摘要
目的应用Bac-to-Bac杆状病毒表达系统表达小鼠可溶性IL-5α受体(Soluble IL-5 receptorα,sIL-5Rα)蛋白,并对其抗原性进行鉴定。方法应用Vector NTI AdvanceⅡ软件优化小鼠IL-5Rα基因胞外区密码子后合成序列,将sIL-5Rα克隆至pFastbacI中,构建重组穿梭质粒pFastbacI-sIL-5Rα,转化感受态大肠杆菌DH10BacTM,构建重组杆粒Bacmid-sIL-5Rα,转染sf9细胞,通过异源基因重组获得杆状病毒,采用Reed-Muench法检测扩增后病毒滴度;将P3、P4代病毒以0.05 MOI感染sf9细胞,SDS-PAGE鉴定sIL-5Rα蛋白的表达,表达产物应用阳离子交换层析法进行纯化,并对纯化的sIL-5Rα蛋白进行SDS-PAGE及Western blot鉴定。结果重组杆粒Bacmid-sIL-5Rα经PCR鉴定,证明构建正确;P3代重组杆状病毒的病毒滴度为1.7×107pfu/ml;表达及纯化的sIL-5Rα蛋白在相对分子质量约43 000处可见特异蛋白条带,感染的sf9细胞裂解上清可与兔抗小鼠IL-5Rα多克隆抗体发生特异性反应。结论Bac-to-Bac杆状病毒表达系统成功表达了小鼠可溶性IL-5Rα受体蛋白,为进一步研究其生物学功能及在哮喘模型动物中的治疗作用奠定了实验基础。
Objective To express mouse soluble IL-5 receptor（SIL-5R）in Bac-to-Bac system and identify its antigenicity. Methods The codon of extracellular region of mouse IL-5Rα was optimized by using Vector NTI Advance Ⅱ software, based on which the IL-5Rα was synthesized and cloned into vector pFastbacI.The constructed recombinant shuttle plasmid pFastbacI-sIL-5Rα was transformed to E.coli DH10Bac TM.The constructed recombinant bacmid was transfected to sf9 cells to obtain baculovirus by heterologous gene recombination.The baculovirus was propagated and determined for titer. The sf9 cells were infected with baculovirus of passages 3（P3）and 4（P4）at a MOI of 0.05,and determined for expression of sIL-5Rα.The expressed product was purified by cation exchange chromatography and identified by SDS-PAGE and Western blot.Results PCR proved that Bacmid-sIL-5Rαwas constructed correctly.The titer of baculovirus of P3 was 1.7×10 7 pfu/ml.Either of expressed and purified sIL-5Rα showed a specific protein band with a relative molecular mass of 43 000,with which the supernatant of lysed sf9 cells infected showed specific reaction with rabbit anti-mouse IL-5Rα polyclonal antibody.Conclusion Mouse sIL-5Rα protein was expressed successfully in Bac-to-Bac system,which laid an experimental foundation of further study on its biological function and its curative effect on asthma in animal model.
出处
《中国生物制品学杂志》
CAS
CSCD
2013年第5期611-615,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(81070028)
