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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 CHO cell line highly expressing hepatitis B surface antigen (HBsAg). Methods From the pCIneo plasmid, a novel expression vector containing the HBsAgS gene with a modified codon and a dihydrofolate reductase (DHFR) transcription unit with reduced promoter was constructed and transfected into CHO / dhfr- Methotrexate (MTX) gradient compression screening and monoclonal screening to obtain high expression of HBsAg in CHO cell lines, and to detect the secretion of HBsAg dynamic. Results The recombinant plasmid pCI-DS was identified by PCR and restriction enzyme digestion. The results showed that the recombinant plasmid pCI-DS3 was constructed correctly and transfected CHO / dhfr- cells. The CHO cell line 3F9 with high expression of HBsAg was screened and the expression level reached 9.21 μg / 106 Cells · 48h. The dynamic of monolayer cell culture showed that 3F9 cells stably and efficiently expressed HBsAg within 40 days. Conclusion The CHO cell line that stably and efficiently expressed HBsAg has been successfully constructed, which has created the conditions for increasing the production of HBsAg.