绵羊肺炎支原体P113蛋白和丝状支原体山羊亚种LppA蛋白共表达质粒对小鼠免疫应答的研究

Effects of co-expression of Mycoplasma ovipneumoniae P113 protein and Mycoplasma mycoides subsp.capri LppA protein on immune response in mice

为研究绵羊肺炎支原体(Mo)P113基因和丝状支原体山羊亚种(Mmc)LppA基因的共表达质粒免疫小鼠后对其免疫系统的影响,本研究构建了真核重组质粒pVAX1-P113-LppA,并经PCR、双酶切及测序鉴定正确后转染MDBK细胞,利用PCR及间接免疫荧光试验(IFA)分别检测P113蛋白和LppA蛋白的表达,结果显示,正确构建了含Mo P113基因及Mmc LppA基因的重组质粒pVAX1-P113-LppA;且该重组质粒在MDBK细胞中能有效转录并表达目的蛋白。分别在0、7 d、14 d时以100μg/只pVAX1-P113-LppA腿部肌肉注射免疫BABL/c小鼠,并设Elution Buffer对照组和空载体pVAX1(100μg)对照组,通过MTT法检测小鼠脾细胞增殖情况,并在初次免疫后28 d利用流式细胞术分析各组小鼠脾细胞中CD4^(+)T淋巴细胞和CD8^(+)T淋巴细胞所占总淋巴细胞数的变化,同时采用的ELISA方法检测小鼠免疫后血清特异性抗体和细胞因子(IL-2、IL-4和IFN-γ)的分泌情况。结果显示,重组质粒组小鼠脾淋巴细胞增殖能力极显著高于对照组和空载体组(P<0.01),且该组小鼠脾CD4^(+)、CD8^(+)T淋巴细胞在免疫28 d时较Elution Buffer对照组和空载体pVAX1(100μg)对照组均极显著上升(P<0.01);该组小鼠免疫后7 d的血清特异性抗体水平极显著升高(P<0.01),在初次免疫后第49 d特异性抗体仍为阳性;血清中IL-2、IL-4和IFN-γ的分泌水平较Elution Buffer对照组和空载体pVAX1(100μg)对照组均呈显著上升趋势(P<0.05),且在首次免疫后28 d时分泌量最高,而后缓慢下降。攻毒试验结果显示,重组质粒pVAX1-P113-LppA对小鼠具有一定的免疫保护能力,小鼠肺部病理损伤明显减轻,发病数量减少。本实验首次表明重组质粒pVAX1-P113-LppA能诱导小鼠产生体液免疫和细胞免疫应答,Mo P113基因和Mmc LppA基因可以作为羊支原体肺炎(MPSG)DNA疫苗的候选基因,该结果为MPSG核酸疫苗的研究与应用提供实验基础。

In order to construct the recombinant plasmid that can simultaneously express the P113 gene of Mycoplasma ovipneumoniae(Mo)and the LppA gene of Mycoplasma mycoides subsp.capri(Mmc),as well as analyze its effect on the immune response of mice,the eukaryotic recombinant plasmid pVAX1-P113-LppA was constructed in this study.The recombinant plasmid was identified by PCR,double enzyme digestion and sequencing,and then transfected into MDBK cells.The expression of P113 protein and LppA protein was detected by PCR and indirect immunofluorescence assay(IFA),respectively.The results showed that the recombinant plasmid pVAX1-P113-LppA was successfully transfected into MDBK cells.The recombinant plasmid pVAX1-P113-LppA containing Mo P113 gene and Mmc LppA gene was constructed correctly.The recombinant plasmid can effectively transcribe and express the target protein in MDBK cells.At 0,7 day and 14 day,100μg of the recombinant plasmid was injected into the leg muscle to immunize BABL/c mice,and the elution buffer control group and the empty vector pVAX1(100μg)control group were included.The proliferation of spleen cells in mice was detected by MTT method,and the changes in the number of CD4^(+)T lymphocytes and CD8^(+)T lymphocytes in the total lymphocytes in spleen cells of mice in each group were analyzed by flow cytometry at 28 day after the first immunization.The ELISA method was used to detect the secretion of serum specific antibodies and cytokines(IL-2,IL-4 and IFN-γ)in mice after immunization.The results showed that the proliferation ability of spleen lymphocytes in the recombinant plasmid group was significantly higher than that in the control group and the empty vector group(P<0.01),and the spleen CD4^(+)/CD8^(+)T lymphocytes in the recombinant plasmid group were significantly higher than those in the elution buffer control group and the empty vector pVAX1(100μg)control group at 28 days post immunization(P<0.01).The serum specific antibody level of mice in this group was significantly increased at 7 day after immunization(P<0.01),and remained positive at 49 day after initial immunization.Compared with elution buffer control group and empty vector pVAX1(100μg)control group,the secretion levels of IL-2,IL-4 and IFN-γin serum increased significantly,and the secretion was the highest at 28 day after the first immunization,and then decreased slowly.The results of challenge test showed that the recombinant plasmid pVAX1-P113-LppA had a certain ability of immune protection in mice,the pathological injury of lung was obviously alleviated and the number of diseases was reduced.The results showed that the recombinant plasmid pVAX1-P113-LppA could induce humoral and cellular immune responses in mice for the first time.Mo P113 gene and Mmc LppA gene could be used as candidate genes for DNA vaccine against Mycoplasmal pneumonia of sheep and goats(MPSG).This study provides basic research data for the research and application of MPSG nucleic acid vaccine.