牛分枝杆菌二价组合及融合DNA疫苗的免疫效果

Immune efficacy of bivalent combinations and double fusion DNA vaccine from Mycobacterium bovis

利用PCR技术和重叠延伸剪接(SOE)技术扩增牛分枝杆菌ag85b、mpb64基因和ag85b-mpb64融合基因,连接真核表达载体pCDNA3.1(+),构建二基因融合(pCDNA-MPB64/Ag85B,pCMA)和二价组合(pCDNA-Ag85B+pCDNA-MPB64,pCA+pCM)DNA疫苗。以牛分枝杆菌卡介苗(BCG)为阳性对照,以pCDNA3.1(+)和PBS为阴性对照,免疫BALB/c小鼠,检测血清特异性抗体水平、脾淋巴细胞增殖情况和IFN-γ及IL-2分泌情况。结果显示:融合DNA疫苗组免疫小鼠血清抗体水平、刺激值(SI值)及IFN-γ和IL-2的分泌水平均明显高于其他各组(P<0.05)。二价组合DNA疫苗组的体液和细胞免疫指标与BCG组相当(P>0.05),而显著高于两阴性对照组(P<0.05)。

The DNA fragments of ag85b,mpb64 and ag85b-mph64 were amplified by PCR and spliced by overlapping extension （SOE） technique. These fragments were inserted into pCDNA3. 1（＋ ） vector to construct two DNA vaccines as follows： fusion of two genes （pCDNA-MPB64/Ag85B, pCMA）,and bivalent combinations （pCD- NA-Ag85B ＋ pCDNA MPB64 ,pCA＋pCM）. BALB/c mice were vaccinated with these DNA vaccines. The mice injected with BCG were positive control and the mice injected with pCDNA3. 1 （ ＋ ） and PBS were negative control. The serum antibodies, the spleen lymphocyte proliferation （SLP） and secreted IFN -γ, IL-2 of spleen were tested. The results showed these index in fusion DNA group were significantly higher than other groups（P〈0.05）. The index in bivalent combinations group were equivalent to group of BCG, and significantly higher than the two control （P〈 0.05）. So our experiment suggested that fusion DNA vaccine represents a promising approach for the prevention of BT.