牛分枝杆菌单基因及双价融合基因DNA疫苗免疫效果的研究

利用PCR技术和SOE技术扩增牛分枝杆菌ag85b、esat-6、hsp65、mpb64基因和ag85b-esat-6、hsp65-esat-6和mpb64-esat-6融合基因，连接真核表达载体pCDNA3.1(+)，构建重组质粒pCA、pCE6、pCH、pCM、pCAE、pCHE和pCME。转染SP2/0细胞，检测目的基因的表达。以各重组质粒和pCDNA3.1(+)及PBS免疫BALB/c小鼠后检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN~γ分泌情况。结果表明，七种重组质粒免疫后小鼠血清抗体水平持续上升，与 pCDNA3.1(+)对照组和PBS对照组相比差异显著 （P<0.05），其中pCA组血清抗体水平明显高于其他六种DNA疫苗免疫组 （P<0.05）；三免两周后，融合基因免疫组的刺激值（SI值）与单基因免疫组相比差异显著（P<0.05），其中以pCME组的SI值最高；PPD刺激后融合基因DNA疫苗免疫组小鼠脾细胞分泌的IFN~γ高于单基因DNA疫苗组（P<0.05），而两对照组则未检测到IFN~γ的产生。本试验成功构建了牛分枝杆菌ag85b、esat-6、hsp65、mpb64单基因和ag85b-esat-6、hsp65-esat-6、mpb64-esat-6双价融合基因DNA疫苗，从而为牛结核病新型疫苗的研制奠定了基础。

Study on immune efficacy of single and double fusion DNA vaccine from Mycobacterium bovis

The DNA fragments of ag85b、esat-6、hsp65、mpb64 and ag85b-esat-6、hsp65-esat-6、mpb64-esat-6 were amplified by PCR and SOE technique.These seven fragments were inserted into pCDNA3.1(+) vector to construct recombinant plasmids pCA、pCE6、pCH、pCM、pCAE、pCHE and pCME. The seven plasmids were transfected into SP2/0 cell in vitro to detect the expression of target genes. BALB/c mice were intramuscularly vaccinated with the seven plasmids and the control vector pCDNA3.1(+) and PBS respectively. The serum antibodies and the spleen lymphocyte proliferation (SLP) and secreted IFN~γ of spleen were tested. The results of indirect ELISA showed the levels of antibodies in all recombinant plasmids groups were significantly higher than the two control groups(P<0.05), and pCA group was significantly higher than the other groups(P<0.05). SLP experiments indicated the SI value induced with PPD in the groups of fusion gene were higher than the single gene groups(P<0.05). The IFN~γ experiments showed the levels of IFN~γ induced with PPD in the groups of pCAE、pCHE and pCME groups were significantly higher than pCA、pCE6、pCH and pCM groups (P<0.05). So the results of our experiments showed ag85b、esat-6、hsp65、mpb64、ag85b-esat-6、hsp65-esat-6 and mpb64-esat-6 DNA vaccines were successfully constructed. They both induced antigen-specific humoral and cellular immune responses in BALB/c mice.So our experiment may be useful for the development of Bovis tuberculosis vaccine.