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目的用GeneSOEing技术构建含日本血吸虫中国大陆株磷酸丙糖异构酶及副肌球蛋白T、B细胞表位的复合表位DNA质粒。方法将一个柔性的氨基酸“接头”插入真核表达质粒载体pcDNA3.1中,构建pcDNA3.1-linker。PCR法扩增磷酸丙糖异构酶表位基因片段(T)。人工合成副肌球蛋白表位的基因片段,退火成双链(P)。分别将T、P片段克隆入改建的载体pcDNA3.1中link-er的上游和下游,构建T-P融合基因;同时还将T、P片段分别克隆入linker的下游和上游,构建P-T融合基因。重组质粒分别转化E.coliXL1-blue,抽提质粒,酶切鉴定。结果两个目的基因片段分别按顺序克隆入真核表达质粒载体pcDNA3.1中,构建成复合表位DNA质粒。结论成功地构建了复合表位DNA质粒,为制备多价表位DNA疫苗奠定了基础。
OBJECTIVE: To construct a DNA complex plasmid containing triosephosphate isomerase and paramyosin T and B cell epitopes from Chinese mainland strains of Schistosoma japonicum using GeneSOEing technique. Methods A flexible amino acid “linker” was inserted into the eukaryotic expression plasmid pcDNA3.1 to construct pcDNA3.1-linker. PCR amplification of triosephosphate isomerase epitope gene fragment (T). Gene fragments of paramyosin epitopes were synthesized and annealed to double-stranded (P). The T and P fragments were cloned into upstream and downstream of link-er in pcDNA3.1 vector to construct T-P fusion gene. At the same time, T and P fragments were cloned downstream and upstream respectively to construct P-T fusion gene. Recombinant plasmids were transformed into E. coliXL1-blue, plasmid extraction, restriction enzyme identification. Results The two target gene fragments were cloned into the eukaryotic expression plasmid vector pcDNA3.1 in sequence to construct the DNA of the complex epitope. Conclusion The construction of the composite epitope DNA plasmid has laid a foundation for the preparation of multivalent epitope DNA vaccine.