重组人骨形态发生蛋白7成熟蛋白在悬浮CHO-S细胞中的稳定表达  

Stable expression of recombinant human bone morphgenetic protein 7 mature protein in CHO cells

作　　者：关诗宇 刘涵[1] 郭凤祥 宋昊[1] 邸丽莹[1] 徐军[1] 于爱东[1] 迟春萍[1] 时成波[1] 李利 曹玉锋[1] 刘畅[1,3] GUAN Shi-yu;LIU Han;GUO Feng-xiang;SONG Hao;DI Li-ying;XU Jun;YU Ai-dong;CHI Chun-ping;SHI Gheng-bo;LI Ii;CAO Yu-feng;LIU Chang(Changchun Institute of Biological Products,Changchun 130012,Jilin Province,China)

机构地区：[1]长春生物制品研究所,吉林长春130012 [2]珠海霍普金斯医药研究院有限公司,广东珠海519031 [3]珠海希望基因医药研究院有限公司,广东珠海519031

出　　处：《中国生物制品学杂志》2020年第5期501-506,共6页Chinese Journal of Biologicals

摘　　要：目的在悬浮CHO-S细胞中稳定表达重组人骨形态发生蛋白7(recombinant human bone morphgenetic protein7,rhBMP7)成熟蛋白。方法应用RT-PCR技术从BALB/c乳鼠股骨组织扩增mbmp7基因,克隆至质粒pMD18-T,通过定点突变3个氨基酸编码序列(G266S、S287N、D359E),得到重组克隆质粒pMD18-mbmp7m3;将mbmp7m3克隆至表达载体pCHO1.0,构建重组表达质粒pCHO-mbmp7m3,转染CHO-S细胞,利用嘌呤霉素和甲氨蝶呤进行两轮加压筛选,有限稀释法筛选单克隆细胞,ELISA法检测rhBMP7成熟蛋白的表达。结果从乳鼠股骨组织扩增的cDNA经测序与GenBank中登录的序列完全一致,点突变后测序与设计完全一致。获得了稳定表达rhBMP7成熟蛋白的单克隆细胞株,最高表达量为202 ng/mL。结论成功利用改造的mbmp7基因在CHO-S细胞中表达了rhBMP7成熟蛋白,为后续该制品工艺开发及产业化奠定了基础。Objective To express recombinant human bone morphgenetic protein 7(rhBMP7)mature protein in CHO-S cells stably.Methods The gene encoding mouse BMP7,mbmp7,was amplified from the femur tissue of suckling mice by RT-PCR and cloned into plasmid pMD18-T,based on which recombinant cloning plasmid pMD18-mbmp7m3 was obtained by site-directed mutation of three amino acids(G266 S,S287 N and D359E)and cloned into expression vector p CHO1.0.The constructed recombinant plasmid pCHO-mbmp7m3 was transfected to CHO-S cells.Two rounds of pressure screening were performed with puramycin and methotrexate,and monoclonal cells were obtained by limiting dilution method.The expression of rhBMP7 mature protein was determined by ELISA.Results The sequence of cDNA amplified from the femur tissue of suckling mice was completely identical to that in GenBank.A monoclonal cell line stably expressing rhBMP7 mature protein was obtained,and the expression level reached 202 ng/mL at most.Construction The rhBMP7 mature protein was successfully expressed in CHO-S cells by using modified mbmp7 gene,which laid a foundation of development and industrial production of the product.

关 键 词：重组人骨形态发生蛋白7 定点突变 中国仓鼠卵巢细胞 

分 类 号：R392[医药卫生—免疫学]