布鲁氏菌P39基因的克隆、原核表达及其糖基化修饰

Cloning,Prokaryotic Expression and Glycosylation of P39 Gene from Brucella

为探究糖基化修饰对布鲁氏菌P39蛋白免疫原性的影响,本试验对布鲁氏菌P39蛋白进行了表达和纯化,并对表达的蛋白进行了甘露六糖修饰。参照GenBank公布的布鲁氏菌P39基因序列设计引物,从布鲁氏菌16M基因组中克隆P39基因片段并连接至pMD19-T载体,转化大肠杆菌DH5α感受态细胞,提取阳性菌株质粒进行酶切等鉴定,鉴定正确后构建重组质粒pGEX-6P-1-P39,对重组蛋白进行诱异表达与条件优化。利用SDSPAGE和Western blotting对诱导表达的目的蛋白进行分析,将鉴定正确的蛋白经GST亲和层析柱纯化。通过EDC/NHS法对纯化的P39蛋白进行甘露六糖修饰,研究甘露六糖修饰后目的蛋白对巨噬细胞吞噬的影响。结果显示,本试验成功克隆了片段大小为1 206bp的目的基因,构建了pGEX-6P-1-P39原核表达载体,在大肠杆菌中成功表达了P39蛋白,该蛋白主要以可溶性形式存在。Western blotting结果显示,在约65ku处有特异性条带。纯化后获得目的蛋白大小为43ku,成功对目的蛋白P39进行了糖基化修饰,得到修饰产物与蛋白的摩尔比为2.3∶1。此外,糖基化修饰可显著提前蛋白激活小鼠巨噬细胞的吞噬作用时间。本试验结果可为研究甘露六糖修饰影响P39蛋白免疫原性的机制提供参考。

In order to investigate the effect of glycosylation modification on the immunogenicity of Brucella P39 protein,the expression and purification of Brucella P39 protein were carried out in this experiment,and the expressed protein was modified with mannose hexasaccharide.The primers were designed according to the Brucella P 39 gene sequence published in GenBank,and the P 39 gene fragment was cloned from the Brucella 16M genome and ligated into pMD19-T vector to transform E.coli DH5αcompetent cells.The positive strain plasmid was extracted and identified by enzyme digestion.After correct identification,the recombinant plasmid pGEX-6P-1-P39 was constructed,and the induction expression and optimization of recombinant protein were analyzed.The target protein induced was analyzed by SDS-PAGE and Western blotting,and the correct protein was purified by GST affinity chromatography.The purified P39 protein was modified by mannose hexasaccharide using EDC/NHS method to study the effect of the target protein on the phagocytosis of macrophages after modification of mannose hexose.The results showed that the target gene with a fragment size of 1 206 bp was successfully cloned,and the prokaryotic expression vector pGEX-6P-1-P39 was constructed.The P39 protein was successfully expressed in E.coli,and the protein was mainly soluble.Western blotting results showed a specific band at approximately 65 ku.After purification,the target protein size was 43 ku,the target protein P39 was successfully glycosylated,and the molar ratio of the modified product to the protein was 2.3∶1.In addition,glycosylation modification could significantly advance the phagocytosis time of mouse macrophages by protein activation.This study could provide a reference for research the mechanism of mannose hexose modification affecting the immunogenicity of P39 protein.