PRV SD株gE基因主要抗原区域原核表达及间接ELISA方法的建立

郑 辉，张 青，邹 敏，董雅琴，刘 爽，范根成，吴发兴，李晓成

（1. 青岛易邦生物工程有限公司，山东青岛 266114；2. 中国动物卫生与流行病学中心，山东青岛 266032）

PRV SD株gE基因主要抗原区域原核表达及间接ELISA方法的建立

郑 辉1，张 青1，邹 敏1，董雅琴2，刘 爽2，范根成1，吴发兴2，李晓成2

（1. 青岛易邦生物工程有限公司，山东青岛 266114；2. 中国动物卫生与流行病学中心，山东青岛 266032）

参考猪伪狂犬病病毒（PRV）SD株gE基因序列，设计1对引物，通过PCR方法扩增一段包含gE主要抗原表位编码区的618 bp片段，将其用BamHⅠ和XhoL I双酶切后，插入到原核表达载体pET-32a（+）中，转化大肠杆菌BL21（DE3），进行表达、SDS-PAGE电泳、Western-blot检测和纯化；利用表达的PRV SD gE蛋白进行gE-ELISA鉴别检测方法研究。结果发现：gE重组蛋白得到了高效表达，融合蛋白的分子量约为42 ku；表达产物存在于上清液中，表达量可达菌体总蛋白的30%以上，并且能被PRV抗体所识别。将纯化后的gE蛋白作为抗原，以辣根过氧化物酶（HRP）标记的兔抗猪IgG为二抗，建立检测PRV抗体的间接ELISA方法。结果显示：确定的抗原最佳包被浓度为36 µg/mL，最佳包被时间为37 ℃、1 h并4 ℃过夜，待检血清（1:50稀释）37 ℃作用1 h，二抗（1:3 000）37 ℃作用1 h。重复性试验、交叉试验证明该方法重复性好、特异性强、灵敏度高。用gE-ELISA和IDEXX gE-ELISA同时对采集的92份猪血清进行平行检测，发现该方法的特异性为85.71%，敏感性为90.63%，二者符合率为89.13%。该方法为我国猪伪狂犬病的鉴别监测提供了一种可供选择的技术手段。

猪伪狂犬病病毒；gE基因；原核表达；间接ELISA；鉴别

Abstract：A pair of primers were designed according to the sequence of the isolate PRV SD. A segment containing major episode coding area of gE gene was amplified by PCR method，which was 618 bp in length. The PCR product was inserted into the expression vector pET-32a（+）after being cleaved by BamHⅠand XhoL I. And then the recombinant plasmid was formed and transformed into BL21（DE3）. A recombination protein was expressed，identified by SDS-PAGE and Western-blot，and then purified. Based on that，differentiation ELISA for PRV gE antibody was studied. The results showed that a recombination protein of 42ku was expressed efficiently in forms of soluble，with expression level over 30%of total bacteria protein. The protein could be identified by PRV specific antibody. An indirect ELISA for PRV antibodies was established with the purified recombination protein as the coating antigen and HRP-conjugated rabbit anti-porcine IgG as the secondary antibody. The optimal reaction condition was that the antigen was coated with concentration of 36 µg/ml under 37 ℃ for 1 hour and then under 4℃ overnight；the samples were 1:50 diluted and incubated under 37 ℃ for 1 hour，the secondary antibody was 1:3 000 diluted and incubated under 37 ℃for 1 hour. All tests of the method indicated that ithad a good repeatability，specificity and sensitivity. 92 porcine serums were tested by the gE-ELISA and IDEXX gEELISA，which showed the new method had a specificity of 85.71%，sensitivity of 90.63% and coincidence of 89.3%.This study provided technical support for PRV differentiation test in our country.

Key words：Pseudorabies virus；gE gene；prokaryotic expression；indirect-ELISA；discrimination

猪伪狂犬病（PR）是由伪狂犬病病毒（PRV）感染猪引起的一种急性传染病，可导致妊娠母猪繁殖障碍（流产，产死胎、弱仔），公猪不育，新生仔猪出现神经症状、腹泻并大量死亡，育肥猪则表现呼吸困难、生长停滞等[1]。……