四种分子伴侣促进牛支原体膜蛋白在大肠杆菌中可溶性表达研究

Soluble expression promotion of Mycoplasma bovine membrane protein by four types molecular chaperones in Escherichia coli

关键词:  分子伴侣  牛支原体  膜蛋白  可溶性表达  原核表达系统

Key words:  molecular chaperone  Mycoplasma bovis  membrane protein  soluble expression  prokaryotic expression system

摘要:

为研究分子伴侣是否可以促进牛支原体膜蛋白在大肠杆菌中以可溶性形式表达及表达的蛋白是否具有活性，应用原核表达和Western blot方法进行检测，结果表明：1）牛支原体一个膜蛋白M1的截短片段在大肠杆菌表达系统中以包涵体形式表达，改变诱导时间、温度以及诱导剂浓度均未改变其表达形式；2）将4种分子伴侣pG-KJE8、pGro7、pG-Tf2和pTf16分别与含有目的蛋白的重组质粒在表达工程菌（BL21）中共表达，当加入终浓度为1.0 mmol/L IPTG以及0.5 mg/mL L-阿拉伯糖或5.0 ng/mL四环素的诱导剂，37℃诱导5 h，发现分子伴侣pG-Tf2和pG-KJE8能显著提高M1截短片段的可溶性表达，其他2种分子伴侣未改变M1的表达形式；3）可溶性表达的M1截短片段与牛支原体阳性血清可以发生特异性反应。因此，研究发现2种分子伴侣可以使牛支原体膜蛋白在大肠杆菌表达系统中以可溶性形式表达，但未改变其生物活性，该研究结果可为建立牛支原体有效的血清学诊断方法及亚单位疫苗的研制奠定基础。

Abstract:

To study whether molecular chaperones could promote the expression of Mycoplasma bovis membrane protein in soluble form in Escherichia coli and the expressed protein was active or not, prokaryotic expression and Western blot methods were adopted in this study. The results showed that:1) The truncated membrane fragment of M1 from M. bovis was expressed in inclusion body in E. coli expression system. Its expression form remained unchanged by changing induction time, temperature or induction agent doses; 2) Four types of molecular chaperone, pG-KJE8, pGro7, pG-Tf2 and pTf16, were respectively combined with recombinant plasmid and expressed in BL21 with the final concentrations of 1.0 mmol/L IPTG and 0.5 mg/mL L-Arabia sugar or 5.0 ng/mL tetracycline inducer, After 5 h induction at 37℃, it was found that molecular chaperones pG-Tf2 and pG-KJE8 significantly increased the soluble expression of truncated M1 fragment, and the other 2 chaperones didn't change the expression of M1; 3) The truncated M1 fragment had specific reaction with the positive serum of Mycoplasma. In conclusion, the four types of molecular chaperones promoted the soluble membrane protein of M. bovis expressed in E. coli expression system, and didn't change its bioactivity. This result laid foundation for establishing effective serological diagnosis method and producing the subunit vaccine of M. bovis.