旋毛虫ES抗原特异性蛋白两个结构基因的序列分析及重组质粒的构建

目的：获取旋毛虫ＥＳ抗原的结构基因，对其鉴定和克隆，并研制旋毛虫基因重组抗原。方法：先用反转录ＰＣＲ技术获取目的基因，经序列测定和酶切分析后，再用重组ＤＮＡ技术分别将目的基因与融合表达载体ｐＥＸ３１Ｃ、ｐＥＸ３１Ｂ及表达载体ｐＢＶ２２０连接，评价在大肠杆菌中的表达效果和鉴定表达产物的特异性。结果：获得了编码ＥＳ抗原特异性蛋白成分的两个结构基因（０．７ｋｂ和０．９５ｋｂ），其序列与文献报道的稍有差异。共构建３个重组质粒并在大肠杆菌中表达出相应分子量大小的重组蛋白，均能被猪旋毛虫病阳性血清所识别，但非融合蛋白的特异性强于融合蛋白。在相同条件下，融合蛋白的表达量高于非融合蛋白，而表达蛋白的分子量大小与表达水平呈负相关性。结论：在大肠杆菌中表达的３种重组蛋白是研制旋毛虫基因重组抗原的良好候选抗原蛋白。

SEQUENCING OF TWO STRUCTURAL GENES ENCODING SPECIFIC PROTEINS IN ES ANTIGEN FROM TRICHINELLA SPIRALIS AND CONSTRUCTION OF RECOMBINANT PLASMIDS

AIM: To identify and clone structural genes encoding ES antigen from T.spiralis for preparing gene recombinant antigen of T.spiralis. METHOD: RT PCR technique was used to gain the target genes. After sequencing and restriction enzyme digestion, the genes were respectively cloned into the fusion expression vectors pEX31C, pEX31B and another expression vector pBV220 by using recombinant DNA techniques. Their expression level in E.coli was evaluated and the specificity of the expression products was also identified. RESULTS: Two structural genes encoding the specific proteins in ES antigen were obtained(0 7 kb and 0 95 kb). Compared with those reported previously, the sequences exhibited some differences. Three recombinant plasmids were constructed. It was shown that the corresponding recombinant proteins were expressed in E.coli containing the plasmids by SDS PAGE electrophoresis. All the recombinant proteins could be recognized by sera from swine infected with T.spiralis, but the specificity of non fusion protein was stronger than that of fusion protein. The expression level of the fusion protein was higher than that of the non fusion protein, and the molecular weight of the expression proteins was in negative correlation with the expression level under the same condition. CONCLUSION: Three recombinant proteins expressed in E.coli are candidate antigenic proteins for preparing gene recombinant antigen of T.spiralis.