中华微生物学和免疫学杂志
中華微生物學和免疫學雜誌
중화미생물학화면역학잡지
CHINESE JOURNAL OF MICROBIOLOGY AND IMMUNOLOGY
2011年
4期
345-349
,共5页
王浩%马驰%崔莲仙%巴德年%何维
王浩%馬馳%崔蓮仙%巴德年%何維
왕호%마치%최련선%파덕년%하유
流感病毒血凝素蛋白%昆虫杆状病毒表达系统%BaculoGold system%Bac-to-Bac system%血凝试验
流感病毒血凝素蛋白%昆蟲桿狀病毒錶達繫統%BaculoGold system%Bac-to-Bac system%血凝試驗
류감병독혈응소단백%곤충간상병독표체계통%BaculoGold system%Bac-to-Bac system%혈응시험
Influenza virus haemegglutinin%Baeularvirus expression system%BaculoGold system%Bac-to-Bac system%Haemegglutinination test
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,进而获得具有牛物学活性的目的 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(1-11N1),人工合成完整HA基因序列;分别利用杆状病毒表达系统BaculoGold system和Bac-to-Bac system,在昆虫细胞中表达目的 基因HA;经亲和层析纯化及Western blot鉴定,红细胞血凝试验检测HA蛋白的生物学活性.结果 获得测序正确的HA基因,分别克隆到pAcGP67B(BaculoGold system)和pFAST Bacl(Bac-to-Bac system)载体,经杆状病毒同源重组后转染Sf9细胞,Western blot鉴定显示,BaculoGold system表达HA蛋白是分泌型的,而Bac-to-Bac system是胞内表达肚表达效果优于前者;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-Bac system更适合表达HA蛋白,为流感病毒的相关研究提供了保障.
目的 利用兩種昆蟲桿狀病毒錶達繫統錶達甲型H1N1血凝素(haemegglutinin,HA)蛋白,進而穫得具有牛物學活性的目的 蛋白.方法 選取中國內地第1例2009甲型H1N1確診病例病毒株A/Sichuan/1/2009(1-11N1),人工閤成完整HA基因序列;分彆利用桿狀病毒錶達繫統BaculoGold system和Bac-to-Bac system,在昆蟲細胞中錶達目的 基因HA;經親和層析純化及Western blot鑒定,紅細胞血凝試驗檢測HA蛋白的生物學活性.結果 穫得測序正確的HA基因,分彆剋隆到pAcGP67B(BaculoGold system)和pFAST Bacl(Bac-to-Bac system)載體,經桿狀病毒同源重組後轉染Sf9細胞,Western blot鑒定顯示,BaculoGold system錶達HA蛋白是分泌型的,而Bac-to-Bac system是胞內錶達肚錶達效果優于前者;血凝試驗證實,這兩種錶達繫統錶達的HA蛋白均具有生物學活性.結論 利用桿狀病毒錶達繫統成功錶達齣具有生物學活性的HA蛋白,Bac-to-Bac system更適閤錶達HA蛋白,為流感病毒的相關研究提供瞭保障.
목적 이용량충곤충간상병독표체계통표체갑형H1N1혈응소(haemegglutinin,HA)단백,진이획득구유우물학활성적목적 단백.방법 선취중국내지제1례2009갑형H1N1학진병례병독주A/Sichuan/1/2009(1-11N1),인공합성완정HA기인서렬;분별이용간상병독표체계통BaculoGold system화Bac-to-Bac system,재곤충세포중표체목적 기인HA;경친화층석순화급Western blot감정,홍세포혈응시험검측HA단백적생물학활성.결과 획득측서정학적HA기인,분별극륭도pAcGP67B(BaculoGold system)화pFAST Bacl(Bac-to-Bac system)재체,경간상병독동원중조후전염Sf9세포,Western blot감정현시,BaculoGold system표체HA단백시분비형적,이Bac-to-Bac system시포내표체두표체효과우우전자;혈응시험증실,저량충표체계통표체적HA단백균구유생물학활성.결론 이용간상병독표체계통성공표체출구유생물학활성적HA단백,Bac-to-Bac system경괄합표체HA단백,위류감병독적상관연구제공료보장.
Objective To express functional haemegglutinin(HA)protein in two different bacularvirus expression systems.Methods The whole open reading frame of A/Sichuan/1/2009(H1N1)HA was obtained by synthesis,and the HA protein were expressed in insect cells by two different bacularvius expression systems:BaculoGold system and Bac-to-Bac 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 amplifyjng by re-infection of Sf9 cells,the HA protein was detected in supematant 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 expressed 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(H1N1)HA protein.
