捻转血矛线虫H11部分功能域基因的原核表达与分析

Expression and analysis of partial functional domains of H11 gene from Haemonchus contortus.

生物信息学软件分析表明，捻转血矛线虫（Haemonchus contortus）ZJ株的H11抗原基因含有4个糖基化位点与1个锌结合区，为分析这些功能域在H11天然抗原诱导的免疫保护中的作用，对H11部分基因进行克隆和表达．参照本实验室在GenBank上登录的捻转血矛线虫ZJ株的H11基因序列设计引物，扩增H11开放阅读框670～1710bp的部分基因序列，命名为HPS（H1jpartial sequence）．基因片段与表达载体pET-28b分别以NdeI、XhoI酶切后构建重组表达载体pET28bHPS，并将其转入大肠杆菌BL21中，提取质粒经PCR和酶切鉴定获得阳性质粒并对其进行测序．结果表明，扩增得到的目的基因片段为1041bp，与已发表的ZJ株和国外株参考序列比较，核苷酸同源性分别为100％和98．8％将重组表达载体用IPTG诱导表达，表达产物利用SDS-PAGE和Western-blotting检测表明，HPS基因在大肠杆菌中成功表达，融合蛋白的分子量约为37．34kDa，诱导6h的蛋白表达量可达到58．9％．

Partial fragment of Hll gene containing several functional domains from Haemonchus contortus was cloned and expressed after bioinformatics analysis of Hll with the soft ware. The fragment of Hll gene from 670 bp to 1710 bp was amplified by means of polymerase chain reaction（PCR） with a pair of primers which was designed according to the published data of our laboratory on GenBank and was named HPS（H11 partial sequence）. Then the HPS gene was ligated to the prokaryotic expression vector pET- 28b and tansformated into E. coli BL21 （DE3）. The positive clones were verified by sequencing. Sequence analysis shows that the gene obtained shared 100% and 98.8% identity to the published full coding sequence of H11 gene from Haemonchus contortus ZJ strain and the reference strain, respectively. And the positive clone was induced by IPTG. The results indicate that the fusion protein was calculated to be about 37.34 kDa by SDS PAGE. The expression quantity of the recombinant fusion protein could reach 58.9% after being induced for 6 h. Western-blotting analysis with anti-his antibodies against the fusion protein showed the recombinant protein shared specificity.