摘要
本试验将编码PCV2 Cap蛋白的基因克隆到pET-32a载体中,构建成重组质粒pET-32a-Cap,并转化到BL21宿主菌中进行诱导表达,通过SDS-PAGE鉴定PCV2 Cap重组蛋白的表达情况,用Ni^+亲和柱纯化重组蛋白,并免疫产蛋鸡来制备抗Cap的多克隆抗体IgY,通过间接ELISA检测抗体滴度和抗Cap IgY抗体与PCV2的结合力。结果显示,Cap蛋白能在原核细胞中成功重组表达,蛋白主要以可溶性存在于细胞裂解液上清中,Ni^+柱纯化的重组蛋白经五次免疫产蛋鸡后卵黄抗体的滴度可达1∶64 000,所制备的IgY抗体与病毒和Cap蛋白的结合力较高。上述结果为研制检测PCV2 Cap蛋白试剂盒奠定了基础。
In the experiment,the PCV2 Cap gene was inserted into the pET-32a vector to construct recombinant plasmid pET-32a-Cap and was expressed in host bacterium BL21. The recombinant Cap protein was identified by SDS-PAGE and was purified by Ni＋ affinity column and then immunized with laying hens to prepare anti-Cappolyclona] antibodies IgY. Antibody titers and binding of IgY against Cap of PCV2 was determined by indirect ELISA. The results revealed that Cap protein was successfully expressed in prokaryotic cells. The recombinant protein was mainly solubly expressed in the supernatant of cell lysate. The titer of the egg yolk antibody was up to 1 ：64 000 and the prepared IgY antibody has higher binding capacity with PCV2 and Cap protein. This study result laid the foundation for the development of a PCV2 Cap protein kit.
出处
《中国兽医科学》
CAS
CSCD
北大核心
2017年第6期727-732,共6页
Chinese Veterinary Science
基金
陕西省国际科技合作基地建设项目(2015SD0018)
