摘要
目的 构建含日本血吸虫相对分子质量为 2 6 0 0 0的抗原 (Sj2 6GST)基因的重组卡介菌(rBCG)疫苗 ,观察其对BALB/c小鼠的免疫保护作用。方法 采用分子生物学技术 ,将Sj2 6GSTcDNA克隆到人结核杆菌热休克蛋白 (HSP) 70启动子下游 ,构成融合基因 ,再将此融合基因亚克隆到E .coli 分枝杆菌穿梭质粒pBCG 2 0 0 0中 ,构成重组质粒pBCG Sj2 6 ,然后转化卡介苗 (BCG) ,构成血吸虫重组BCG Sj2 6GST(rBCG Sj2 6GST)疫苗 ,经热诱导 ,在BCG中表达Sj2 6GST抗原。用rBCG Sj2 6GST疫苗皮下免疫BALB/c小鼠 ,以淋巴细胞刺激指数 (SI)反映细胞增殖能力 ,以NO释放量检测巨噬细胞吞噬活性 ,以ELISA试剂盒检测血清及脾淋巴细胞培养上清的白细胞介素 (IL) 2和干扰素 (IFN) γ的含量。结果 HSP70启动子与Sj2 6GSTcDNA融合基因已被克隆到pBCG 2 0 0 0穿梭表达质粒中 ,相对分子质量为 2 6 0 0 0处可见明显的表达蛋白带 ,其表达量占菌体总蛋白量的 15 %。rBCG Sj2 6GST疫苗以 10 6菌落形成单位 (CFU)经皮下免疫小鼠后 ,其脾SI为 2 .2 6± 0 .43,与对照组 (1.6 1± 0 .2 8) ,载体组(1.48± 0 .30 )和BCG组 (1.42± 0 .2 6 )比较 ,P <0 .0 5 ;巨噬细胞NO的含量为 35 7nmol/ml± 84nmol/ml,与对照组 (183nmol/ml± 33nmol/ml)和?
Objective To construct recombinant BCG vaccine bearing Schistosoma japonicum 26Ku glutathione S transferase (Sj26GST) gene and determine its immunogenicity on BALB/c mice. Methods Using techniques of molecular biology, human mycobacterium tuberculosis HSP70 promoter and Sj26GST gene were linked to produce a fused gene. The fused gene was cloned into an E.coli Mycobacterium shuttle plasmid pBCG 2000 to construct an E. coli Mycobacterium expression shuttle plasmid pBCG Sj26 that could express Sj26GST gene. Then, the pBCG Sj26 was introduced by electroporation into mycobacterium bovis BCG to construct a recombinant BCG vaccine bearing Sj26GST gene ( rBCG Sj26GST). The expression of Sj26GST gene in BCG was induced by heating. The lymphocyte stimulating index (SI), macrophage activity and IL 2, IFN γ levels of the serum and culture supernatant of spleen lymphocytes were tested after immunization of BALB/c mice with rBCG Sj26GST vaccine. Results The fused gene of HSP70 promoter and Sj26GST cDNA was inserted into an E. coli Mycobacterium shuttle expression plasmid by analysing electrophoresis results on PCR products using plasmid pBCG Sj26 as a templet. The content of rSj26GST contained 15% of total bacterial protein of BCG. The SI of the experimental group was 2.26±0.43, which was significantly higher than those in the control group (1.61±0.28, P <0.05), vector group (1.48±0.30, P <0.05) and BCG group (1.42±0.26, P <0.05). The macrophage NO level of the experimental group was (357.42±84.11) nmol/ml which was significantly higher than those in the control group (183 nmol/ml±33 nmol/ml, P <0.01) and vector group (203 nmol/ml±56 nmol/ml, P <0.01). The serum IL 2 level of the experimental group was (267 pg/ml±130 pg/ml), which was significantly higher than those in the control group (45 pg/ml±15 pg/ml, P <0.01) and vector group (52 pg/ml±29 pg/ml, P <0.05). Compared with the control group, the serum IFN γ level increased by 20%, the IL 2 level of the culture supernatant of spleen lymphocytes increased by 44%. Conclusions The foreign gene encoding Sj26 GST can be expressed in BCG. rBCG Sj26GST vaccine may induce stronger immune response in BALB/c mice than in control, vector and BCG groups.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2000年第6期407-410,共4页
National Medical Journal of China
基金
总理基金!(94Y19)
国家自然科学基金!(39480 022)资助项目
