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

Study on Immune Efficacy of Single and Double Fusion DNA Vaccine from Mycobacterium bovis

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,7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6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。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。

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 ag85 b, esat-6 ,hsp65, mpb64 ,ag85 b-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 this experiment may be useful for the development of Boris tuberculosis vaccine.