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

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。

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

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.