高效表达乙型肝炎表面抗原的CHO工程细胞株的构建  

Construction of Recombinant CHO Cell Strain for High Expression of HBsAg

作　　者：时成波[1] 徐军[1] 王堃[1] 贾重来 徐冬冬[1] 盛军[1] 

机构地区：[1]长春生物制品研究所,长春130062 [2]北京天坛生物制品股份有限公司疫苗二室,北京100024

出　　处：《中国生物制品学杂志》2009年第12期1172-1175,共4页Chinese Journal of Biologicals

摘　　要：目的构建高效表达乙型肝炎表面抗原(HBsAg)的CHO工程细胞株。方法从pCIneo质粒出发,构建含有改造了稀有密码子的HBsAgS基因和启动子弱化的二氢叶酸还原酶(DHFR)转录单位的新型表达载体,利用脂质体转染CHO/dhfr-细胞,经3轮氨甲喋呤(MTX)梯度加压筛选和单克隆筛选,获得高效表达HBsAg的CHO工程细胞株,并对HBsAg的分泌动态进行检测。结果所构建的新型表达载体pCI-DS经PCR及酶切鉴定,证明构建正确,转染CHO/dhfr-细胞后,经筛选得到高效表达HBsAg的CHO工程细胞株3F9,表达量达9.21μg/106个细胞·48h。单层细胞培养动态表明,3F9株细胞能在40d内稳定高效表达HBsAg。结论已成功构建稳定高效表达HBsAg的CHO工程细胞株,为提高HBsAg的产量创造了条件。Objective To construct a recombinant CHO cell strain for high expression of HBsAg. Methods Based on plasmid pCIneo, a novel expression vector was constructed, containing a HBsAg gene of which the rare codon was modified and a DHFR transcription unit of which the promoter was attenuated. CHO / dhfrcells were transfected with the constructed expression vector in mediation of liposome, based on which a recombinant CHO cell strain for high expression of HBsAg was screened by three cycles of MTX gradient pressure screening followed by monoclonal screening, and determined for secretion of HBsAg. Results Both PCR and restriction analysis proved that novel expression vector pCI-DS was constructed correctly. A recombinant CHO cell strain 3F9 was obtained after transfection of CHO / dhfrcells with pCI-DS, in which the expression level of HBsAg reached 9. 21 μg / 106 cells every 48 hours. The dynamics of 3F9 cell monolayer culture showed stable and high expression of HBsAg within 40 d. Conclusion A recombinant CHO cell strain for high expression of HBsAg was successfully constructed, which provided a condition for increasing the yield of HBsAg.

关 键 词：乙型肝炎表面抗原 中国仓鼠卵巢细胞 高效表达 

分 类 号：Q789[生物学—分子生物学]