犬细小病毒NY株VP2蛋白多克隆抗体的制备与鉴定

试验旨在制备犬细小病毒（canine parvovirus，CPV）VP2蛋白多克隆抗体。构建pET28a-CPV-VP2重组表达质粒转化大肠杆菌BL21（DE3）感受态细胞，经IPTG诱导，SDS-PAGE鉴定融合蛋白的表达，纯化目的蛋白与佐剂混合、乳化制备后作为免疫原，免疫家兔制备VP2蛋白多克隆抗体；采用免疫过氧化物酶单层细胞染色法（immunoperoxidase monolayer assay，IPMA）检测抗体的免疫活性、抗体滴度、病毒滴度及中和活性。结果表明，重组VP2蛋白（rVP2）以包涵体形式存在，分子质量约为72 ku；所制备的多克隆抗体滴度为1 600倍，病毒滴度为10⁷ TCID₅₀ /mL，中和效价为1∶2 884，该抗体与体外培养的CPV呈特异性反应，CPV VP2蛋白的多克隆抗体免疫活性和特异性良好，且中和活性高，为CPV基因工程疫苗的研究和临床治疗奠定了基础。

Preparation and Identification of Canine Parvovirus NY Strain VP2 Protein Polyclonal Antibody

This study was aimed to prepare canine parvovirus (CPV) VP2 protein polyclonal antibody.The recombinant expression vector pET28a-CPV-VP2 was constructed and transfromed into E.coli BL21 (DE3),the expression of recombinant proteins was induced by IPTG from which the fusion protein was identified by SDS-PAGE.The target protein was purified and emulsify with adjuvant,the prepared immunogen was inoculated into rabbit by subcutaneous injections to prepare of VP2 protein specific polyclonal antibody.The immuno-activity,titers,neutralization titers of the prepared polyclonal antibody were determined by immunoperoxidase monolayer assay (IPMA).The results showed that the expressed recombinant protein VP2 (rVP2) existed in the form of inclusion body with a molecular weight of 72 ku.The prepared polyclonal antibody titer was 1 600 dilution,the virus titer was 10⁷ TCID₅₀/mL,the neutralizing titer was 1∶2 884.The antibodies showed specific reaction with CPV.In conclusion,rVP2 specific polyclonal antibody showed wonderful immunocompetence,specificity and neutralizing activity,providing foundation for the development of genetic vaccine and clinical therapeutic method.