抗汉坦病毒核衣壳蛋白单链抗体基因的克隆与鉴定

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．coli BL-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5000bp和750bp，与预期值一致；重组质粒PCR产物大小为750bp，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人表达载体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。

Gene cloning and identification of scFv against nucleocapsid protein of hantavirus

Objective To clone the selected gene of the single-chain fragment of the variable region （scFv） against the nucleocapsid protein （NP） antigen of hantavirus into the prokaryotic expression vector pGEX-6p-1 to construct a recombinant expression plasmid pGEX-6P-1-scFv. Methods T7 phage DNA containing the gene for the scFv against the hantavirus NP antigen was extracted. The scFv gene was amplified with template to T7 DNA by PCR and was then digested with BamH Ⅰ and Sal Ⅰ. The obtained fragment was then ligated into the expression vector pGEX-6p-1 that had been digested with BamH Ⅰ and Sal Ⅰ. After the recombinant DNA was transformed into competent E. coli BL-21 （DE3）, a positive recombinant plasmid was selected preliminarily by agarose gel electrophoresis. The recombinant plasmid was identified by PCR, digestion of BamH Ⅰ and Sal Ⅰ and DNA sequencing. Results The recombinant pGEX-6P-I-scFv was digested into two fragments of 5 000 bp and 750 bp by BamH Ⅰ and Sal Ⅰ as we had expected. The PCR product of the recombinant plasmid was 750 bp as we had expected. The scFv gene in the recombinant plasmid was sequenced and compared with the scFv gene in T7 phage DNA and was found to corresponded with the later. The results show that the gene for the scFv against NP was cloned into the expression vector pGEX-6p-1. Conclusion Prokaryotic expression recombinant pGEX-6P-1-scFv, which contained the gene for the scFv against the hantavirus NP antigen was constructed success-fully.