马铃薯A病毒重组CP多克隆抗体的制备及其在DAS-ELISA检测中的应用

利用重组CP作抗原制备出马铃薯A病毒的多克隆抗体,并将其用于DAS-ELISA检测。以p ET22b(+)为起始载体,构建了PVA-CP基因的原核表达载体p ET22b-ACP,重组菌BL21(p ET22b-ACP)经IPTG诱导表达出了分子量为30 k Da的特异性重组CP。利用高纯度重组CP为抗原免疫家兔,制备出了效价为1∶512 k的抗血清。以PVA重组CP为抗原,用间接ELISA与直接ELISA分别测定重组CP纯化抗体(Ig G)及其碱性磷酸酶标记物(Ig G-AP)的活性,用DAS-ELISA测定2种抗体的活性,目测及OD值测定结果表明3种ELISA测定反应都呈阳性。以PVA病毒阳性标准物为抗原,在上述3种ELISA测定中也呈阳性反应,重组CP多克隆抗体及其酶标抗体与PVA有较强的反应信号。利用重组CP制备的多克隆抗体及其酶标抗体达到了马铃薯A病毒DAS-ELISA检测的要求。

Preparation of Polyclonal Antibody Against Recombinant CP of Potato virus A and Its Application to DAS-ELISA Detection

The purpose was to prepare polyclonal antibody against recombinant CP of Potato virus A,and apply it to DAS-ELISA.Taking pET22b(+) as the initial vector,CP gene prokaryotic expression vector of Potato virus A (pET22b-ACP) was constructed,the recombinant strain BL21 (pET22b-ACP) was induced with IPTG and specific recombinant CP of 30 kDa was obtained.Antiserum with the titer of 1:512k was prepared by using high-purity recombinant CP as antigen to immune rabbits.Using PVA recombinant CP as antigen,visual inspection and OD value measurement results indicated that reactions were respectively positive in indirect ELISA assay with the purified antibody (IgG) against the recombinant CP,in direct ELISA assay with the alkaline phosphatase-conjugated IgG (IgG-AP),and in DAS-ELISA determination with IgG against the recombinant CP and its AP-conjugated IgG.When PVA positive standard substance was used as antigen,reactions were also positive respectively in the three kinds of ELISA detections mentioned above.The polyclonal antibody against PVA recombinant CP and its enzymelabeled antibody could meet the requirements of DAS-ELISA detection of Potato virus A.