伪狂犬病病毒<i>gE</i>基因主要抗原表位区的原核表达及间接ELISA方法的建立

吉艺宽; 王雨; 程艺; 张宝石; 向柯宇; 琚春梅

doi:10.7671/j.issn.1001-411X.2015.04.002

伪狂犬病病毒gE基因主要抗原表位区的原核表达及间接ELISA方法的建立

Prokaryotic expression of main antigen epitope of pseudorabies virus gE gene in Escherichia coli and establishment of indirect ELISA

摘要

摘要:
目的为了区分伪狂犬病病毒疫苗免疫猪和自然感染猪，建立快速诊断的方法.
方法利用PCR方法从伪狂犬病病毒中扩增出糖蛋白E(gE)基因的主要抗原表位区，用BamHⅠ和Hind Ⅲ双酶切后，插入原核表达载体pET-32a(+)中，构建重组表达质粒pET-gE184，并转化至大肠埃希菌Escherichia coli BL21(DE3)，经IPTG诱导后进行SDS-PAGE电泳和Western-blot检测，用纯化后的gE184蛋白作为包被抗原，建立间接gE184-ELISA检测方法.
结果和结论 用该方法检测290份临床猪血清样本，并与商品化ELISA试剂盒检测结果比较，两者的符合率为93. 1%.

Abstract:
Objective To distinguish pseudorabies virus (PRV) -infected pigs from those vaccinated with glycoprotein E (gE) -negative vaccine, the method of rapid diagnosis was established.
Method The gE gene fragment of PRV containing the main antigen region was amplified by PCR and cloned into the pro karyotic expression vector pET32a (+) to establish the recombinant plasmid pET-gE184. The recombi nant plasmid was transformed into Escherichia coli BL21 (DE3). After being induced with IPTG, the tar get protein was identified by SDS-PAGE and Western-blot. Wells of ELISA plates were coated with puri fied recombinant protein gE184 to establish the indirect gE184-ELISA.
Result and conclusion Two hundred ninety clinical porcine sera samples have been detected by this ELISA. Compared with commer cial ELISA Kit, the coincidence rate was 93.1%.

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