捻转血矛线虫肌动蛋白基因的克隆与表达及其DNA疫苗的构建

Gene cloning,expression and DNA vaccine construction of Haemonchus contortus actin

根据秀丽新杆线虫等其他几种线虫的肌动蛋白(Actin)开放阅读框(ORF)设计简并引物,以捻转血矛线虫总RNA为模板,合成cDNA,用RT-PCR方法扩增出约为1 100 bp的DNA片段。将该片段克隆到T载体后进行序列测定和分析,结果表明该基因的ORF为1 131 bp,与秀丽新杆线虫、新杆状线虫、肿孔古柏线虫等的同源性高达86%以上,推导出其蛋白质序列含有376个氨基酸,与胎生网尾线虫、秀丽新杆线虫、新杆状线虫、马来丝虫等的肌动蛋白相似性均为98%以上,说明成功克隆了捻转血矛线虫actin基因。将捻转血矛线虫肌动蛋白基因的ORF克隆到pET-28a(+)中,构建了原核表达载体,用IPTG进行诱导表达。SDS-PAGE分析发现,该基因获得了表达,且以包涵体的形式存在,融合蛋白相对分子质量约46×103。以此重组蛋白为抗原,用自然感染捻转血矛线虫山羊血清为第一抗体进行Western blot分析,结果出现1条特异性条带,表明捻转血矛线虫Actin蛋白在自然感染过程中能被宿主的免疫系统识别,可能是一种天然抗原。用纯化重组Actin蛋白免疫小鼠,制备抗血清,以该血清为第一抗体进行Western blot,结果发现该血清能识别捻转血矛线虫成虫蛋白谱中相对分子质量约为42×103的蛋白条带。将捻转血矛线虫actin基因亚克隆到真核表达载体pVAX1中,构建了DNA疫苗pVAX1-ACT,动物免疫试验结果表明该DNA疫苗在机体内获得了表达。

A pair of degenerate primers was designed based on the sequences of actin gene open reading frames（ORF）of Caenorhabditis elegans and other nematodes.And reverse transcriptase-polymerase chain reaction（RT-PCR）was performed,using total RNA extracted from adult H.contortus as the template.The result showed that a DNA fragment of 1 100 bp was amplified.Then the PCR product was cloned into T vector and sequenced.Followed by the online BLAST analysis,we found that the length of this gene was 1 131 bp,which encodes a protein of 376 amino acids.The nucleotide and amino acid sequences of this new gene were highly homologous with these of other nematodes such as C.elegans,C.briggsae and Cooperia oncophora.The actin gene of H.contortus was cloned to pET-28a（＋）and transformed into BL21（DE3）.The recombinants were identified and induced by IPTG.The results of SDS-PAGE showed that the actin gene was expressed as a fusion protein weighing 46×103,mainly in the inclusion body.It was found in the Western blot,that the recombinant Actin could be recognized by the serum of the goat infected with H.contortus.This may indicate that the natural Actin in H.contortus could be released and stimulate the host immune system.After the purification of the recombinant Actin,it was administered to the mice.The anti-Actin serum was obtained and used as first antibody in the Western blot.We found that the 42×103 natural Actin in this parasite could react with the antibody.In this paper,the Actin DNA vaccine against H.contortus was constructed by subcloning the actin gene into the vector pVAX1.Mice were immunized by the DNA vaccine pVAX1-ACT and the expression of this vaccine in the body was detected by RT-PCR and Western blot.The results showed that the DNA vaccine was expressed in the muscle cells,successfully.