HCMV pp65 DNA疫苗诱导小鼠的细胞免疫应答

Study on the cellular immune response induced by DNA vaccine with the pp65 gene of HCMV in mice

目的 构建含HCMV pp65336～ 50 7位氨基酸的重组真核表达质粒 pcDNA3 1 pp65作为HCMVpp65基因疫苗 ,观察其诱导小鼠的细胞免疫应答情况。方法 以 6～ 8周龄♀Balb/c小鼠为动物模型 ,试验组和对照组各 6只。试验组于股四头肌注射pp65DNA疫苗 ,对照组注射空载体pcDNA3 1。 2组小鼠分别在 0d、5d、3周注射该DNA疫苗 2 0 0μg。在末次免疫后 3d ,无菌取小鼠脾细胞 ,并用ConA和重组HCMV pp65336～ 50 7作为抗原刺激 ,吉氏液染色观察T淋巴细胞的形态学变化 ,并计算转化率。MTT法对脾脏的特异性杀伤细胞率和淋巴细胞的增殖指数进行测定 ,间接ELISA定量检测脾细胞上清中IFN γ的含量。结果 pp65336～ 50 7抗原刺激的DNA疫苗免疫鼠脾细胞体积变大 ,胞浆增多 ,核仁增多 ;淋巴细胞增殖试验示pp65336～ 50 7抗原能特异性刺激免疫鼠脾细胞增殖 ;试验组IFN γ较对照组增高 ,其含量为 80 0ng/L。与对照组比较 ,差异均具显著性 (P<0 0 5)。试验组及对照组脾细胞杀伤率分别为 :56 0 5 %±1 3 64 %、9 85 %± 2 0 6 % ,试验组脾细胞CTL活性比对照组增高 (P <0 0 5)。

Objective Recombinent eukaryotic expression plasmid s of pcDNA3 1-pp65 encoding pp65 336～507 proteins of HCMV were constructed as gene vaccines to observe specific cellular immune responses in mice induced by DNA immunization. Methods The DNA vaccine (pcDNA3 1-pp65 336～507 ) and control plasmid(pcDNA3 1) was injected into the Balb/c mice via muscles. Three booster injections were given on the fifth day and in the th ird week following the first injection. The transformation percentage of spleen cells was tested by Gimsa staining. The proliferation response of spleen cells was tested by MTT method.HCMV pp65 336～507 specific cytotoxic T-lymphocy te (CTL) activity was measured by MTT assay. IFN-γ was detected by ELISA. Results The spleen cells of Balb/c mice were stimulated to transform and proliferate by pp65 336～507 .The pp65 336～507 specific CTL from immunized mice was 56 05%±13 64%,and that of control groups was 9 85%±2 06%;The CTL activity in the immunized mice was remarkably higher than thatof control groups(P <0 0 5 ) .Conclusion The results in dicate that the DNA vaccineofpc DNA3 1 pp65 3 3 6～ 50 7/HCMV hasstrongan tigenecit yincellul arimmunity.