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根据GenBank中细粒棘球绦虫EgA31序列(GenBank登录号为AF067807)设计引物,以细粒棘球绦虫mRNA为模板,RT-PCR扩增EgA31基因,将其克隆入pUCm-T载体,转化大肠埃希菌(E coli)DH5α,筛选阳性克隆,经BamH I、Sac I双酶切和PCR鉴定,获得阳性重组质粒pUCm-T/EgA31,并将测序正确的片段连接表达载体,成功构建重组质粒pET30a-EgA31。经序列分析和同源性比较,以及对其编码产物进行B细胞和T细胞表位分析,结果表明PCR扩增的特异条带为636 bp,与预期相符,与GenBank已知基因序列同源性为100%。编码产物B细胞和T细胞联合表位预测,氨基酸区域可能在32～79、79～95、105～124和141～154位。
The primers were designed according to the EgA31 sequence of GenBank (GenBank accession number AF067807) in GenBank. The EgA31 gene was amplified by RT-PCR from E. coli and cloned into pUCm-T vector to transform into E.coli The positive clones were screened by DH5α. The recombinant plasmid pUCm-T / EgA31 was obtained by digestion with restriction endonucleases BamH I and Sac I and the positive recombinant plasmid pUCm-T / EgA31. The correct fragment was ligated to the expression vector and the recombinant plasmid pET30a -EgA31. Sequence analysis and homology comparison, as well as B cell and T cell epitope analysis of the encoded products, showed that the specific band amplified by PCR was 636 bp, which accorded with the expectation and had homology with GenBank known gene sequence It is 100%. The encoded product B cells and T cell epitopes predict that the amino acid region may be 32-79, 79-95, 105-124 and 141-154.