为探索隐孢子虫病免疫预防的新途径,本文对猴源人隐孢子虫Cryptosporidium hominis P23抗原基因进行了克隆、表达及其免疫活性的测定.采用RT-PCR方法对猴源C.hominis P23抗原基因进行克隆,构建重组质粒pGEX 4T 1 P23,并用IPTG诱导其在E.Coli Rossetta中表达;采用SDS PAGE检测其表达效果;采用Dot ELISA和淋巴细胞转化试验分别检测其与抗体反应特性和淋巴细胞增殖情况.结果表明:所克隆的P23抗原基因大小为342 bp;重组质粒表达蛋白的大小为40 kDa(含26 kDa的融合蛋白GST),以1、5、10 μg剂量的重组蛋白rP23均能刺激鼠脾脏淋巴细胞增殖,并能够与感染猴血清中的抗体明显地发生反应.%To develop a new method for immunoprophylaxis against cryptosporidiosis, the antigen P23 gene of Cryptosporidium hominis was cloned and expressed, and its immunogenicity was detected. The p23 gene of C. hominis was amplified by RT-PCR, and the recombinant plasmid of pGEX-4T-1-P23 was then constructed and expressed in Escherichia coli by IPTG induction. The expressed protein was detected by SDS-PAGE. Lymphocyte transformation assay was applied to detect lymphocyte proliferation after stimulation by the recombinant protein, and Dot-ELISA was performed to detect its reactivity towards Abs. The results indicated that the size of successfully cloned p23 gene was 342 bp, the molecular weight of recombinant protein expressed by IPTG induction was about 40 kDa. The lymphocyte transformation assay revealed that the recombinant protein, in a dosage of 1 , 5 and 10 μg could stimulate mice spleen lymphocyte to proliferate, and Dot-ELISA showed the recombinant protein could markedly react to Abs originated from serum of monkey infected by C. hominis .
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