幽门螺杆菌尿素酶C基因的克隆表达及其免疫原性的鉴定

Cloning, expression and immunogenicity of Helicobacter pylori ureC

目的 利用 PCR方法扩增 Hp ure C基因 ,将其克隆入表达载体 p BV2 2 0中以表达 Hp ure C重组蛋白 ,并验证其抗原性和免疫原性 .方法 用 PCR方法从 Hp临床株中扩增Hp ure C基因 ,经 Eco R 和 Bam H 双酶切后克隆入 p GEM-3Zf(- )中 ,用全自动测序仪进行双向测序 .然后将目的片段克隆入表达载体 p BV2 2 0中 ,温度诱导表达 Hp ure C重组蛋白 .用 Western Blot实验验证表达蛋白的抗原性 ,用免疫动物实验验证其免疫原性 .结果 用 PCR方法从 Hp临床株中扩增得到了一段 1173bp的 Hp ure C基因片段 ,对其进行测序 ,经BL AST分析证明所得 DNA序列与 Hp标准菌株 M6 0 398的同源性为 95 % .通过温度诱导 ,获得了 Mr为 4 4× 10 3的 Hpure C重组蛋白质 .经 Western Blot实验和免疫动物实验证实表达的蛋白质有较强的抗原性和免疫原性 .结论 成功地在E.coli中表达了 Hp ure C重组蛋白并证明其有较强的抗原性和免疫原性 .

AIM To express Hp ure C gene in E.coli , a fragment from Hp ure C which was amplified by the polymerase chain reaction (PCR) and was cloned into E.coli expressing plasmid vector pBV220. METHODS The 1173 bp gene fragment was amplified by the polymerase chain reaction. The template DNA was purified from Hp clinical strain. The gene fragment was digested by Bam HⅠ and Eco RⅠ and cloned into pGEM 3Zf ( ) plasmid. Its sequence was determined by autosequencing instrument in two directions. Then the gene fragment was cloned into pBV220. Subsequently the DH5α was transformed by pBV220/ureC and was induced by heat at 42℃. Antigenicity of the expressed protein was confirmed by Western Blot. Immunogenicity of the expressed protein was confirmed by immune animal. RESULTS 95% of the DNA sequence we got is the same as that of Hp strain M60398. A M r 44 000 recombinant protein was induced by heat at 42℃. The molecular mass of the expressed ure C gene coincides with what is expected. It proved that the recombinant ureC protein had antigenicity and immunogenicity.CONCLUSION Recombinant H.pylori urease C protein is expressed in E.coli and the expressed protein has the antigenicity and immunogenicity of ureC.