title: [Rapid expression and preparation of the recombinant fusion protein sTNFRII-gAD by adenovirus vector system].
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-TNFalpha 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 TNFalpha'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.
authors.affiliation: Zhejiang Provincial Key Laboratory for Technology & Application of Model Organisms, Wenzhou Medical College, Wenzhou 325035, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Lu
authors.forename: Dan
authors.initials: D
authors.lastname: Liu
authors.forename: Xiaoren
authors.initials: X
authors.lastname: Zhang
authors.forename: Xuerong
authors.initials: X
authors.lastname: Liu
authors.forename: Wei
authors.initials: W
authors.lastname: Shen
authors.forename: Gang
authors.initials: G
authors.lastname: Zheng
authors.forename: Yunfan
authors.initials: Y
authors.lastname: Liu
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Dong
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Wu
authors.forename: Jimin
authors.initials: J
authors.lastname: Gao
mesh_terms: D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism*; D052242:Adiponectin / Q000096:biosynthesis* / Q000235:genetics; D000818:Animals; D002460:Cell Line; D005822:Genetic Vectors / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D047889:Receptors, Tumor Necrosis Factor, Type II / Q000096:biosynthesis* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D052242:Adiponectin; D047889:Receptors, Tumor Necrosis Factor, Type II; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins