犬钩虫特异性抗原基因的克隆与表达

Cloning and Expression of Specific Antigen Genes of Ancylostoma caninum

目的 寻找可诱发宿主保护性免疫力的犬钩虫特异性抗原。 方法 用犬钩蚴免疫获得保护性免疫力的家犬血清 ,免疫筛选犬钩虫 期幼虫 λZAP c DNA文库 ,阳性克隆基因经测序 ,在质粒 PUC18、PET2 8中进行一系列亚克隆 ,重组 p ET2 8C质粒诱导表达并对表达产物进行 SDS- PAGE和 Western blotting分析。 结果 从 c DNA文库中筛获 5个相同的阳性克隆 ,携带的犬钩虫抗原 (Ac Ag)外源基因片段在原核体系 (p ET2 8C)中表达分子量为 4 3k Da的融合蛋白。 Western blotting分析该重组蛋白可与筛库的犬血清反应。 结论 Ac Ag为新的犬钩虫特异性抗原 ,其基因与美丽纤杆线虫 (Caenorhabditis elegans)基因 unc- 89同源性为 35 %。该抗原诱发宿主保护性免疫力及作为疫苗的潜力值得进一步研究。

Objective To search for the gene encoding specific antigen of Ancylostoma caninum that induces host′s protective immunity. \ Methods\ A lambda ZAPⅡ cDNA library of A.canium was screened with sera from dogs \{immunized\} subcutaneously with hookworm larvae(L3). After sequencing, insert gene (AcAg) from positive clones was \{ligated\} into PUC18 and PET28C. Recombinant pET28C plasmid was induced to expressed protein in the E.coli BL21. The \{characteristic\} of recombinant protein is analyzed by SDS\|PAGE and Western bloting assay. \ Results \ Five positive \{clones\} were obtained, and proved to be the same. The insert gene (AcAg) in pET28C vector expressed a recombinant \{protein\} of about 43 kDa. Using Western bloting assay, this protein was recognized by the sera from dog immunized with \{Ancylostoma\} caninum third\|stage infected larvae and used for screening library. \ Conclusion \ The AcAg, which \{exhibits\} 35% homologous to Caenorhabditis elegans gene unc\|89, is a novel specific antigen of A.caninum. Its ability to elicit the \{protective\} immunity and the probability of the recombinant protein as a vaccine need to be further evaluated.