鸡IL-17A真核表达质粒的构建及其对IBD rVP2亚单位疫苗的免疫佐剂效应

Construction of chicken IL-17A eukaryotic expression vector and its immune adjuvant effect

为探究鸡白介素-17A(IL-17A)真核表达质粒对鸡传染性法氏囊病(IBD)rVP2亚单位疫苗的免疫佐剂效应,本研究采用RT-PCR方法扩增鸡IL-17A基因,将其克隆于原核表达载体pET-28a中,构建重组质粒pET-28a-chIL-17A,将其转化BL21(DE3)大肠杆菌,诱导表达重组蛋白rchIL-17A,通过SDS-PAGE检测后,经His色谱层析柱纯化rchIL-17A,纯化蛋白免疫小鼠制备多克隆抗体,并通过ELISA鉴定。同时将鸡IL-17A基因克隆于真核表达载体pVAXI中构建重组质粒pVAX-chIL-17A,将pVAX-chIL-17A肌肉注射鸡7 d后经western blot检测其的表达。将21日龄SPF鸡随机分为4组,分别为对照组、pVAX-chIL-17A组、rVP亚单位疫苗组、pVAX-chIL-17A+rVP亚单位疫苗组,免疫后通过ELISA检测各组鸡血清中IBD病毒(IBDV)特异性抗体水平(0、14 d、28 d、42 d和56 d)和IL-2、IL-4、IL-6、IL-17A、IFN-γ及TNF-α的分泌水平(28 d),通过流式细胞术检测各组鸡脾细胞中CD4+T和CD8+T细胞亚群的比例(14 d和28 d)。RT-PCR扩增结果显示获得了约为420 bp的目的片段,并构建原核表达质粒后经诱导纯化后得到了rchIL-17A;将其免疫小鼠后经ELISA检测,结果显示获得了其多克隆抗体。将构建的重组质粒pVAX-chIL-17A免疫鸡后,western blot分析结果显示,注射部位肌肉组织中目的蛋白正确表达。将SPF鸡分组免疫后,ELISA检测结果显示,pVAX-chIL-17A+IBD rVP2亚单位疫苗组各时间点的IBDV特异性抗体水平均显著或极显著高于rVP2亚单位疫苗单独免疫组(P<0.05、P<0.01、P<0.001);pVAX-chIL-17A单独及与rVP2亚单位疫苗共同免疫28 d后,鸡血清中IL-2、IL-6、IL-17A及TNF-α的分泌水平相较其它组显著提高(P<0.05、P<0.01),而IFN-γ和IL-4分泌水平无明显变化。流式细胞术检测结果显示,与对照组相比,单独免疫pVAX-chIL-17A 14 d及28 d后鸡脾细胞中CD4+T和CD8+T细胞的占比均极显著增加(P<0.01、P<0.001);与rVP亚单位疫苗单独免疫组相比,pVAX-chIL-17A与rVP2亚单位疫苗共同免疫14 d及28 d后鸡脾细胞中CD4+T和CD8+T细胞的占比均显著增加(P<0.001);pVAX-chIL-17A与rVP2亚单位疫苗共同免疫28 d后鸡脾细胞CD4+T/CD8+T细胞比值极显著高于rVP2亚单位疫苗单独免疫组(P<0.001)。本研究首次证实pVAX-chIL-17A可以显著增强IBD基因工程亚单位疫苗(rVP2)的免疫水平,为新型高效鸡免疫佐剂的研发奠定了基础。

In order to explore the immune adjuvant effect of chicken interleukin-17A(IL-17A)eukaryotic expression vector on chicken infectious bursal disease rVP2 subunit vaccine,chicken IL-17A gene was amplified and cloned into the prokaryotic expression vector pET-28a.The recombinant plasmid was transformed into BL21(DE3)Escherichia coli,and the induced expression of the recombinant protein(rchIL-17A)was detected by SDS-PAGE.Antiserum was prepared by immunizing mice with purified rchIL-17A.Meanwhile,the recombinant plasmid pVAX-chIL-17A was constructed by cloning the chicken IL-17A gene into the eukaryotic expression vector pVAXI.Seven days after intramuscular injection into chickens,the expression of pVAX-chIL-17A was detected by western blot.The 21-day-old SPF chickens were randomly divided into 4 groups,designated as the control group,the pVAX-chIL-17A group,the rVP2 subunit vaccine group,and the pVAX-chIL-17A+rVP2 subunit vaccine group,respectively.After immunization,the serum levels of infectious bursal virus(IBDV)-specific antibodies and the secretion levels of IL-2,IL-4,IL-6,IL-17A,IFN-γand TNF-αwere detected by ELISA.The ratio of CD4+T and CD8+T cell subsets in splenocytes was detected by flow cytometry.A target fragment with the length of approximately 420bp was amplified by RT-PCR,and a prokaryotic expression plasmid was constructed.After induction and purification,rchIL-17A was obtained,and its polyclonal antibody was identified by ELISA.After immunizing chickens with the constructed recombinant plasmid pVAX-chIL-17A,Western blot analysis showed that there were obvious bands at the position as expected in the muscle tissue of the injection site.After immunization of the SPF chickens,the ELISA test results showed that at each time point the levels of IBDV specific antibodies from the group of co-immunization with pVAX-chIL-17A and rVP2 subunit vaccine were significantly or extremely significant higher than those in the rVP subunit vaccine alone immunization group(P<0.05,P<0.01,P<0.001).After immunization with pVAX-chIL-17A alone or with rVP2 subunit vaccine for 28 days,the secretion levels of IL-2,IL-6,IL-17A and TNF-αwere significantly increased(P<0.05,P<0.01),and secretion level of IFN-γand IL-4 did not change significantly.The results of flow cytometry showed that after 14 and 28 days after immunization with pVAX-chIL-17A alone,the percentage of CD4+T and CD8+T cells in spleen cells was significantly increased compared with the control group.Compared with the group immunized with rVP2 subunit vaccine alone,the percentage of CD4+T and CD8+T cells in spleen cells was significantly increased after 14 and 28 days after co-immunization with pVAX-chIL-17A and rVP2 subunit vaccine.After 28 days after co-immunization with pVAX-chIL-17A and rVP2 subunit vaccine,the CD4+T/CD8+T cell ratio was significantly higher than that of the rVP2 subunit vaccine alone group.The results showed that pVAX-chIL-17A could significantly enhance the immune response induced by IBD gene engineering subunit vaccine(rVP2).This study provides a research basis for the study and development of a new type of high-efficiency chicken immune adjuvant.