猪腺病毒3型Hexon蛋白多克隆抗体制备与免疫活性鉴定

本研究旨在制备猪腺病毒3型(PADV-3)Hexon蛋白多克隆抗体。试验构建重组原核表达载体pET28a-Hexon,IPTG诱导重组蛋白的表达并进行Western blotting鉴定,目的蛋白与佐剂混合、乳化制备免疫原,免疫家兔制备Hexon蛋白多克隆抗体。采用免疫过氧化物酶单层细胞染色法(IPMA)检测抗体的免疫活性与抗体滴度。结果显示,Hexon蛋白原核表达以包涵体形式存在,分子质量约为105ku;真核细胞表达的Hexon蛋白均定位于HEK293细胞质内,细胞核内无分布;IPMA测定所制备的多克隆抗体滴度为1∶1 600,该抗体与体外培养的PADV-3及稳定表达Hexon蛋白的细胞均呈特异性反应。结果表明本试验制备的PADV-3型Hexon蛋白多克隆抗体免疫活性和特异性良好。

Preparation and Immuno-activity Identification of Polyclonal Antibodies against Porcine Adenovirus Serotype 3 Hexon Protein

This study was aimed to prepare polyclonal antibodies against porcine adenovirus serotype 3 (PADV-3) Hexon protein.The recombinant prokaryotic expression plasmid pET28a-Hexon was constructed and transformed into BL21 (DE3), 1 mmol/L concentration of IPTG was used to induce the target gene expression which was then analyzed by SDS-PAGE and Western blotting.The expressed proteins were emulsified with freund''s adjuvant, the mixture was used to immunize the rabbit to prepare the polyclonal antibodies against PADV-3 Hexon protein.The immunocompetence and titers of the antibody were identified by immunoperoxidase monolayer assay (IPMA).The results showed that prokaryotic expressed Hexon protein existed as inclusion bodies, with a molecular weight of 105 ku, eukaryotic expressed Hexon protein was only observed in the cytoplasm of HEK293 cells.PADV-3 Hexon-specific polyclonal antibody showed perfect activity which recognized the PADV-3 propagated in PK-15 cells and the recombinant Hexon protein expressed in HEK293 cells specifically, it was IPMA antibody positive when diluted in 1:1 600 ratio.The prepared PADV-3 Hexon polyclonal antibodies showed wonderful activity and specificity.