犬CTLA-4胞外区的分子克隆、表达和对犬细小病毒VP2的免疫佐剂作用

Immunoadjuvant effect of the extracellular domain of canine CTLA-4 on the VP2 protein of canine parvovirus

摘要:

【目的】探索犬细胞毒性T细胞相关抗原-4(cytotoxic T lymphocyte-associated antigen-4, CTLA-4)胞外区作为免疫佐剂的可行性。【方法】根据已发表序列设计引物, 用RT-PCR扩增CTLA-4胞外区编码序列, 用PCR扩增犬细小病毒(canine parvovirus, CPV)VP2蛋白主要抗原表位基因片段VP2S, 将VP2S克隆入含和不含CTLA-4胞外区基因片段的原核表达质粒pQE-31; 用获得的重组质粒pQE-CTLA-4-VP2S和pQE-VP2S转化大肠杆菌, 并进行诱导表达; 用相同剂量的重组蛋白VP2S和CTLA-4-VP2S免疫小鼠, 用间接ELISA和血凝抑制试验比较两个免疫组的抗体水平。【结果】经过30次循环PCR扩增后, 琼脂糖凝胶电泳显示预期大小的扩增产物; 序列测定结果显示, 克隆的毕格犬CTLA-4胞外区与已发表序列的核苷酸同源性为99.2%, 氨基酸序列同源性为98.4%, 结合B7分子的六肽基序(MYPPPY)无变化; VP2S与已发表CPV VP2的核苷酸序列同源性为99%, 氨基酸序列同源性为98.6%; 经IPTG诱导后, 两种重组大肠杆菌表达预期的29kDa VP2S和42kDa CTLA-4-VP2S重组蛋白, 两者均能被CPV抗血清识别; 间接ELISA和血凝抑制试验结果显示, CTLA-4-VP2S免疫组的抗体产生时间为初免后第2周, 抗体高峰期为初免后第4周, 而VP2S免疫组的抗体产生时间为初免后第4周, 抗体高峰期为初免后第5周, 两个试验组高峰期ELISA抗体效价和血凝抑制抗体效价分别相差100倍和10倍。【结论】犬CTLA-4胞外区可作为分子佐剂促进CPV VP2蛋白抗体的产生。

Abstract:

[Objective] Investigation into the adjuvant effect of the extracellular domain of canine cytotoxic T lymphocyte-associated antigen 4 (CTLA-4). [Methods] We first amplified the cDNA for the extracellular domain from blood lymphocytes using RT-PCR and then amplified the VP2S gene fragment for major antigenic epitopes of the VP2 protein of canine parvovirus (CPV) using PCR. After sequence analysis, we inserted the VP2S fragment into expression vector pQE-31 with or without the coding sequence for the extracellular domain of canine CTLA-4. After transformation of the two recombinant vectors into E. col, we studied recombinant protein expression by isopropyl b-D-1-thiogalactopyranoside (IPTG) induction. Finally, we immunized BALB/c mice with the same dose of the purified protein VP2S or CTLA-4-VP2S and compared their antibody responses by enzyme-linked immunosorbant assay (ELISA) and haemagglutination inhibition (HI) assay. [Results] After 30 cycles of amplification, agarose gel electrophoresis revealed expected PCR products for both gene fragments. Sequence analysis showed that the amplified extracellular domain was 99.2% identical to previously published sequence without mutation in the hexapeptide motif (MYPPPY) for B7 molecule binding. After IPTG induction, the vector-transformed E. coli expressed expected 29-kDa VP2S protein and 42-kDa CTLA-4-VP2S protein. Western blotting showed that both VP2S and CTLA-4-VP2S proteins were recognized by CPV-specific antiserum. After two times of immunizations, the VP2S-specific antibody appeared from week 2 and reached the highest level at week 4 in CTLA-4-VP2S-immunized mice. In VP2S-immunized mice, however, the specific antibody appeared from week 4 and reached the highest level at week 5. The CTLA-4-VP2S-immunized mice had a 100-fold higher ELISA antibody and 10-fold higher HI antibody compared to VP2S-immunized mice. [Conclusion]The extracellular domain of canine CTLA-4 had strong immunoadjuvant effect on its fused protein.