AIM: To construct a prokaryotic expression vector for HCV core-Ant and to study its effect on the transdifferenliation of hepatic stem cells. METHODS: The HCV core gene was obtained by PCR of two primers templating with each other and T-vector cloning method. The positive clone was identified and analyzed by the restriction enzyme digestion and sequencing, respectively. The recombinant vector pGEM T-ant was used to insert Ant and HCV core cDNA into restriction endonuclease enzyme and ligated with T4 ligase. The fusion gene fragments HCV core-ant was recloned to pET2Sa( + ) and the DNA sequence was confirmed by the dideoxy chain termination method. The recombinant vector was transformed and expressed in E. coli BL21 ( DE3 ) under induction of IPTG. After purification, the antigenicity of purified protein was detected by ELISA. Then HCV core and HCV core-Ant were transfected into NIH 3T3 cells, and the effect was measured by immunohistochetnistry. RESULTS: The prokaryotic expression vector carrying HCV core-Ant was confirmed by the restriction enzyme digestion and sequencing. The recombinant plasmid pTE28/ HCV core-Ant was identified by the digestion of restriction enzyme and agarose gel electrophoresis and the results conformed theoretical values. SDS-PAGE analysis showed that relative molecular mass of the expressed product by pET28a( + )/HCV core-Ant was 2.73 × 103. It also showed good antigenicity. The expression of HCV core-Ant gene in NIH 3T3 cells was confirmed by immunohistochemistry. CONCLUSION: The plasmid pTE28 containing the expression box of HCV core-Ant is successfully constructed by molecular cloning and recombination techniques in vitro, which lays basis for further study on transdifferentiation effects of HCV core on cells and carcinogenici-ty of HCV.
NETL
NSTL
万方数据
维普
