[犬钩虫特定抗原基因的克隆与表达]
[Cloning and expression of specific antigen genes of Ancylostoma caninum].
作者信息
Guo Xiang-rong, Xue Hai-chou, Li Tie-hua, Liu Sen
机构信息
Institute of Parasitic Disease, Chinese Center for Disease Control and Prevention, Shanghai 200025.
出版信息
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi. 2003;21(1):12-5.
OBJECTIVE
To search for the gene encoding specific antigen of Ancylostoma caninum that induces host's protective immunity.
METHODS
A lambda ZAP II cDNA library of A. caninum 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 blotting 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 blotting 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.
目的
寻找编码犬钩虫特异性抗原的基因，该抗原可诱导宿主产生保护性免疫。
方法
用经钩虫幼虫（L3）皮下免疫的犬血清筛选犬钩虫的λZAP II cDNA文库。测序后，将阳性克隆的插入基因（AcAg）连接到PUC18和PET28C中。重组pET28C质粒在大肠杆菌BL21中诱导表达蛋白。通过SDS-PAGE和Western印迹分析重组蛋白的特性。
结果
获得5个阳性克隆，经证实为同一克隆。pET28C载体中的插入基因（AcAg）表达了一种约43 kDa的重组蛋白。通过Western印迹分析，该蛋白可被经犬钩虫第三期感染幼虫免疫的犬血清识别，并用于筛选文库。
结论
AcAg与秀丽隐杆线虫基因unc-89具有35%的同源性，是犬钩虫一种新的特异性抗原。其诱导保护性免疫的能力以及重组蛋白作为疫苗的可能性有待进一步评估。
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