鸡传染性贫血病毒VP2基因的原核表达及兔抗血清的制备

发布时间：2019-04-22 19:34

【摘要】：为了用大肠杆菌原核表达系统表达鸡传染性贫血病毒(CIAV)VP2基因片段,并制备其兔抗血清,试验以CIAV M9905株基因组DNA为模板,通过PCR扩增VP2基因,与p ET28a(+)载体连接后转化DH5α获得阳性重组子,将其转化E.coli BL21(DE3)菌,IPTG诱导表达出His-VP2重组蛋白,经Ni-NTA树脂纯化后免疫新西兰白兔制备兔抗血清,采用IFA和Western-blot检测该血清的特异性,ELISA测定其效价。结果表明:重组His-VP2融合蛋白的分子质量约为36 ku,以包涵体形式存在。制备的兔抗VP2血清效价在1∶6 400以上,可特异性结合真核表达的VP2蛋白,表明其反应原性较好。说明试验成功表达VP2蛋白,并制备出兔抗血清,可用于该蛋白与病毒其他蛋白之间的相互作用的进一步研究。
[Abstract]:In order to express (CIAV) VP2 gene fragment of chicken infectious anemia virus by E. coli prokaryotic expression system and prepare rabbit antiserum, VP2 gene was amplified by PCR using genomic DNA of CIAV M9905 strain as template. After being ligated with p-ET28a () vector, DH5 伪 was transformed into positive recombinant plasmid, then transformed into E.coli BL21 (DE3) strain. IPTG was induced to express His-VP2 recombinant protein. After purification with Ni-NTA resin, rabbit antiserum was prepared by immunizing New Zealand white rabbits. The specificity of the serum was detected by IFA and Western-blot, and the titer of the serum was determined by ELISA. The results showed that the molecular weight of the recombinant His-VP2 fusion protein was about 36 ku, in the form of inclusion body. The titer of rabbit anti-VP2 serum was more than 1:6 400, which could specifically bind to VP2 protein expressed in eukaryotic cells, which indicated that the antiserum was of good antigenicity. The results indicated that the VP2 protein was successfully expressed and rabbit antiserum was prepared, which could be used for further study of the interaction between the protein and other proteins of the virus.
【作者单位】：徐州医科大学形态学实验教学中心;中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/禽免疫抑制病科技创新团队;徐州医科大学病原生物学与免疫学教研室/江苏省免疫与代谢重点实验室;
【基金】：国家肉鸡产业技术体系项目(CARS-42-G07) 徐州医科大学优秀人才启动基金项目(D2015004)
【分类号】：S852.65

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