利用两种真核昆虫表达系统表达可溶性甲型H1N1血凝素蛋白

Expression of soluble H1N1 haemegglutinin in two distinct insect baculovirus expression systems

目的利用两种昆虫杆状病毒表达系统表达甲型H1N1血凝素（haemegglutinin，HA）蛋白，进而获得具有生物学活性的目的蛋白。方法选取中国内地第1例2009甲型H1N1确诊病例病毒株A／Sichuan／1／2009（H1N1），人工合成完整HA基因序列；分别利用杆状病毒表达系统BaculoGoldsystem和Bac—to-Bacsystem，在昆虫细胞中表达日的基因HA；经亲和层析纯化及Westernblot搭定，红细胞血凝试验检测ttA蛋门的生物学活性。结果获得测序正确的HA基因，分别克隆到pAcGP67B（Baculo Goldsystem）和pFASTBacl（Bac-to-Bacsystem）载体，经杆状病毒同源重组后转染Sf9细胞，Westernblot鉴定硅示，BaculoGoldsystem表达HA蛋白是分泌型的，而Bac—to—Bacsystem是胞内表达且表达效果优于前者；血凝试验证实，这两种表达系统表达的HA蛋白均具有生物学活性。结论利用杆状病毒表达系统成功表达出具有生物学活性的HA蛋白，Bac—to-Bacsystem更适合表达HA蛋白，为流感病毒的相关研究提供了保障。

Objective To express functional haemegglutinin （HA） protein in two different bacu-larvirus expression systems. Methods The whole open reading frame of A/Sichuan/1/2009（ HINI ） HA was obtained by synthesis, and the HA protein were expressed in insect eells~ by two different bacularvius ex- pression systems： BaeuloGold system and Bac-to-Bae system. Soluble HA protein was identified by Western blot and haemegglutination test. Results The correct full length of HA gene was obtained and cloned into pAcGP67B and pFAST Bacl vectors, respectively. After 3 rounds of virus amplifying by re-infection of SO ceils, the HA protein was detected in supernatant of BaculoGold system and in intracellular of Bac-to-Bac system which is much better than the former. Purified HA protein was positive not only identified by Western blot, but also detected by haemegglutinin test. Conclusion Functional HA protein was successfully ex- pressed in two distinct bacularvirus expression systems, of which the Bac-to-Bac bacularvirus expression system is more suitable for expression of A/Sichuan/1/2009（ H1 N1 ） HA protein.