幽门螺杆菌5种候选疫苗抗原基因的克隆、表达及抗原性的鉴定

目的:构建含人Hpylori5种候选疫苗抗原Lpp20,HspA,UreaseA,CagA,UreaseB的编码基因的重组质粒并研究其抗原性.方法:应用PCR技术从Hpylori染色体中扩增编码Lpp20,HspA,UreaseA,CagA,UreaseB的基因片段,将其T-A克隆和测序,并与GenBank公布的其他Hpylori菌株基因序列比较,再将目的基因克隆至融合表达载体pGEX-4T-1上中进行表达,用GST亲和层析对其进行纯化,纯化产物用于对29株小鼠抗Hpylori-全菌单克隆抗体(mAb)的鉴定及与Hpylori感染患者血清进行Westernblot分析.结果:扩增的Lpp20,HspA,UreaseA,CagA,UreaseB基因全长分别为528bp,351bp,675bp,855bp,1704bp(GenBank登录号分别为DQ106902,DQ141574,DQ141577,DQ141575,DQ141576),与GenBank公布的其他菌株的核酸序列的同源性在95%-99%,表达Lpp20,HspA,UreaseA,CagA,UreaseB融合蛋白的相对分子质量分别约为48000,41000,52000,60000,91000Da,29株小鼠抗Hpylori全菌mAb中针对Lpp20,HspA,UreaseA,CagA,UreaseB抗原的分别为4,5,5,1,6株,5种抗原的纯化产物均可被Hpylori感染患者血清特异性识别.结论:重组表达的Lpp20,HspA,UreaseA,CagA,UreaseB均具有较好的抗原性.

Cloning, expression and antigenicity identification of five candidate vaccine antigen genes of human Helicobacter pylori

AIM: To construct a recombinant plasmid contai- ning genes encoding Lpp20, HspA, UreaseA, CagA, UreaseB from H pylori, express it in E.coli and explore the antigenicity. METHODS: The genes, encoding Lpp20, HspA, UreaseA, CagA, and UreaseB, were amplified from H pylori chromosomal DNA by polymerase chain reaction (PCR), and then T-A was cloned and sequenced. The target genes cloned into pGEX-4T-1 fusion expression vector were ex- pressed in E.coli and purified by GST-affinity chromatography. The purified products were used to identify 29 strains of mouse monoclonal antibodies (mAbs) against H pylori, and the anti- genicity of the products was analyzed by West- ern blot with serum of H pylori-infected patients. RESULTS: Lpp20, HspA, UreaseA, CagA, and UreaseB fragments were 528 bp, 351 bp, 675 bp, 855 bp, and 1704 bp in length, respectively (Gen-Bank submission No. DQ106902, DQ141574, DQ141577, DQ141575, and DQ141576, respec- tively), and the nucleotide homology was 95%-99% with other H pylori strains. Lpp20, HspA, UreaseA, CagA, and UreaseB fusion protein were expressed with molecular weights of 48 000, 41 000, 52 000, 6 0000, and 91 000 Da in E.coli respectively. Of 29 anti-H pylori mouse mAbs, there were 4, 5, 5, 1, and 6 strains against Lpp20, HspA, UreaseA, CagA, and UreaseB. Western blot proved that fi ve recombinant pro- teins were specifi cally recognized by the serum of H pylori-infected patients. CONCLUSION: Five recombinant proteins, Lpp20, HspA, UreaseA, CagA, and UreaseB, preserve original antigenicity.