肠炎沙门菌SPI-19编码的hcp基因的克隆、表达及多抗制备

Cloning and Prokaryotic Expression of Hcp Gene Encoded by SPI-19 in Salmonella enteritidis and Its Polyclonal Antibody Preparation

利用PCR方法扩增2株肠炎沙门菌参考株CMCC(B)50336和SD-2 SPI-19毒力岛内的hcp基因,将其序列鉴定后克隆入原核表达载体pET-28a(+),构建重组质粒pET28a-hcp,并转化至大肠杆菌BL21(DE3)中,用IPTG诱导重组细菌高效表达;表达产物以包涵体的形式存在,变性条件下用镍亲和柱纯化得到重组蛋白,以每只50μg剂量免疫小鼠3次,间接ELISA测定抗体效价,用Western blot验证免疫血清的特异性。测序结果显示,2个参考株hcp基因的核苷酸序列与已发表序列的同源性为100%。SDS-PAGE显示20 ku重组蛋白,主要以包涵体形式存在,通过镍亲、层析和尿素变性/复性获得了可溶性的纯化蛋白;免疫小鼠第5周抗体效价达1∶8 000,且能特异性检测出SPI-19 hcp+肠炎沙门菌和鸡伤寒沙门菌中表达的Hcp蛋白。上述结果表明,制备的外源性重组蛋白具有较好的免疫原性和反应原性,为进一步研究Ⅵ型分泌系统Hcp蛋白在肠炎沙门菌中的功能提供了重要的生物素材。

The hcp gene encoded by SPI-19 in Salmonella enteritidis reference strains CMCC（B）50336 and SD-2 were amplified by PCR and sequenced,then inserted into the multiple clone sites of pET-28a（＋） prokaryotic expression vector. The positive recombinant plasmid was screened and transformed into E.coli BL21 （DE3）. After being inducted with IPTG, the expected 20 ku engineered protein from recombinant E.coli carrying pET28-hcp was detected by SDS-PAGE and Ni-NTA purification system. ICR mice were immunized for three times with 50 μg purified recombinat antigen per time and the specific serum antibody against the protein was tested by indirect ELISA, the specificity of irnnmne serum was eonfirmed by Western blot. The results showed that nueleotide sequence of hcp gene of CMCC（B）50336 anti SD-2 shared 100% homology with other hcp genes of GenBank database. SDS-PAGE analysis showed that the recombinant protein was 20 ku. ELISA titer of immune serum reached at 1：8 000. The specificity of immune serum to Hcp protein was determined in SPI-19 hcp-positve S.enteritidis and S.Gallinarum. These results suggested that recombinant Hcp had good specificity,and could provide important biomaterials for further study of protein function in Salmonella enteritidis.