犬细小病毒NY株VP2蛋白多克隆抗体的制备与鉴定

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

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72ku;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。

This study was aimed to prepare canine parvovirus （CPV）VP2 protein polyclonal anti-body.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 ad-j uvant,the prepared immunogen was inoculated into rabbit by subcutaneous inj ections 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.