猪繁殖与呼吸综合征病毒N基因和流感病毒HA基因的联合表达及应用

Coexpression and Application of N Gene of Porcine reproductive and respiratory syndrome virus and HA gene of Influenza Virus

参照已公布的流感病毒血凝素基因(HA基因)及猪繁殖与呼吸综合征病毒(PRRSV)基因组序列,设计并合成一对引物P1、P2,以RT-PCR方法扩增出PRRSV的ORF7片段(约410bp),其中含HA基因主要核苷酸序列(33bp)。用BamH Ⅰ、Xho Ⅰ分别对扩增出的片段及pET32a质粒进行酶切,连接后构建了重组质粒pETHN并转化到BL21(DE3)宿主菌中诱导表达。用纯化后的表达产物与流感病毒血凝素单抗及乳胶建立了诊断猪繁殖与呼吸综合征(PRRS)的乳胶凝集试验。检测结果显示:该方法有良好的特异性及敏感性,与IDEXX公司FLISA检测试剂盒符合率达93.8％。

From to the nucleotide sequences of haemoagglutinin gene of Influenza virus and Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) VR2332 in GenBank, a pair of primers which includes the main sequences of haemoagglutinin (HA) gene(33bp) was designed to amplify N gene (ORF7)of PRRSV by RT-PCR. The amplified fragment and pET-32a plasmid were digested by BamHI and XhoI. A recombinant plasmid named pETHN was constructed and transformed into BL21(DE3). Consequently, the target protein was expressed by IPTG induction, and the purified fusion protein was obtained by His-binding purification kit. As a result latex-agglutination test was set up and plenty of serum samples were tested by the method. The results showed the method had same sensitivity and specificity and had 93.8% accord rate compared with ELISA (IDEXX) diagnosis kit.