PcDNA3.1-sTNFRⅠ重组载体体外表达产物生物学活性的检测

Bioassay of the soluble human tumor necrosis factor receptor Ⅰ recombinant plasmid expression in vitro

目的 检测构建的PcDNA3 1 sTNFRⅠ真核表达载体在体外转染哺乳动物细胞后能否有效表达目的产物 ,表达的目的产物是否具有生物学活性 ,为探索sTNFRⅠ基因治疗牙周炎奠定一定的实验基础。方法 ELISA法检测PcDNA3 1 sTNFRⅠ转染的CHO细胞培养上清液中sTNFRⅠ的表达 ,MTT法检测表达产物是否具有抑制肿瘤坏死因子α(TNF α)对L92 9的细胞毒作用的能力。结果 重组质粒转染的CHO细胞上清液中表达的sTNFRⅠ量超过 10 0 0ng/L ,显著高于对照组 (10 9 6 0± 5 32 )ng/L ,P <0 0 0 1,表达的sTNFRⅠ能中和TNF α对L92 9的细胞毒作用。结论 PcDNA3 1 sTNFRⅠ重组质粒在体外导入哺乳动物细胞后能够表达具有相应生物学活性的目的产物。

Objective To detect the expression of recombinant plasmid PcDNA3.1-sTNFRⅠin vitro and evaluate the bioactivity of expressed sTNFRⅠ.Methods CHO cells were transfected with recombinant plasmid PcDNA3.1-sTNFRⅠ by liposome. sTNFRⅠin cell culture supernatant was detected by ELISA and sTNFRⅠblockage of TNF-αcytotoxicity in L929 cells evaluated by MTT assay.Results The expression of sTNFRⅠin transfected cell culture supernatant was higher than control groups( P <0.001). The expressed sTNFRⅠ could significantly neutralize TNF-αcytotoxicity in L929 cells.Conclusions These results showed that the recombinant plasmid PcDNA3.1-sTNFRⅠcan be expressed in mammalian cells and the recombinant sTNFRⅠhas biological function.