly in inclusion body and expressed in the highest level at 4.5h of induction. The purified protein and polyclonal antibody were obtained successfully. ELISA manifested the titer of polyclonal antibody was over 1∶512 000. The high specificity was testified by Western blot. Conclusions The successful expression and purification of NS5ATP1 fusion protein and the preparation of NS5ATP1 specific polyclonal antibody will be valuable for the study on the biological function of NS5ATP1.