非洲猪瘟病毒p54蛋白的高效表达及在ELISA中的应用  

Studies on the Efficient Expression of African Swine Fever Virus p54 Protein and Its Antigenic Analysis in ELISA

作　　者：龚振华[1] 王丽萍[1,2] 臧京帅[2] 张康[1,2] 刘春菊[1] 吴晓东[1] 张启迪[2] 秦晓冰[2] 陈琳琳[2] 单虎[2] 王树双[1] 王志亮[1] 

机构地区：[1]中国动物卫生与流行病学中心,青岛266032 [2]青岛农业大学动物科技学院,青岛266109

出　　处：《畜牧兽医学报》2013年第11期1832-1837,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：公益性行业科技专项-非洲猪瘟等重大外来动物疫病防控技术研究(200903037)

摘　　要：本研究旨在高效原核表达非洲猪瘟病毒(ASFV)p54蛋白,并进一步将其用于ELISA试验研究。参考非洲猪瘟病毒Con09/Bzz020株P54基因(E183L)的核苷酸序列,通过序列优化后合成P54基因,克隆至表达载体pET-30c(+),构建重组质粒pET-30c(+)-p54。结果显示,pET-30c(+)-p54有完整的阅读框架,可高效表达分子量约20.7ku的p54蛋白,该蛋白表达稳定,具有可溶性,易于纯化,纯度高达900μg·mL-1,所表达的p54蛋白的氨基酸序列与Con09/Bzz020株p54蛋白氨基酸序列相同;Western blot试验显示,表达的p54蛋白能与ASFV阳性血清发生特异性反应;ELISA试验显示,p54蛋白针对ASFV阳性血清和阴性血清的P/N值为4.67。研究结果证实p54蛋白表达产量高,易于纯化,具有较好的抗原性,可用于非洲猪瘟ELISA诊断。In this study, p54 protein of African swine fever virus （ASFV） was prokaryotically ex- pressed efficiently, and the expressed p54 protein was tested to be specific to ASFV in ELISA. A sequence-optimized P54 gene was synthesized with reference Of P54 gene （E183L） sequence of ASFV Con09/Bzz020 strain, the synthesized ASFV P54 gene was cloned into pET-30c（＋） and the recombinant plasmid pET-30c （＋）-p54 was obtained. Nucleotide sequence analysis of pET 30c（＋）-p54 showed that the synthesized sequence-optimized P54 gene was cloned successfully in- to pET-30e（＋） with the right reading frame. The p54 fusion protein with approximately 20. 7 kDa was expressed efficiently, solubly and stably, and easily to be purified with the purity at 900 μg mL-1, the expressed p54 protein had the same amino acid sequence as the p54 protein of Con09/Bzz020, the expressed p54 protein was tested to be specific to anti-ASFV serum in West- ern-blot and had a P/N value of ASFV at 4. 67 in ELISA. The results suggested that the expressed p5,t protein with high yield was easy to be purified and was specific to anti-ASFV sera, and it could be used for diagnosis of ASFV by ELISA.

关 键 词：非洲猪瘟病毒 p54蛋白 ELISA 

分 类 号：S852.659.1[农业科学—基础兽医学]