牛分枝杆菌esat-6和mpb70-mpb83基因DNA疫苗的免疫效果

Immunogenicity of DNA vaccine containing esat-6 gene or mpb70-mpb83 fusion gene from Mycobacterium bovis

用PCR技术和重叠延伸剪接技术获得牛分枝杆菌esat-6基因和mpb70-mpb83融合基因,连接真核表达载体pcDNA3.1(+),构建了重组质粒pCE6和pC70-83-E6。分4组免疫小鼠:pCE6组、pC70-83-E6组、pcDNA3.1(+)和PBS对照组,采用间接ELISA法检测免疫小鼠血清特异性抗体水平,MTT法检测免疫小鼠脾淋巴细胞增殖情况和IFN-γ分泌情况。结果表明,2重组质粒免疫组小鼠的血清抗体水平持续上升,而2对照组始终维持在较低水平,且pC70-83-E6组小鼠的抗体水平高于pCE6组。经PPD刺激后,pCE6组和pC70-83-E6组小鼠的SI值与2对照组均差异显著(P<0.05),2重组质粒免疫组间差异不显著(P>0.05);2重组质粒免疫小鼠脾细胞产生的IFN-γ均显著高于2对照组(P<0.05),且pC70-83-E6组明显高于其他3组(P<0.05)。证实本试验构建的2种牛分枝杆菌DNA疫苗可有效诱导实验动物产生体液免疫和细胞免疫应答。

DNA fragments of esat-6 and mpb70-mpb83 obtained from genomic DNA of Mycobacterium boris ValleeⅢ by PCR and SOE（splicing by overlapping extension） were inserted into pCDNA3.1（＋） vector to construct recombinant plasmids pCE6 and pC70-83-E6, respectively. Four groups of BALB/c mice were vaccinated intramuscularly with pCE6, pC70-83-E6, pcDNA3.1 （＋） and PBS, respectively. Serum antibodies were detected by indirect ELISA, and spleen lymphocyte proliferation（SLP） and IFN-γsecreted by spleen cells were tested by MTT. The indirect ELISA showed that levels of antibodies in groups pCE6 and pC70-83-E6 were higher than those in the other groups,and group pC70-83-E6 was higher than group pCE6. SLP experiment indicated that stimulus value of groups pCE6 and pC70-83-E6 was higher than that of groups peDNA3.1（＋） and PBS, and there was no significant difference between groups pCE6 and pC70-83-E6. Levels of IFN-γin group pCE6 and pC70-83-E6 were higher than those in the two controls, and level of IFN-γ in group pC70 83 E6 was significantly higher than those in the others. The results showed that DNA vaccine containing esat-6 gene or mpbTO-mpb83-esat-6 gene could induce both antigenγspecific humoral and cellular immune response in BALB/c mice.