小反刍兽疫病毒(PPRV)N蛋白的表达与ELISA检测方法的建立

Expression of peste des petits ruminants virus N protein and development of indirect ELISA

通过对下载的16株小反刍兽疫病毒(PPRV)N基因片段进行序列分析,合成了PPRV N基因。用NP1和NP2引物扩增。纯化的PPRV N基因在T4DNA连接酶的作用下,和PET32-a载体进行连接反应构建重组质粒pET-a-PPRV-N。经诱导表达和纯化后,得到了大量的PPRV N蛋白。用PPRV N蛋白制备免疫血清,经国际标准血清标化后作为参考阳性血清,同时以PPRV N蛋白为抗原建立间接ELISA(I-ELISA)方法。对山东省的467份羊血清检测,使用S/P(样品值/阳性值)平均值加3倍标准差的方法,计算出其临界点为0.28。应用间接ELISA和针对H蛋白的单抗为基础的C-ELISA检测来自于疫区的69份血清,结果两者的符合率为79.7%。

PPRV N gene was synthesized by analyzing 16 strains PPRV′s N gene.Using primer NP1 and NP2 PPRV N gene was amplified.The purified PPRV-N gene and plasmid PET32-a were sequenced by T4 DNA polymerase to construct plasmid pET-a-PPRV-N.Abundant PPRV N protein is expressed by inducing.Using PPRV N protein prepares immuno-sera,and produce positive control sera by standardizing the immuno-sera by reference positive sera of IAH（institute of animal health）.Indirect ELISA test was developed by using PPRV N protein as coating antigen.467 sera of sheep and goats of Shandong province were detected,and sample′s S/P value（sample′s OD450/reference positive serum′s OD450） was calculated to acquire cut-off value by S/P′s average adding 3 times standard deviation with the threshold value of 0.28.Using PPRV-N indirect ELISA and C-ELISA which based on PPRV-H protein′s mono-antibody 69 sera of PPRV infected area were detected.With the accordance rate of 79.7% between I-ELISA and C-ELISA.