捻转血矛线虫HLJ株H11基因克隆及部分胞外域表达

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

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。

A pair of primers were designed and synthesized according to sequence of Haemonchus contortus published in GenBank. The H1 lgene of Haemonchus contortus HLJ strain was amplified by RT-PCR. The sequences analysis indicated that the Hll gene consists of 2 919 bp, and the ORF encoding a protein of 972 amino acid residues. The homoJogy 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 Hll 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 Hll 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 inE. coil 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,