青海湖裸鲤IL-8的原核表达及诱导条件优化

Construction of Prokaryotic Expression System and Optimization of Expression Conditions of IL-8 in Gymnocypris przewalskii przewalskii

白细胞介素-8(interleukin-8,IL-8)是一种CXC家族趋化因子,主要通过趋化炎症细胞参与机体免疫反应。本研究通过RT-PCR技术,扩增得到含一对酶切位点的青海湖裸鲤IL-8成熟蛋白DNA序列(gpIL-8),经酶切连接至pET30a(+)质粒得到原核表达重组质粒pET30a-gpIL-8,将重组质粒转化入大肠杆菌BL21后成功诱导得到分子量约13.7 kD的重组蛋白,重组蛋白以包涵体和可溶性蛋白两种形式存在。通过对影响蛋白表达量的诱导物浓度和诱导时间进行优化,发现包涵体重组蛋白表达量随诱导时间增加而增加,随诱导物浓度增加呈先增加后减少的趋势,在0.4 mmol/L IPTG诱导8 h时表达量最高。可溶性重组蛋白表达量总体上随诱导时间的增加呈先增加后减少趋势,随诱导物浓度的增加表达量无明显改变,在0.4 mmol/L IPTG诱导6 h时即可表达高浓度的可溶性重组蛋白。因可溶性蛋白有利于后续实验,故利用最佳诱导条件诱导后对可溶性蛋白进行纯化,得到高纯度的IL-8重组蛋白,测得浓度达101.5μg/mL,满足后续实验要求。本研究为青海湖裸鲤IL-8多克隆抗体制备,及其功能、作用机制研究提供了理论依据。

Interleukin-8(IL-8)is one of CXC chemokines playing the important role in mediating inflammatory response and being involved in immune response.In this study,the primers with two restriction enzyme cutting sites were used to clone the complete DNA sequence of IL-8 mature protein of Gymnocypris przewalskii przewalskii.Then the gpIL-8 gene was cloned into the prokaryotic expression vector pET30 a(+)to construct the recombinant vector pET30 a-gpIL-8.After that,the recombinant plasmid was transformed into E.coli BL21 strain to the expression of target protein.A molecular weight of 13.7 kD recombinant protein which existed in forms of inclusion body and soluble protein was successfully expressed by induction.The concentration of inducer and the induction time affecting the expression level of recombinant protein were optimized,the expression level of inclusion body increased with the augment of induction time,while the expression level firstly increased and then decreased with the augment of the concentration of inducer,and the expression level was the highest at 0.4 mmol/L IPTG for 8 h.On the whole,the expression level of soluble recombinant protein firstly increased and then decreased with the augment of the induction time,while the expression level of soluble protein didn’t change obviously with the augment of the concentration of inducer,and the expression level was the highest at 0.4 mmol/L IPTG for 6 h.Owing to soluble protein being beneficial to subsequent experiments,it can use the best induction conditions to purify soluble recombinant protein of gpIL-8,a concentration of 101.5μg/mL,which can meet the requirements of the subsequent experiments.The results laid a foundation for the preparation of IL-8 polyclonal antibody,and the research of function and mechanism of IL-8 in Gymnocypris przewalskii przewalskii.