日本血吸虫抗原表位编码基因重组质粒的高效构建与鉴定

Efficient construction and identification of the recombinant plasmid carrying genes coding to Schstosoma japonicum antigen epitope peptides

目的探索用一种新方法—胶内连接法,快速构建日本血吸虫抗原表位编码基因重组表达载体,高效克隆及表达日本血吸虫抗原表位,用于疫苗候选分子的筛选。方法将pUMCV质粒载体用SalI和EcoRI双酶切,酶切产物于低熔点胶中电泳,紫外灯下割取含酶切载体的条带。取一定量割取的低熔点胶,直接将其与合成的单个抗原表位编码基因或者是两个不同抗原表位编码基因进行连接。连接产物转化感受态菌DH5α。挑选阳性克隆进行双酶切、电泳及测序鉴定。结果单个抗原表位或两个不同的表位编码基因被成功地插入真核表达载体。结论采用胶内连接法直接在低熔点胶存在的条件下进行插入基因片段与质粒载体的连接,快速高效地获得了多个单一或组合抗原表位编码基因重组质粒,为进一步筛选日本血吸虫疫苗分子奠定了基础。

By means of a new method, gel-ligation method, the recombinant expression plasmid carrying genes coding to Schistosoma japonicum antigen epitope peptide was rapidly and efficiently constructed. In this method, the plasmid pUMCV was digested with restriction endonucleases Sal I and EcoRI, and the digested products were subjected to ehctrophoresis in low-melting point agarose gel. Then, the digested plasmid in gel was sliced under ultra-violet lamp. Single or multiple gene cDNA coding to antigen epitope peptide was ligated together,and the ligated product was directly inserted into digested plasmid in the presence of low-melting point agarose gel. The recombinant plasmid was then transformed to E. coli DH5α strain, and the colonies were picked out and characterized by restriction endonuclease digestion and sequence analysis. It was demonstrated that the cDNAs coding to S. japonicum antigen epitope peptide could be inserted successively into vector plasmid. It is evident that the gel-ligation method is proved to be a simple and efficient method to make recombinant constructs, which would provide a new tool for the rapid and convenient cloning and further screening of vaccine candidate epitope of S. japonicum.