泛素基因介导下的猪繁殖与呼吸综合征病毒M基因对小鼠的免疫试验  

Effects of fusion gene of ubiquitin and Porcine Reproductive and Respiratory Syndrome virus M gene on the immune response in inoculated mice

作　　者：张维军[1] 童铁钢[1] 白宇[1] 王群[1] 徐树兰[1] 孙庆歌[1] 田野[1] 杨涛[1] 童光志[2] 吴东来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所,兽医生物技术国家重点实验室,农业部兽医公共卫生重点开放实验室,哈尔滨150001 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《微生物学报》2009年第6期799-806,共8页Acta Microbiologica Sinica

基　　金：国家"863计划"(2006AA10A203);黑龙江省自然科学基金重点项目(ZJN-0602-01)~~

摘　　要：【目的】获得猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome virus,PRRSV)M蛋白基因及其与泛素(Ubiquitin,Ub)基因融合的真核表达质粒,并进一步研究Ub对M基因免疫效果的影响。【方法】利用RT-PCR技术以PRRSV CH-1a株和BALB/c小鼠脾脏组织总RNA为模板,分别扩增出PRRSVM蛋白基因与小鼠的Ub基因,利用SOE技术将PRRSVM基因与小鼠Ub基因进行融合,最终构建真核表达质粒pVAX1-M与pVAX1-U-M。以两种真核表达质粒DNA肌肉免疫BALB/c小鼠后,分别检测体液免疫反应与细胞免疫反应,比较M单基因与Ub-M融合基因DNA免疫所诱生的免疫应答强度【结果】IFA试验表明,pVAX1-M与pVAX1-U-M均能BHK-21细胞内成功表达目的基因;两种重组质粒免疫小鼠后均可诱生PRRSVELISA抗体与细胞免疫反应,但是pVAX1-U-M诱导的细胞免疫反应明显高于pVAX1-M,差异显著(P<0.05);而其诱导的抗体水平明显低于pVAX1-M,二者差异也显著(P<0.05)。【结论】泛素在一定程度上可以促进PRRSVM基因诱导细胞免疫反应,但对体液免疫未见到同样作用。[ Objective] To evaluate the effects of the fusion gene of ubiquitin （Ub） and Porcine Reproductive and Respiratory Syndrome virus （PRRSV） M gene on the immune response in inoculated mice. [ Methods] Mouse Ub gene and PRRSV M gene were amplified by RT-PCR from BALB/c mice spleen cells and PRRSV Ch-la strain, respectively, and the M and Ub gene （U- M） was fused by SOE PCR. Therefore, pVAX1-U-M and pVAX1-M recombinant plasmid were constructed for eukaryotic expression.[ Results] The fusion U-M and M protein expressions were verified in transfected BHK-21 cells by indirect fluorescence assay. Furthermore, both pVAX1-M and pVAX1-U-M induced specific humoral and cellar immune responses against PRRSV in the recombinant plasmid injected mice. However, pVAX1-U-M was able to induce higher level of T cell response then that of pVAX1-M （ P 〈 0.05）, but lower level of antibody （ P 〈 0.05）. [ Conclusion] Expression of U-M fusion gene had ability to enhance specific T cell response against PRRSV, but no effect on stimulation of humoral response in inoculated mice.

关 键 词：猪繁殖与呼吸综合征病毒 M基因 泛素 体液免疫 细胞免疫 DNA疫苗 

分 类 号：S852.5[农业科学—基础兽医学]