腺病毒载体介导的重组蛋白sTNFRII-gAD快速表达和制备

Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system

摘要:

利用腺病毒载体高效感染哺乳动物细胞及表达外源基因的特性，建立一种快速高效表达及制备重组蛋白的方法，并用该方法获得sTNFRII-gAD (可溶性肿瘤坏死因子受体II-脂联素球部融合蛋白) 蛋白纯品。首先用携带EGFP基因的腺病毒载体rAd5-EGFP以不同的腺病毒用量 (MOI) (0~1 000) 感染BHK21c022细胞，观察比较其转导效率和细胞毒性。用AdMax腺病毒载体系统制备携带融合基因sTNFRII-gAD的重组复制缺陷型5型腺病毒rAd5-sTNFRII-gAD。用rAd5-sTNFRII-gAD以不同的MOI (0~1 000) 感染BHK21c022细胞，收取上清进行Western blotting分析，比较上清中sTNFRII-gAD蛋白的表达量。在此基础上，用rAd5-sTNFRII-gAD以MOI 100感染大量培养的BHK21c022细胞，在无血清培养条件下反复多次收取培养上清，经过硫酸铵浓缩、分子筛柱层析、透析等步骤浓缩和纯化sTNFRII-gAD融合蛋白，并体外测定该融合蛋白拮抗TNFa的活性。结果获得了携带sTNFRII-gAD融合基因的重组腺病毒rAd5-sTNFRII-gAD；用rAd5-EGFP感染BHK21c022细胞结果表明，随着腺病毒用量的增高，表达EGFP蛋白的BHK21c022细胞数量和亮度明显增加；MOI在0~100之间被感染的BHK21c022细胞未表现出明显的细胞毒性，MOI为1 000时可观察到细胞变圆和少量死亡现象。Western blotting分析结果表明，随着腺病毒用量的增高，培养上清中sTNFRII-gAD融合蛋白表达量明显增加，以MOI为1 000时最高。在此基础上，我们用MOI为100的rAd5-sTNFRII-gAD感染5个转瓶培养的BHK21c022细胞以制备sTNFRII-gAD融合蛋白。每个转瓶加无血清培养液100 mL，每48 h收获上清1次并换液，反复6次收取培养上清共约3 L，经过纯化获得了约11 mg的sTNFRII-gAD融合蛋白。体外活性测定实验表明，获得的sTNFRII-gAD融合蛋白能有效拮抗TNFα对L929细胞的杀伤作用。腺病毒载体/BHK21细胞表达系统是一个简便、高效、通用的表达系统，利用该系统成功制备了有生物学活性的sTNFRII-gAD融合蛋白。该表达系统的特点是生产规模易于放大，适应无血清培养，可以反复多次收获目标蛋白。

Abstract:

We expressed and prepared the recombinant fusion protein sTNFRII-gAD consisted of soluble TNF receptor II and the globular domain of adiponectin by Adenovirus Vector System in mammalian BHK21c022 cells. First we used the adenovirus vector containing EGFP gene (rAd5-EGFP) to infect BHK21c022 cells at different MOI (from 0 to 1 000), and then evaluated their transduction efficiency and cytotoxicity. Similarly, we constructed the replication-deficient adenovirus type 5-sTNFRII-gAD (rAd5-sTNFRII-gAD). We collected the supernatants for Western blotting to determine the optimal MOI by comparing the expression levels of sTNFRII-gAD fusion protein, 48 h after the BHK21c022 cells were infected by rAd5-sTNFRII-gAD at different MOIs (from 0 to 1 000). Then, we chose rAd5-sTNFRII-gAD at MOI 100 to infect five bottles of BHK21c022 cells in 100 mL of serum-free chemically defined media 100 mL, harvested the supernatant every 48 h for 6 times, and condense and purify sTNFRII-gAD fusion protein by ammonium sulfate salt-out and size-exclusion chromatography, respectively. Finally, we analyzed anti-TNFa activity of sTNFRII-gAD fusion protein on L929 cells in vitro. The results showed that the number of BHK21c022 cells expressing EGFP protein was increased significantly with the increase of MOI. However, some cells died at MOI of 1 000 while there was no significant cytotoxicity at MOI from 0 to 100. Western blotting analysis showed that the more adenoviruses, the higher expression of sTNFRII-gAD fusion protein in the supernatant with the highest expression at MOI 1 000. We successfully obtained about 11 mg bioactive and purified sTNFRII-gAD fusion protein at last. The in vitro assay demonstrated that the sTNFRII-gAD fusion protein was potent to antagonize TNFα’s cytotoxicity to L929 cells. Put together, we established a recombinant adenovirus vector/BHK21 cell expression system, characteristic of the efficient serum-free culture and easy scaling-up.