【作者】 尹恒；

【导师】 余兴龙；

【作者基本信息】 湖南农业大学 ， 预防兽医学， 2008， 硕士

【摘要】 参照GenBank上公布的高致病性PRRSV毒株基因序列,设计了扩增N、M基因的特异性引物,并应用RT-PCR方法扩增出约372bp与约525bp目标基因片段。对扩增片段及pET-28a（+）与pBV220质粒分别用EcoRⅠ、SalⅠ两种酶进行消化,随后用T4DNA连接酶连接,将连接产物转化到BL₂₁（DE3）LysS中,经双酶切鉴定与测序分析后表明已构建成功重组质粒pET（28a）N与pBV220M对N基因表达重组菌于37℃下培养至OD值0.5～0.6时用IPTG进行诱导表达,经SDS-PAGE电泳及Western-blotting分析表明重组菌在诱导后可以表达特异性的N蛋白,该重组蛋白分子量约为17kD。将M基因表达重组菌30℃下培养至OD值0.4～0.6时迅速升温至42℃诱导4～6小时经SDS-PAGE电泳及Western-blotting分析表明该重组菌在诱导后可以表达特异性的M蛋白,该重组蛋白分子量约为22kD。在以上实验基础上以重组N、M表达蛋白作为包被抗原,初步建立了检测PRRS抗体的间接ELISA方法。实验确定了N、M蛋白最佳抗原包被浓度分别为0.5μg/ml与1μg/ml,血清稀释度分别为1:40;经重复性试验、交叉试验、等试验,结果表明所构建的方法重复性好、特异性强、敏感度高;将建立的rN-ELISA、rM-ELISA方法与美国IDEXX公司PRRSV抗体检测试剂盒相比较,其敏感性、特异性、和符合率经统计学分析,无显著性差异。更多还原

【Abstract】 Acoording to the high pathogenic PRRSV nucleotide sequences published in Genbank, two pairs of primes for N and M genes were designed, respectively to amplify the corresponding target genes by RT-PCR. Then the amplified fragment and plasmid pET-28(a) and pBV220 were respectively digested by Ecol I and Sal I, the following ligation reaction was operated. Finally the recombinant plasmid was constructed, designated PET-28N and pBV220M. The recombinant plasmid of PET-28N and pBV220M were respectively transformed into the host cell BL21(DE3). Positive clones were screened and identified by PCR and enzyme-digestion. Except termination codon sequencing results showed N and M genes contained 372bp and 525bp nucleotides.The recombinant plasmid were transformed successfully into the host cell BL21(DE3), the expression procedure was optimized. The results of SDS-PAGE and West-blotting indicated that the N and M protein were expressed successfully. The results that the recombinant N and M protein molecule weight were about 17 kD and 22 kD respectively.Using the purified recombinant protein, an indirect ELISA for detection of anti-PRRSV antibodies was developed and its optimal reactions were determined: the optimal coationg concentration was 0.5μg/ml(N) and 1.0gμ/ml(M); the optimal dilution of serum examined was 1:40. The results showed that the indirect N-ELISA and M-ELISA methods had advantages of good repetition, high sensibility and strong specificity by repeated test and crossing test. Compared with the IDEXX test kit, the sensitivity, specificity and coincidence of the established methods were no significant difference by statistics analysis.更多还原