犬细小病毒NY株VP2蛋白的原核表达及反应原性鉴定

Prokaryotic Expression and Reactogenicity Identification of Canine Parvovirus NY Strain VP2 Protein

犬细小病毒(CPV)编码的VP2基因容易发生变异,导致病毒嗜性和致病性的改变。为制备一株高致病性的CPV毒株(NY株,基因型为2a型)重组VP2蛋白,构建重组原核表达载体pET28a-CPVVP2,转化E.coli BL21(DE3)表达菌株,通过不同温度、不同IPTG浓度和不同诱导时间等条件的优化进行表达蛋白,采用SDS-PAGE和Western blot进行蛋白鉴定。结果表明,重组CPV-NY毒株VP2蛋白(rVP2)的分子质量约为72ku,在不同温度条件下均以包涵体形式存在,在37℃条件下以终浓度为0.2mmoL/L IPTG诱导4h表达量达到最高。rVP2不仅与His标签mAb反应,也能与CPV特异性阳性血清反应,说明rVP2具有良好的反应原性,为CPV抗体检测试剂盒和基因工程疫苗的研究奠定了基础。

Canine parvovirus （CPV） encoded VP2 gene shows frequent genetic variation, which contributes to the tropism and pathogenicity changes of CPV. This study aimed to prepare the recombinant VP2 pro- tein （rVP2） encoded by a highly pathogenic CPV strain （NY strain） by prokaryotic expression system. A recombinant plasmid pET28a-CPV-VP2 was constructed and transfected into E. coli 13L21 （DE3）, rVP2 was expressed at different temperatures, or with different IPTG concentrations and with different induc- tion time, SDS-PAGE and Western blot were used to analyze expression and antigenicity of rVP2. The re- suits showed that the molecular weight of the rVP2 was about 72 ku, the protein existed in the form of in- clusion body under different conditions. The expression levels showed the highest with 0.2 mmoL/L final concentration of IPTG, induced for 4 h at 37 ℃. rVP2 can not only react with His-tag monoclonal antibody but also can react with specific positive serum of CPV, rVP2 showed good reactogenicity. This study laid the foundation for the development of CPV antibody detection kits and genetic vaccine.