绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒的构建及对小鼠细胞免疫应答的影响

为探究绵羊肺炎支原体（Mycoplasma ovipneumoniae，Mo）pcDNA3.1-TBP30-Hsp70融合表达质粒对小鼠细胞免疫应答影响，本试验构建了绵羊肺炎支原体pcDNA3.1-TBP30-Hsp70融合表达质粒。用已构建的pMD19T-P30和pMD19-Hsp70质粒为模板，采用基因定点突变（SDM）原理设计引物，应用SOE-PCR扩增目的基因片段，并将其定向克隆至表达载体pcDNA3.1（+），构建重组质粒pcDNA 3.1（+）-TBP30和融合重组质pcDNA3.1（+）-TBP30-Hsp70。使用pcDNA3.1-TBP30、pcDNA3.1-TBP30-Hsp70、pcDNA3.1（+）和Elution Buffer对小鼠进行免疫，应用ELISA试剂盒检测小鼠血清中细胞因子白细胞介素-2（IL-2）、IL-4、干扰素-γ（INF-γ）分泌水平。结果显示，pcDNA3.1-TBP30-Hsp70酶切后可见大小分别约为1 413 bp的目的基因片段和5 400 bp的载体条带。与空白对照组和pcDNA3.1（+）组相比，免疫重组质粒组均可引起小鼠血清中细胞因子INF-γ、IL-2和IL-4分泌水平的增强，与空白对照组和pcDNA3.1（+）组相比差异显著或极显著（P<0.05；P<0.01）；而空白对照组和空质粒组之间差异不显著（P>0.05）；免疫pcDNA3.1-TBP30和pcDNA3.1-TBP30-Hsp70组小鼠血清IL-2、INF-γ和IL-4终分泌量增加，表明重组质粒组可刺激小鼠血清中IL-2、INF-γ和IL-4的变化，并且在时间上都呈现出先增多后减少的规律。本试验结果表明，重组质粒pcDNA3.1（+）-TBP30-Hsp70免疫小鼠后，IL-2和INF-γ分泌水平的升高，增强了机体的细胞免疫功能，进而调节机体细胞免疫影响T细胞和巨噬细胞的分泌，从而提高机体细胞免疫能力；IL-4分泌水平升高，促进机体Th2向Th1分化，维持Th1的优势状态，增强了机体的细胞免疫功能。本试验结果为绵羊肺炎支原体基因工程疫苗的研制提供了参考依据。

Construction of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 Fusion Plasmid and Its Effect on Cellular Immune Response in Mice

To investigate the effect of the fusion expression plasmid of Mycoplasma ovipneumoniae (Mo) pcDNA3.1-TBP30-Hsp70 on the cellular immune response in mice,a fusion expression plasmid of Mycoplasma ovipneumoniae pcDNA3.1-TBP30-Hsp70 was constructed.Using the constructed plasmids pMD19T-P30 and pMD19-Hsp70 as templates,primers were designed based on the principle of gene site-directed mutagenesis (SDM).The target gene fragments were amplified by SOE-PCR and directionally cloned into the expression vector pcDNA3.1(+) to construct pcDNA3.1(+)-TBP30 and the fusion plasmid pcDNA3.1(+)-TBP30-Hsp70.Mice were immunized with pcDNA3.1-TBP30,pcDNA3.1-TBP30-Hsp70,pcDNA3.1(+) and Elution Buffer.Serum levels of cytokines interleukin-2 (IL-2),IL-4 and interferon-γ (INF-γ) were detected by ELISA kit.The results showed that after digestion of pcDNA3.1-TBP30-Hsp70,the target gene fragments with the size of 1 413 bp and the vector bands with the size of 5 400 bp were visible.Compared with the blank control and the pcDNA3.1(+) groups,the immune recombinant plasmid groups could increase the secretion levels of cytokines INF-γ,IL-2 and IL-4 in the serum of mice.Compared with the blank control and the pcDNA3.1(+) groups,the difference was significant or extremely significant (P<0.05;P<0.01),but there was no significant difference between the blank control and blank plasmid groups (P>0.05).The final secretion of IL-2,INF-γ and IL-4 in serum of mice immunized with pcDNA3.1-TBP30 and pcDNA3.1-TBP30-Hsp70 increased,which indicated that the recombinant plasmids could stimulate the changes of IL-2,INF-γ and IL-4 in serum of mice,and showed the regularity of increasing and decreasing in time.The results showed that after immunizing mice with recombinant plasmid pcDNA3.1(+)-TBP30-Hsp70,the secretion levels of IL-2 and INF-γ increased,which enhanced the cellular immune function of the organism,and then regulated the cellular immunity of the organism to affect the secretion of T cells and macrophages,thereby improving the cellular immunity of the organism.Th2 differentiated into Th1,maintains the dominant state of Th1 and enhanced the cellular immune function of the body.The results provided a reference for the development of genetic engineering vaccine against Mycoplasma ovipneumoniae.