犬细小病毒VP2亲水性编码区的表达与免疫原性分析

Expression of VP2 Hydrophilic Coding-region of Canine Parvovirus and Analysis of Immunogenicity

目的:探讨犬细小病毒VP2亲水性编码区的免疫原性,为进一步研究基因工程亚单位疫苗奠定基础。方法:利用蛋白质分析软件Protean对已克隆的犬细小病毒VP2基因序列进行分析,选择亲水性好、抗原性强的293-520位氨基酸区域(命名为VP2S)作为靶序列,然后以已有VP2序列作为模版通过PCR扩增的方法获得VP2S,将VP2S克隆入pQE-31载体获得pQE-31-VP2S;将pQE-31-VP2S的原核表达产物经Western-blotting确认后免疫小鼠,用血凝抑制试验测定抗体水平。结果:293～520位氨基酸区域的亲水性好、抗原性强;重组质粒pQE-31-VP2S可成功表达大约29KDa的能被CPV抗血清识别的VP2S;VP2S能诱导小鼠产生高滴度的血凝抑制(HI)抗体(25)。结论:VP2S具有较强的免疫原性,能作为基因工程亚单位疫苗进行开发研究。

Objective： To investigate the Immunogenicity of VP2 hydrophilic coding-region of canine parvovirus and lay foundation for further study of Subunitvaccine. Method： Protein analysis software-PROTEAN was used to find VP2 major antigenic epitopes according to hydrophile and antigen character of VP2 amino acids and then the corresponding region of nucleotide was amplified by PCR and cloned into prokaryotic expression vector pQE-31. After transformation, the recombinant protein was analyzed by isopropyl i3-D-1 thiogalactopyranoside （ITPG） induction. Finally, mice were immunized with VP2S and the antibody were measured by hemagglutination inhibition （HI） assay. Result： The hydrophilic and antigenic region from 293 to 520 amino acids of the VP2 protein of canine parvovirus, called VP2S, was located. After amplification the prokaryotic expression vector pQE-31-VP2S was obtained. The protein with molecular weight of 29kD, were detected in the lysates of the recombinant E.coli by IPTG induction. Western-blotting assay showed that the recombinant proteins were recognized by CPV antiserum. And VP2S-immunized mice appeared hemagglutination inhibition （HI） antibody compared to the control, which demonstrated the VP2S had good immunogenicity. Conclusion： These results indicated that the VP2S had good immunogenicity.