百日咳毒素亚基基因的克隆与表达及其重组亚基的免疫学特性  

Cloning and expression of subunit genes of pertussis toxin and its immunological evaluation

作　　者：韩峰[1] 刘德铮[1] 肖詹蓉[1] 项金忠[1] 张鹏飞[1] 

机构地区：[1]卫生部北京生物制品研究所

出　　处：《中华医学杂志》1998年第4期257-258,共2页National Medical Journal of China

摘　　要：目的从百日咳包特杆菌ＣＳ株中克隆百日咳毒素（ＰＴ）及其各亚基的基因，并探讨在昆虫细胞中表达百日咳毒素亚基基因片段的可能性及表达产物的生物学特性。方法采用ＰＣＲ技术进行克隆，并通过限制性内切酶和Ｓｏｕｔｈｅｒｎｂｌｏｔ进行分析鉴定。利用重组杆状病毒技术，在昆虫细胞Ｓｆ９中表达各亚基基因，并通过免疫荧光实验进行分析鉴定，通过ＥＬＩＳＡ实验进行重组蛋白的免疫学特性评价。结果获得百日咳毒素及其各亚基的基因，并在昆虫细胞中表达了重组亚基。经体外聚合后的各重组亚基混合物与抗天然完整ＰＴ免疫血清的反应性和诱生特异性抗ＰＴ抗体的能力都明显高于各重组亚基单体混合物，含有Ｓ１重组亚基混合物诱生特异性抗ＰＴ抗体水平又明显高于不含Ｓ１重组亚基混合物的水平。说明百日咳毒素亚基单体仅具有很弱的诱生特异性抗ＰＴ抗体的能力，且抗完整ＰＴ的抗血清对百日咳毒素亚基单体也缺乏很好的识别作用。结论百日咳毒素诱导产生特异性抗体的能力依赖于聚合体的空间构型，而且其主要抗原决定簇位于聚合体的Ｓ１亚基上。Objective To clone the gene encoding pertussis toxin (PT) from Bordetella Pertussis CS strain and five genes encoding subunits of PT, to investigate the possibility of expressing the genes encoding for mature methionyl subunits of PT in insect cell and to evaluate the immunological characteristics of recombinant subunits. Methods The genes encoding for PT and its subunits were cloned by PCR and confirmed by restriction enzyme digestion and Southern blot. Using recombinant baculovirus technology, the recombinant subunits were expressed in Sf9 insect cells and analyzed by immunofluorescence assay. The immunological properties of recombinant subunits were analyzed by ELISA. Results The genes encoding for PT and its subunits were cloned and expressed in Sf9 cells. The level of specific antibodies against nature PT induced by five recombinant subunits polymerized was higher than that unpolymerized and polymerized by four recombinant subunits (without S1) in mice. The results indicate that PT subunits had weak ability to induce specific antibodies against PT, and also anti nature PT sera had poor recognition to PT subunits. Conclusion The ability of PT to induce specific antibody is conformation dependent of intact PT, and antibody induction domains are located dominantly in S1 subunit of intact PT.

关 键 词：百日咳 百日咳毒素 亚基因 克隆 基因表达 疫苗 

分 类 号：R516.603[医药卫生—内科学] R392－33[医药卫生—临床医学]