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

作　　者：宫强[1] 刘思国[1] 王春来[1] 王勇[1] 刘建东[1] 迟磊[1] 赵昆[1] 周媛媛[1] 常月红[1] 云孟克[1] 孔宪刚[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室动物细菌病研究室,哈尔滨150001

出　　处：《中国生物工程杂志》2007年第2期47-52,共6页China Biotechnology

基　　金：国家科技攻关计划"奶牛主要疫病防治关键技术研究与产业化开发课题"资助项目(2002BA518A04)

摘　　要：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.

关 键 词：牛分枝杆菌 单基因 融合基因 DNA疫苗 

分 类 号：Q785[生物学—分子生物学]