微小隐孢子虫三个基因主要抗原表位区的串联表达及其抗原性分析

TANDEM EXPRESSION OF MAJOR EPITOPE DOMAIN OF CRYPTOSPORIDIUM PARVUM THREE ANTIGEN GENES AND ITS ANTIGENICITY ANALYSIS

应用多个抗原表位预测软件对微小隐孢子虫CP15、P23和CP15/60三个子孢子表面抗原的氨基酸序列进行T细胞表位预测及分析,从中选取了三个抗原表位富集的基因片段,利用重叠延伸PCR(gene splicing by overlapping extension PCR,SOE PCR)将该三个基因片段串联在一起,各基因片段之间以柔性氨基酸(GGGGS)碱基序列链接,得到的拼接片段命名为CpTm。将目的基因克隆到原核表达载体pET-28a(+)上,构建重组表达质粒pET-CpTm,并转化到大肠杆菌BL21(DE3)中进行诱导表达,将纯化的重组蛋白免疫BALB/c小鼠制备多克隆抗体。结果成功地构建了CpTm串联基因并在大肠杆菌中以可溶形式高效表达,质谱分析表明重组表达蛋白包含了上述三个抗原的氨基酸序列。Western blot分析显示该重组蛋白能被牛抗微小隐孢子虫阳性血清及隐孢子虫鼠基因型CP15、P23、CP15/60基因重组表达蛋白免疫兔血清识别,制备的抗血清能被重组蛋白特异性识别,表明表达的重组蛋白具有较好的反应原性和免疫原性,为多表位疫苗的研制奠定了基础。

The T cell epitopes of the sporozoite surface antigen CP15, P23 and CP15/60 of Cryptosporidium parvum were predicted with several antigen epitope prediction software. Three immunodominant gene regions were selected and connected by gene spliced by overlap extension PCR （SOE PCR） with a flexible linker （Gly4Ser） inserted between each other fragments. The fusion gene was named CpTm and subcloned into the prokaryotic expression vector pET28a（＋）. The recombinant plasmids pET-CpTm were transformed into E.coli BL21（DE3） and induced by IPTG. The recombinant protein expressed was purified and used to immune BALB/c mice. The results revealed that the recombinant prokaryotic expression plasmid of the tandem gene CpTm was successfully constructed and expressed efficiently in the form of soluble fusion protein in E.coli. Mass spectrometric analysis showed that the recombinant protein contained the amino acid sequences of the three antigens. Western blot analysis indicated that the recombinant protein could be specifically recognized by serum from cattle infected with C. parvum, rabbit immuned with recombinant CP15, P23, CP15/60 protein and mice immuned with the recombinant protein, respectively. The results indicated that the recombinant protein had good reactogenicity and immunogenicity. The present study provides ftmdamental information for further research of multiepitope vaccine