hTNFα/CHO基因工程细胞的构建及其分泌蛋白功能的检定

Construction of h TNFα/CHO genetic engineered cell and verification of its protein secretion function

目的构建能够持续分泌人肿瘤坏死因子(h TNFα)的中国仓鼠卵巢细胞(CHO)基因工程细胞h TNFα/CHO细胞系,并且观察其分泌功能的变化。方法应用载体GV141构建携带h TNF基因表达的质粒;采用脂质体转染法,经G418筛选稳定转染细胞株;通过Real-Time PCR鉴定其基因表达,采用ELIAS法鉴定其蛋白分泌情况。结果经序列比对,证明GV141-h TNFα表达载体构建成功,Real-Time PCR证明转染细胞系包含h TNFα目的基因,ELIAS鉴定结果证明该细胞系可以在一定代数稳定持续内分泌h TNFα。结论构建的h TNFα/CHO细胞系可以在一定代数内稳定持续地分泌人肿瘤坏死因子。

Objective To construct the genetic engineered cell line which can continuously secrete human tumor necrosis factor（ h TNFα） in Chinese Hamster Ovary cells（ CHO cells）,and observe the change of its protein secretion function. Methods Constructed plasmid which carries h TNFα gene expression through vector GV141. Selected stable transfection cell lines by G418 and transfection with lipofectamine. Identified its gene expression with Real-Time PCR,and identified its protein secretion by ELISA. Results GV141-h TNFα expression vectors were constructed successfully which were proved by sequence alignment. Real-Time PCR proved that it contained h TNFα gene in h TNFα / CHO cell line. ELIAS identification results showed that the cell lines can continuously secrete h TNFα within a certain cell propagation. Conclusion The h TNFα / CHO cell line can continuously secrete human tumor necrosis factor within a certain cell propagation.