hCGβ真核表达载体的构建和瞬时表达筛选  

Construction of eukaryotic expression vector of human chorionic gonadotropin beta subunit(hCGβ) and hCGβ transient expression in Hela cell

作　　者：张富春[1] 王妮[2] 陈幼珍[2] 沈孝宙[2] 曹咏清[2] 王宾[3] 

机构地区：[1]新疆大学生物系分子生物学重点实验室,新疆乌鲁木齐830046 [2]中国科学院动物所计划生育生殖生物学国家重点实验室,北京100080 [3]宾夕法尼亚大学医学院病理系,费城19104

出　　处：《生物技术》2002年第5期3-5,共3页Biotechnology

基　　金：国家计生委"九五"攻关项目 ( 96- 90 4 - 0 1 - 0 2a);新疆教委科研基金资助项目 ( 1 9990 1 1 )

摘　　要：为探讨人绒毛膜促性腺激素 β亚基 (hCGβ)基因避孕疫苗的可能性。利用DNA重组技术将hCGβ的基因片段连接到真核表达载体pCMV4上 ,酶切分析鉴定正确构建了质粒DNApCMV4 -hCGβ。将质粒DNApCMV4 -hCGβ通过脂质体转染的方法导入体外培养的Hela细胞 ,双抗夹心ELISA法检测质粒DNApCMV4 -hCGβ在Hela细胞瞬时表达系统中特异性的表达了hCGβ。结果表明hCGβ表达含量 2 4h为 6 78ng/ml,4 8h为 1 5 2 4ng/ml,说明在体外瞬时表达了特异性hCGβ的pCMV4 -hCGβ真核表达载体能够作为DNA疫苗使用 ,为pCMV4 -hCGβ质粒DNA免疫小鼠进行DNA避孕疫苗的研究奠定了基础。Human chorionic gonadotropin beta subunit (hCGβ) has been considered as a promising candidate antigen for contraceptive vaccine. In this study, the complete hCGβ cDNA fragment was cloned into an eukaryotic expression vector-pCMV4 to generate the prototypic DNA vaccine for this purpose. The constructed pCMV4-hCGβ were characterized by restriction digestions and transient expressions in Hela cells. The expressed level of hCGβ was assessed by Sandwich ELISA assays with specific anti-hCGβ antibody and comparing in a standard concentration curve of purified hCGβ. The results shown that the levels of hCGβ were 6.78 ng/ml supernatant at 24 hours and 15.24 ng/ml at 48 hours after pCMV4-hCGβ transfection, respectively. This expression construct may be used to determine its potential for development of a contraceptive DNA vaccine in further study.

关 键 词：HCGΒ 真核表达载体 构建 瞬时表达 筛选 

分 类 号：R394.8[医药卫生—医学遗传学]