摘要
目的探讨汉坦病毒H8205株包膜糖蛋白G2-IL-2嵌合基因(IL-2-G2)在中国仓鼠卵巢(CHO)细胞获得稳定表达可溶性融合蛋白的可行性。方法将人源IL-2基因与汉坦病毒H8205株包膜糖蛋白G2的cDNA分别克隆于真核表达载体pcDNA3.1-HisB,构建重组真核表达载体pcDNA3.1-HisB-IL-2-G2,在脂质体介导下,将其导入CHO细胞,通过G418筛选获得阳性克隆,并继续培养6周,然后用原位杂交、ELISA、SDS-PAGE电泳方法检测IL-2-G2基因在CHO细胞中的稳定表达情况。结果经原位杂交和SDS-PAGE电泳证实,转染pcDNA3.1-HisB-IL-2-G2的CHO细胞内有IL-2-G2的mRNA转录,且在培养上清和CHO细胞胞质中有融合蛋白的表达,经人IL-2 ELISA检测试剂盒检测证实在培养上清和细胞裂解物中所表达的融合蛋白有IL-2特性。结论在脂质体介导下,外源性IL-2-G2嵌合基因能够成功导入CHO细胞并获得稳定表达。
Objective To examine the effectiveness of the stable expression of the fusion gene of membrane glycoprotein G2 gene of hantavirus H8205 strain and IL-2 gene in China Hamster Oophorus cell （CHO）. Methods pcDNA3. 1-HisB-IL-2-G2 was constructed using gene clone and recombined techniques. By using lipofeetamine, CHO was transfected with pcDNA3.1- HisB-IL-2-G2. The positive cell clones were selected with G418. The stable transfection and expression of pcDNA3.1-HisB-IL- 2-G2 were determined by in situ hybridization, ELISA and SDS-PAGE eleetrophoresis. Results The two fragments digested from pcDNA3.1-HisB-lL-2-G2 by Hind Ⅲ and Xho I represented 5.5 kb and 1.9 kb respectively by eleetrophoresis, which were confirmed to be the vector and the IL-2-G2 gene fragments inserted originally, indicating that the construction of peDNA 3.1-HisB-IL-2-G2was successful. Abundant IL 2 G2 stable expression in CHO transfeeted with pcDNA3.1 HisB-IL-2-G2 was confirmed by in situ hybridization, human IL-2 ELISA kit and SDS-PAGE electrophoresis. Conclusion By using lipofectamine, IL-2-G2gene can be transferred and stably expressed in CHO.
出处
《华中科技大学学报（医学版）》
CAS
CSCD
北大核心
2006年第2期163-165,共3页
Acta Medicinae Universitatis Scientiae et Technologiae Huazhong
基金
国家自然科学基金资助项目(No.30170819)
