副猪嗜血杆菌重组P2蛋白的高效表达及间接ELISA方法的建立

High level expression of P2 protein of HPS strain HB070214 and establishment of ELISA diagnosis based on the recombinant fusion protein

根据GenBank中副猪嗜血杆菌(HPS)的P2蛋白基因序列,设计合成1对特异性引物,用P2-PCR方法从HB070214株中克隆了1 077bp的P2蛋白基因。将P2蛋白基因亚克隆到原核表达载体pET-32a中构建了重组表达质粒pET-P2,在IPTG的诱导下成功表达了相对分子质量约为60 500的可溶性融合蛋白P2-His;经SDS-PAGE和凝胶薄层扫描分析,融合蛋白的表达量约占细菌总蛋白量的30%。将融合蛋白加入镍琼脂糖凝胶树脂层析柱,经300mmol/L咪唑磷酸盐缓冲液洗脱,其纯度可达92.5%。用Western blotting分析纯化后的融合蛋白,显示良好的生物学活性。利用纯化融合蛋白作包被抗原及优化ELISA反应条件,建立了可检测抗HPS P2蛋白抗体的间接ELISA方法(P2-ELISA),并用所建立的P2-ELISA与国外进口的HPS检测试剂盒Synbiocits-ELISA对196份临床送检血清进行平行检测,阳性符合率为93.4%。结果表明,本试验建立的P2-ELISA与Synbiocits-ELISA具有同样高的特异性。

A pair of specific primers was designed and synthesized according to the published P2 gene sequence of HPS from GenBank（ZP_02477753）,and the P2 gene（1 077 bp） of HPS strain HB070214 was amplified by P2-PCR from a template of total HPS DNA.The nucleotide sequence of P2 gene was determined（GU323686）.The P2 gene was cloned into pET-32a vector to generate the recombinant expressing plasmid pET-P2.The PCR-generated plasmids was verified by sequencing of the entire insert.Then pET-P2 was transformed into the host cell BL21（DE3） and the expression procedure was optimized.The P2-His fusion protein with relative molecular mass 60 500 was successfully obtained with the induction of 1.0 mmol/ L IPTG.The P2-His fusion protein could specifically react to antiserum against HPS by Western blotting.The P2-His fusion protein was further purified by nickel-agarose gel resin chromatographic column with 300 mmol/L imidazole phosphate elution buffer and the purity of P2-His was up to 92.5%.The P2-His fusion protein was used as a packaging antigen to establish a novel HPS P2-ELISA diagnosis assay.The comparison between P2-ELISA and kit Synbiocits-ELISA showed that the positive result s of two methods had 93.4% agreement by detecting 196 sera samples,indicating that the indirect P2-ELISA was the same specific and sensitive as kit Synbiocits-ELISA.