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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。
Objective To explore a new method - in-gel method to rapidly construct the recombinant expression vector of Schistosoma japonicum antigen epitope encoding gene, and to clone and express the antigenic epitope of Schistosoma japonicum efficiently for the screening of vaccine candidate molecules. Methods pUMCV plasmid vector was double digested with SalI and EcoRI. The digested product was electrophoresed on a low melting point gel. The strips containing the digested vector were excised by UV light. Take a certain amount of low-melting plastic cut off, directly with the synthesis of a single epitope gene or two different epitope-encoding genes to connect. The ligation product was transformed into competent bacteria DH5α. Positive clones were selected for double enzyme digestion, electrophoresis and sequencing identification. As a result, a single antigenic epitope or two different epitope-encoding genes were successfully inserted into a eukaryotic expression vector. Conclusion The intramolecular inoculation method was used to directly insert the gene fragment into the plasmid vector in the presence of low melting point gel, and multiple single or combined epitopes encoding gene recombinant plasmids were obtained quickly and efficiently. In order to further screen the molecular markers of Schistosoma japonicum vaccine Foundation.