捻转血矛线虫HLJ株H11基因克隆及部分胞外域表达

根据GenBank上发表的捻转血矛线虫核苷酸序列设计引物,采用RT-PCR方法克隆捻转血矛线虫HLJ株H11基因。结果表明,H11ORF核苷酸长为2 919 bp,编码972个氨基酸残基。经分析,捻转血矛线虫HLJ株H11与X94187、AY247714、AY819650同源性分别为99.14%、98.49%和99.59%;H11基因含有4个糖基化位点和1个Zn结合功能域;4个糖基化位点分别位于99~102 aa、227~230 aa、549~552 aa和858~861 aa处;Zn结合功能域位于376~385 aa处。参照捻转血矛线虫HLJ株H11序列设计引物,扩增H11ORF 451~2 919 bp一段胞外域序列,构建包含3个糖基化位点和1个Zn结合功能域pGEX-6P-1-H11原核表达质粒,在大肠杆菌表达菌Rosetta(DE3)中进行表达。SDS-PAGE结果表示,融合蛋白分子质量约为118 ku,以包涵体形式存在。免疫印迹实验未出现目的条带,说明H11确是一种隐蔽抗原。

Cloning of the H11 gene and expressed of partial ECD of Haemonchus contortus HLJ strain

A pair of primers were designed and synthesized according to sequence of Haemonchus contortus published in GenBank.The H11gene of Haemonchus contortus HLJ strain was amplified by RT-PCR.The sequences analysis indicated that the H11 gene consists of 2 919 bp,and the ORF encoding a protein of 972 amino acid residues.The homology of the nucleotide sequence of H11 gene between X94187,AY247714 and AY819650 were 99.14%,98.49%,and 99.59%,respectively.The H11 gene containing four glycosylation sites and one zinc-binding domain.The four glycosylation sites are located in 99-102 aa,227-230 aa,549-552 aa,858-861 aa,and the zinc-binding domain is lied in 376-385 aa.The ORF of H11 gene from 451 bp to 2 919 bp was amplified by means of polymerase chain reaction(PCR) with a pair of primers which was designed according to sequence of H11 gene of Haemonchus contortus HLJ strain.The prokaryotic expression plasmid pGEX-6P-1-H11 were constructed including three glycosylation sites and one zinc-binding domain,and expressed in E.coli Rosetta(DE3).The result of SDS-PAGE analysis showed that was successfully expressed with the form of inclusion body and molecular weight of fusion protein was about 118 ku.