汉坦病毒Hunan03株S基因克隆、表达及核蛋白免疫原性分析

Cloning and expression of hantavirus nucleoprotein gene of Hunan03 and its immunogenicity

目的构建汉坦病毒Hunan03株核蛋白基因原核重组表达载体,在大肠杆菌中进行核蛋白表达,研究核蛋白的免疫性及免疫反应性。方法设计特异性扩增汉坦病毒Hunan03株S基因完整开放阅读框(ORF)的引物,RT-PCR扩增,产物克隆到pGM-T载体,转化感受态细胞TOP10,应用蓝白斑筛选、酶切、PCR鉴定,定向克隆到pGEX-6p-2原核表达载体,转化Ecoli.BL21StarTM(DE3),IPTG诱导表达,SDS-PAGE、Western blot对重组蛋白进行鉴定。应用Glutathione Sepharose 4B纯化柱纯化重组蛋白,免疫新西兰兔,建立间接ELISA法对核蛋白的免疫原性与免疫反应性进行评价。结果 PCR扩增S基因ORF区域产物大小约1 306bp,重组载体pGEX-6p-2-S经双酶切、PCR、测序鉴定提示构建成功;在37℃,IPTG浓度为0.8mmol/L诱导5h的条件下,表达出最高量的相对分子量约74kDa的GST-NP融合蛋白。建立的间接ELISA法检测GST-NP融合蛋白免疫后的新西兰兔血清,IgM抗体滴度达1∶8 000,IgG抗体滴度达1∶16 000。结论成功构建了高效表达的汉坦病毒S基因重组表达载体,获得了纯度较高具有较好的免疫原性与免疫反应性的核蛋白,为后续汉坦病毒单克隆抗体的制备奠定了基础。

We cloned the hantavirus nucleoprotein gene and expressed it in E.coli for laboratory diagnosis.The whole open reading frame（ORF）of hantavirus Hunan03 strain S gene was amplified by RT-PCR after designing specific primers,and the PCR products was cloned into the pGM-T vector and transformed into competent cell of TOP10 and identified by the assay of blue-white spot screening,PCR and enzyme digestion.Then,we directed cloned the S gene into the prokaryotic expression vector of pGEX-6p-2and the recombinant plasmid of pGEX-6p-2/S was transformed into the competent cell of E.coli BL21StarTM（DE3）and induced by IPTG.We identified the expression product by SDS-PAGE and Western-blot.Results showed that the PCR product of S gene was about 1 306 bp.The recombinant plasmid of pGEX-6p-2/S was constructed successfully after being identified by PCR and double enzyme digestion.Under the condition of 37 ℃ and 0.8mmol/L IPTG induction,the pGEX-6p-2/S has expressed a 74 kDa fusion GST-NP protein.The successful expression of the recombinant prokaryotic plasmid pGEX-6p-2/S will benefit to the laboratory diagnosis of hantavirus infection.