四环素调控型猪鼻支原体蛋白P37表达细胞系的建立

Establishment of a stable and regulable 293 cell line expressing mycoplasma hyorhinis protein P37

目的建立四环素调控型猪鼻支原体蛋白P37的表达细胞系,为研究p37对细胞表型的影响及相关机制奠定基础。方法构建pcDNA5/FRT/TO-p37重组质粒,酶切鉴定后用脂质体方法将质粒与重组酶表达载体pOG44共转染Flp-In-T-REx-293细胞,用Hygromycin和Blasticidin双药筛选出阳性克隆,分别提取细胞总RNA和总蛋白,应用反转录-聚合酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)和Western印迹对其表达进行鉴定,并确定四环素诱导的最适时间和浓度。通过噻唑蓝(methyl thiazolyl tetrazolium,MTT)检测P37对细胞增殖的影响。结果转染pcDNA5/FRT/TO-p37的Flp-In-T-REx-293细胞克隆能成功表达P37蛋白(相对分子质量为43.5×103),且能将该蛋白分泌至胞外。四环素对其表达显示出明显的调控作用,不加四环素的细胞不表达P37蛋白。四环素质量浓度在2mg/L并作用60h其表达水平即可达到最高峰。P37的表达对细胞增殖具有一定的促进作用。结论已建立受四环素调控的P37高效表达细胞系,为进一步研究p37的功能和作用机制提供了实验细胞模型。

Objective： To establish a stable cell line, which can express P37 protein of mycoplasma hyorhinis and be regulated by tetracycline, for investigating the effect of p37 on phenotype of cells and its mechanism. Methods： Recombinant plasmid PcDNA5/FRT/TO-p37 was constructed and cotransfected with pOG44 into Flp-In-T-REx-293 cells by lipofectamine. Positive clones were screened with Hygromycin and Blasticidin. RT-PCR and Western blot were used to exam the mRNA and protein expression in selected clones. The expression level at different inducing times and concentrations of tetracycline were examined. MTT assay was used to observe the effect of P37 on proliferation of 293 cells. Results： P37 protein, which is 43.5 × 10^3, was expressed in the selected clone as well as secreted from cells. Tetracycline showed a good regulation on the expression of P37 protein, which was not detectable without tetracycline induction. When induced with 2 mg/L tetracycline for 60 hours, the P37 protein expression reached maximum level. Cell growth was promoted after being transfected with p37. Conclusion： A stable cell line expressing P37 regularly was established, which provides a good cell model for studying p37 function and its molecular mechanism.