摘要
目的构建携带结核分枝杆菌PPE68基因的重组卡介苗(BCG),并检测其诱导小鼠的免疫应答情况。方法将带有分枝杆菌复制子OriM的穿梭质粒pBudCE4.1/PPE68/OriM电转化入BCG中,PCR鉴定重组BCG。用鉴定正确的PPE68/OriM重组BCG免疫BALB/c小鼠,每2周免疫1次,共3次,末次免疫后2周采血,分离血清,检测血清中IgG2a、IFNγ和IL-12水平;取脾、肺,分离脾淋巴细胞,检测经特异性蛋白刺激后淋巴细胞的增殖水平;进行脾CD4+和CD8+T淋巴细胞计数,计算CD4+/CD8+比值;检测脾、肺荷菌量并观察组织病理变化。结果 PPE68/OriM重组BCG经PCR鉴定构建正确;其免疫小鼠后,可诱导小鼠血清中IgG2a、IFNγ和IL-12水平显著增加(P<0.05),脾淋巴细胞增殖显著(P<0.05),CD4+和CD8+T细胞百分比增加,CD4+/CD8+T细胞比值降低,脾、肺均无菌落生长且未见明显病理改变。结论成功构建了PPE68基因重组BCG,其免疫BALB/c小鼠能明显提高小鼠的Th1型免疫效应,有利于机体抗结核菌感染。
Objective To construct a recombinant BCG with PPE68 gene and determine the immune response induced in mice.Methods Shuttle plasmid pBudCE4.1 / PPE68 / OriM containing replicon OriM of mycobacterium was transformed to BCG by electrotransformation.The recombinant BCG was identified by PCR,and the correct one were used for immunization of BALB / c mice,once 2 weeks for 3 times.Serum samples were collected 2 weeks after the last immunization and determined for IgG2a,IFNγ and IL-12 levels.Spleens and lungs of mice were collected,and lymphocytes were isolated from spleens and determined for proliferation level after stimulation with specific protein.The CD4＋ and CD8＋ T lymphocytes were counted,based on which CD4＋ / CD8＋ ratio in spleen was calculated.The numbers of bacteria-bearing as well as pathological changes of spleen and lung were observed.Results PCR proved that recombinant BCG with PPE68 / OriM was constructed correctly.The IgG2a,IFNγ and IL-12 levels in sera of immunized mice increased significantly（P 〈 0.05）.The lymphocytes in spleen increased significantly（P 〈 0.05）,and the percentages of CD4＋ and CD8＋ T lymphocytes increased significantly,while the CD4＋ / CD8＋ ratio decreased.No bacterial growth or pathological change was observed in spleen or lung of mice.Conclusion Recombinant BCG with PPE68 gene was successfully constructed,which enhanced Th1 immune response in BALB / c mice and was beneficial to prevention of infection with Mycobacterium tuberculosis.
出处
《中国生物制品学杂志》
CAS
CSCD
2012年第6期665-668,675,共5页
Chinese Journal of Biologicals
基金
国家自然科学基金(30771922
30901280)
