Molecular cloning and characterization of gene virD[STHX]4 in Helicobacter pylori / 临床检验杂志

ABSTRACT

Objective@#To investigate the function of virD4 gene in Helicobacter pylori clinical strain SBK. @*Methods@#The virD4 gene segment was obtained through T-A cloning method. The prokaryotic expression vector pET-28a(＋)-virD4 was constructed and transformed into E. coli Rosetta for the expression by induction of IPTG. The recombinant proteins were obtained and purified by KCl dyeing with gel cutting method, and identified via SDS-PAGE analysis. The purified recombinant virD4 protein was used to immunize mice to produce polyclonal antibodies. The titer of the polyclonal antibodies was tested by ELISA and the antigenic specificity was identified by western blot. The purified recombinant virD4 proteins were co-cultured with GES-1 cells followed by detecting the expression of inflammatory cytokines secretion and the ability of cell proliferation. @*Results@#The full length of virD4 gene was 1 728 bp. The sequence shared quite high homology with the virD4 gene of isolate Shi470. The recombinant prokaryotic expression plasmid pET-28a(＋)-virD4 was successfully constructed. The recombinant virD4 proteins were obtained by IPTG induction and purified via KCl dyeing method. SDS-PAGE showed that the relative molecular weight of recombinant virD4 protein was 63 000. The purified proteins were used to immunize mice to obtain the anti-virD4 polyclonal antibodies with the titer 512 000. The reaction between anti-virD4 polyclonal antibodies and recombinant virD4 proteins was highly specific. The recombinant virD4 protein induced inflammatory cytokines secretion and promoted GES-1 cell proliferation. @*Conclusion@#The virD4 gene was successfully cloned and highly expressed in prokaryotic expression system, and its antibodies were prepared. The recombinant virD4 protein can induce cytokine secretion and cell proliferation.