捻转血矛线虫Hc38重组蛋白的表达纯化及抗原性鉴定

[目的]通过表达和纯化捻转血矛线虫新功能基因He38最大开放阅读框ORF1,确定完整的Hc38蛋白天然表达形式、分子量大小和免疫学活性鉴定,为研究该基因功能和基因工程应用奠定基础.[方法]参照本实验室在GenBank上登录的捻转血矛线虫ZJ株的He38基因序列(AY594263)设计引物,PCR扩增目的基因并克隆到表达载体中,重组表达载体转化大肠杆菌后IPTG诱导表达,SDS-PAGE和薄层扫描检测分析表达产物,通过亲和层析柱对重组蛋白进行纯化,并用Western bloting和ELISA方法检测其抗原性.[结果]Hc38基因在大肠杆菌中高效表达,表达的融合蛋白相对分子量约为68 KD,表达量占菌体蛋白总量的30;,亲和层析柱纯化出目的蛋白,并能被捻转血矛线虫病绵羊阳性血清和重组表达产物免疫小鼠血清识别,具有反应原性.[结论]实现了Hc38基因的原核表达和纯化,验证了Hc38基因在虫体内的表达形式和抗原性.

Expression Purification and Antigenic Identification of Recombinant He38 Protein of Haemonchus contortus

【Objective】To express,purify and characterize recombinant protein of Haemonchus contortus Hc38 gene.【Method】Hc38 gene mostly ORF1 was cloned by RT-PCR with a set of primers designed on complete cDNA(AY594283) with the total RNA extracted from H.contortus adults.After cloning and sub-cloned into pET28a and transformed into E.coli Rosetta(DE3).Recombinant expression Escherichia coli was induced by IPTG,SDS-PAGE、Western-bolt were employed for analyzing the recombinant protein.【Result】pET28a-Hc38 recombinant protein was expressed successfully and its relative molecular weight of expressed fusion protein was about 68 KD,accounting for 30% of total bacteria protein,and could be recognized by positive sera against H.contortus.【Conclusion】Hc38 recombinant protein was expressed and purified successfully.In addition,its size in H.contortus adults and antigenicity were identified by western bolt.