肠炎沙门菌鞭毛蛋白的原核表达纯化及其多克隆抗体的制备

Prokaryotic expression,purification and polyclonal antibody preparation of FliC protein of Salmonella enteritidis

利用PCR技术成功扩增到了肠炎沙门菌鞭毛蛋白fliC基因,将其连接到原核表达载体pET-32a(+)中,构建原核重组表达质粒pET-32a(+)-fliC。将pET-32a(+)-fliC转化到大肠杆菌BL21(DE3)菌株中,并在30℃下用终浓度为1mmol/L IPTG进行诱导表达9h。表达的重组蛋白通过镍离子亲和层析和分子筛的方法进行纯化,获得的蛋白用SDS-PAGE方法进行分析。对纯化的重组蛋白进行定量和内毒素测定后,用其免疫SPF鸡,制备超免疫血清。纯化的FliC蛋白与其受体之间的相互作用通过流式细胞仪进行分析。结果显示,成功扩增到了长度为1518bp的全长fliC基因,该基因编码的蛋白在30℃下以可溶性形式表达,经过镍离子亲和层析与分子筛的方法获得的FliC蛋白的纯度高,其内毒素的含量低于0.5EU/mL,并能够结合该蛋白在细胞上的受体。用纯化的重组蛋白免疫鸡获得的阳性血清的效价为1∶12 800。上述结果为进一步研究FliC蛋白的免疫学功能奠定了基础。

The fliCgene of Salmonella enteritidis was successfully amplified using PCR and inserted into pET-32a（＋）vector to obtain the recombinant plasmid pET-32a（＋）-fliC.The expression of FliC protein was induced with 1mmol/L IPTG for 9hat 30℃after the transformation of the recombinant plasmid pET-32a（＋）-fliCinto E.coli BL21（DE3）.The recombinant FliC protein was purified with using Ni-NTA and Superdex 200 column and analyzed using SDS-PAGE.After quantification and detection of endotoxins,the FliC protein was submitted to immunize SPF chickens to produce hyper-immune sera.The interaction between the purified FliC protein and its receptors was analyzed with FACS.The results showed that the fliCgene with 1 518 bp in length was successfully cloned.The FliC protein was expressed in soluble form at 30℃.The highly purified FliC protein,in which the endotoxin was lower than 0.5EU/mL,was obtained using Ni-NTA and Superdex 200 column and able to bind to its receptors on cells.The titer of positive chicken sera against the FliC protein was 1∶12 800.These results will facilitate to explore immune functions of the FliC protein in future.