破伤风毒素C片段的基因克隆、表达、纯化及免疫原性分析  

Gene cloning, expression and purification of tetanus toxin fragment C and characterization of its immunogenicity

作　　者：谭亚军[1] 雷殿良[1] 张庶民[1] 

机构地区：[1]中国药品生物制品检定所血清室,北京100050

出　　处：《中华微生物学和免疫学杂志》2004年第3期222-225,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的　对破伤风毒素C片段进行基因克隆、重组表达、蛋白纯化和免疫原性分析。方法　应用PCR技术直接从破伤风梭状芽孢杆菌的质粒DNA中扩增出 1370bp的破伤风毒素C片段(简称TTC)基因 ,将此基因片段插入到表达载体pET 2 2b(+)中 ,并在大肠杆菌BL2 1(DE3)中表达。用阴离子交换层析和金属离子螯合亲和层析方法进行蛋白纯化后 ,参照《中国生物制品规程 2 0 0 0年版》的免疫攻击实验方法测定其效价。结果　经SDS PAGE分析 ,重组蛋白的表达量占菌体总蛋白的15 %。免疫印迹实验证实该重组蛋白是破伤风毒素C片段抗原。纯化得到纯度为 96 .5 %的重组蛋白 ,纯化回收率达 4 0 %。免疫攻击实验测定其效价为 18.70 6IU/mg,ED50 为 2 0 μg。经加强免疫 ,1μg免疫剂量即能产生足够的抗体保护动物免受破伤风毒素的攻击。结论　所获得的重组蛋白具有良好的免疫原性 。Objective To clone, express, purify and analyse the immunogenicity of the fragment C of tetanus toxin. Methods The fragment C gene of tetanus toxin was amplified from Clostridium tetani plasmid DNA by PCR. It was inserted into the high expression vector pET 22b(+) and expressed in E.coli BL21(DE3). The expressed protein was purified by two steps of anion exchange chromatography and Ni 2+ chelate affinity chromatography respectively. Immunogenicity of the purified protein was tested according to the recommended method of the Chinese requirement of biological products, 2000 edition. Results The result of SDS PAGE showed that a specific recombinant protein with molecular weight of 55kD was expressed and accounted for 15% of the total bacterial protein. Western blot analysis indicated this product had certain antigenicity. The final purity was 96.5% and the recovery rate was 40%. The calculated valence and ED 50 were 18.706IU/mg and 20μg respectively. Multiple immunized mice with expressed protein at the dosage of 1μg produced enough antibodies that were able to protect mice against a challenge with tetanus toxin. Conclusion The results confirmed that fragment C gene is an immunoprotective antigen gene of tetanus toxin and thus constructs a basis for studying genetic engineering vaccine in the future .

关 键 词：破伤风毒素 基因克隆 蛋白纯化 免疫原性 PCR技术 基因工程疫苗 

分 类 号：R392[医药卫生—免疫学]