犬细小病毒SH14株VP2基因原核表达及免疫原性分析

PROKARYOTIC EXPRESSION AND CHARACTERIZATION OF VP2 OF CANINE PARVOVIRUS SH14 STRAIN

根据犬细小病毒(Canine Parvovirus,CPV)SH14株基因序列设计引物,采用PCR方法扩增CPV VP2基因,经Bam HⅠ、XhoⅠ双酶切后,将其克隆至p GEX-4T-1载体,构建重组表达载体p GEX-4T-VP2,经酶切鉴定和测序验证正确后转化大肠杆菌BL21中进行诱导表达,应用SDS-PAGE法检测蛋白的表达。然后,使用纯化的重组VP2蛋白制备其兔源多克隆抗体,分别应用蛋白免疫印迹、间接免疫荧光检测该抗体的免疫原性。结果表明,p GEX-4T-VP2能在大肠杆菌中以包涵体形式高效表达,所制备的兔抗CPV-VP2抗体能与重组蛋白和病毒蛋白发生特异性反应。本研究为研发CPV亚单位疫苗、检测技术及致病机理研究等奠定了物质基础。

According to the genome sequence of CPV SH14 strain, a pair of specifi c primers were designed for amplifying VP2 gene in PCR. The resulting PCR product was digested with Bam H Ι and Xho Ι and cloned into p GEX-4T-1 vector to obtain the recombinant plasmid p GEX-4T-VP2. Then, p GEX-4T-VP2 was transformed into E.coli BL21 cells with IPTG induction. The expressed products were identifi ed in SDS-PAGE. Polyclonal antibodies against CPV VP2 were prepared with the purifi ed recombinant VP2 by vaccinating rabbits. The rabbit antibodies were used in Western blot and indirect immunofluorescence to detect their reactivity with the expressed VP2. The results showed that the recombinant VP2 expressed in E.coli BL21 cells reacted positively with rabbit polyclonal antibodies. The availability of the recombinant VP2 has laid the solid foundation for development of subunit vaccine, diagnostic methods and pathogenic mechanism research for CPV.