Cloning and expression of hantavirus nucleoprotein gene of Hunan03 and its immunogenicity
CAI Liang1, 2, ZHANG Hong1, GAO Li-dong1, LIU Jian-gao1, QIN Di1, HE Fang-ling1, LIU Jia-hui1, ZOU Yi-zhou2, LI Jun-hua1
1. Hunan Provincial Center for Disease Control and Prevention,the Key Laboratory of Microbial Molecular Biology of Hunan Province, Changsha 410005, China; 2. School of Basic Medical Science, Central South University, Changsha 410013, China
Abstract: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-2 and the recombinant plasmid of pGEX-6p-2/S was transformed into the competent cell of E. coli BL21 StarTM(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 0.8 mmol/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.
蔡亮, 张红, 高立冬, 刘建高, 覃迪, 何方玲, 刘佳惠, 邹义洲, 李俊华. 汉坦病毒Hunan03株S基因克隆、表达及核蛋白免疫原性分析[J]. 中国人兽共患病学报, 2016, 32(8): 711-716.
CAI Liang, ZHANG Hong, GAO Li-dong, LIU Jian-gao, QIN Di, HE Fang-ling, LIU Jia-hui, ZOU Yi-zhou, LI Jun-hua. Cloning and expression of hantavirus nucleoprotein gene of Hunan03 and its immunogenicity. Chinese Journal of Zoonoses, 2016, 32(8): 711-716.
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