甲型H1N1流感病毒HA基因在昆虫细胞中的截短表达

目的:利用Bac-to-Bac Baculovirus Expression System表达重组HA蛋白,Western blot及IFA方法鉴定其表达。方法:采用PCR方法扩增A/California/04/2009(H1N1)HA基因,将其克隆到pFastBacHT A载体上,重组质粒pFastBacHT-HA经双酶切及测序鉴定正确后,转化阳性重组载体进入E.coli DH10Bac感受态细胞中,通过Bluo-gal蓝白斑筛选、PCR鉴定获得重组转座子rBacmid-HA。从重组转座子中提取rBacmid-HA质粒DNA转染sf 9昆虫细胞,制备重组杆状病毒。重组杆状病毒感染sf 9细胞表达重组蛋白,Western blot及IFA鉴定重组蛋白表达情况。结论:成功构建了甲型H1N1流感病毒HA基因的昆虫杆状病毒表达载体,该表达载体转染昆虫细胞后制备的重组杆状病毒病毒滴度较高,重组杆状病毒表达的重组蛋白经Western blot 及IFA 鉴定后具有良好的免疫反应原性。

Baculovirus Expression of Truncated HA Gene of A/H1N1 Influence Virus

Object: In order to express recombinant HA protein using the Bac-to-Bac Baculovirus Expression System. The expression of the recombinant HA protein was confirmed by Western blot and IFA. Method: A/California/04/2009(H1N1)HA gene was amplified by PCR, and the PCR product was cloned into the pFastBacHT A vector. The recombinant plasmid pFastBacHT-HA was identified by restriction enzyme digestion and gene sequencing. The recombinant plasmid pFastBacHT-HA was subsequently transformed into E. coli DH10Bac component cells. The recombinant rbacmid-HA identified by Bluo-gal blue/white selection and PCR was purified and transfected into sf 9 cells. The P1 virus obtained from sf 9 cells was amplified and stocked. The sf 9 cells infected with the baculovirus was harvested and analyzed for detection of the expression of recombinant protein by Western blot and IFA. Conclusion: The expression vector engineered for expressing A/H1N1 influence virus HA gene was constructed, which was transfected into insect cells, The recombinant baculovirus with higher viral titer was obtained. The recombinant protein was expressed by recombinant baculovirus in the infected sf 9 cell and identified by Western blot and IFA, demonstrating that the truncated HA protein can be recognized by the antibody specific to HA of 2009 H1N1 influenza virus.