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目的将已筛选出的抗汉坦病毒核衣壳蛋白(NP)抗原单链抗体基因克隆入原核表达载体pGEX-6p-1,构建重组表达质粒pGEX-6P-1-scFv。方法提取含抗NP抗原单链抗体基因的T7噬菌体DNA,此为模板,PCR扩增单链抗体基因,BamHⅠ和SalⅠ双酶切,经连接酶与BamHⅠ和SalⅠ双酶切的表达载体pGEX-6p-1连接;重组DNA转化入宿主菌E.coliBL-21(DE3),琼脂糖凝胶电泳初筛选阳性重组质粒,并用PCR法、双酶切法及DNA测序鉴定。结果重组质粒pGEX-6P-1-scFv经BamHⅠ和SalⅠ双酶切,片段大小分别为5 000 bp和750 bp,与预期值一致;重组质粒PCR产物大小为750 bp,与预期值一致;测定重组质粒序列,并与scFv序列比较,结果相一致。抗NP单链抗体基因序列克隆入表达载体pGEX-6p-1。结论成功构建了含抗汉坦病毒NP抗原单链抗体基因的重组原核表达质粒pGEX-6P-1-scFv。
Objective To clone the scFv gene of anti-Hantavirus nucleocapsid protein (NP) antigen into prokaryotic expression vector pGEX-6p-1 and construct recombinant plasmid pGEX-6P-1-scFv. Methods The T7 phage DNA containing the anti-NP antigen single chain antibody gene was extracted and used as a template to amplify the single-chain antibody gene by PCR. The double-digested BamHⅠ and SalⅠ double-digested products were digested with BamHⅠ and SalⅠ, and ligated into pGEX-6p -1. The recombinant DNA was transformed into host strain E.coli BL-21 (DE3). The positive recombinant plasmids were screened by agarose gel electrophoresis at first and then identified by PCR, double enzyme digestion and DNA sequencing. Results The recombinant plasmid pGEX-6P-1-scFv was digested by BamHⅠ and SalⅠ, and the fragment size was 5 000 bp and 750 bp, respectively, which was consistent with the expected value. The PCR product size of the recombinant plasmid was 750 bp, which was consistent with the expected value. Plasmid sequences, and compared with the scFv sequences, the results are consistent. The anti-NP single chain antibody gene sequence was cloned into the expression vector pGEX-6p-1. Conclusion The recombinant prokaryotic expression plasmid pGEX-6P-1-scFv containing anti-Hantaan virus NP antigen single chain antibody gene was successfully constructed.