高致病性禽流感病毒H5N1亚型核蛋白NP真核表达载体的构建、表达与鉴定

Construction of eukaryotic expression vector of avian influenza virus H5N1 nuclear protein (NP) and expression,confirmation of recombinant NP protein in mammalian eukaryotic cells

目的构建高致病性禽流感病毒H5N1亚型核蛋白(NP)的真核表达载体,并在哺乳动物细胞中表达、鉴定其编码重组蛋白NP。方法采用RT-PCR法扩增NP基因,T-A克隆到pMD18-T载体中构建pMD18-T-NP质粒。经PCR、双酶切鉴定后,双酶切阳性质粒与pXJ40-HA载体,电泳后胶回收,连接目的片段,构建pXJ40-HA-NP质粒,经PCR、双酶切、测序分析鉴定为阳性的质粒即为NP蛋白的真核表达载体。转染293T细胞后,采用免疫印迹法(Western blot)鉴定重组NP蛋白的表达。结果成功构建了高致病性禽流感病毒H5N1亚型NP基因的真核表达载体,并在293T细胞中成功表达出分子量为56kD的重组蛋白。结论成功构建的NP蛋白真核表达载体为进一步研究其功能,研发高致病性禽流感病毒H5N1亚型的诊断、治疗方法和疫苗奠定了基础。

Objective To construct an eukaryotic expression vector of high pathogenic avian influenza （HPAI） virus HSN1 nuclear protein （NP） gene ; to express and confirm the expression of NP recombinant protein in 293T cells. Methods The complete coding sequence of NP gene was amplified by RT-PCR and cloned into pMD18-T vector to construct pMD18-T-NP plasmid by T-A cloning, which was validated by PCR and double digestion analysis. Double digestion of pMD18-T-NP and pXJ40-HA plasmids, followed by ligation of gel-purified fragments constructed pXJ40-HA-NP plasmid, which was verified by PCR, double digestion and sequencing analysis, pXJ40-HA-NP plasmid was transfected into ggaT cells. Expression of recom- binant NP protein was confirmed by Western blot analysis. Results Eukaryotic expression vector pXJ40-HA-NP was success- fully constructed. A 56kD recombinant protein was expressed in mammalian eukaryotic cells, which was confirmed by Western blot analysis. Conclusion The successful construction of NP gene eukaryotic expression plasmid should lay a foundation for further study of NP function and lay a solid foundation on development of diagnosis , treatment and vaccines for HPAI virus HSN1 infection.