赖型钩体重组质粒及表达保护性抗原,p68的研究

STUDY OF RECOMBINANT DNA AND P68,AN ANTIGEN EXPRESSED FROM THE RECOMBINANT DNA OF LEPTOSPIRA INTERROGANS SEROVAR LAI

目的 寻找钩体病基因工程疫苗保护性抗原候选。方法 （１） 鸟枪法构建赖型钩体０１７ 株基因库； （２） α互补、 Ｄ Ｎ Ａ 原位杂交筛选重组质粒； （３） Ｓｏｕｔｈｅｒｎ 杂交行 Ｄ Ｎ Ａ 同源性分析； （４） 双脱氧链中止测重组 Ｄ Ｎ Ａ 序列及与外膜蛋白基因 （ｏｍｐ） 匹配； （５） Ｉ Ｐ Ｔ Ｇ 诱导重组子表达； （６） 免疫印迹、 Ｍ Ａ Ｔ、 Ｅ Ｉ Ａ、 Ｍ Ｔ Ｔ 检测表达蛋白抗原特性及 Ｉ Ｌ—２ 、 Ｉ Ｌ—６ 活性； （７） 纯化表达蛋白ｐ６８ 进行豚鼠主动免疫 攻击实验， 观免疫保护性。结果 （１） 重组质粒ｐ Ｄ Ｊ Ｈ２ （ 亚克隆ｐ Ｄ Ｊｔ） 插入片段１９ｋｂ ， 与各致病钩体不同片段 Ｄ Ｎ Ａ 高度同源； （２） 序列测定插入片段实际１８１１ｂｐ ， 推测有２ 个读框 （ Ｏ Ｒ Ｆ） ， 各有启动子、终止密码。查 Ｇｅｎ Ｂａｎｋ Ｅ Ｍ Ｂ Ｌ 无类似序列； （３） 表达蛋白分子量６８ｋ Ｄ （ｐ６８） ； （４） ｐ６８ 是胸腺依赖性 （ Ｔ Ｄ） 抗原，有良好抗原特性， 抗血清效价１ ： ５２４２８８ ， （ Ｅ Ｉ Ａ） 具很强的动物免疫保护作用。结论 （１） ｐ６８ 编码基因是致病钩体保护性抗原基因； （２） ｐ６８ 是致病钩体外膜保护性抗原，

To look for a antigen candidate of gene engineering vaccine for leptospirosis.Characteristic of p68,a protective antigen expressed from recombinant gene of L interrogans serovar Lai strain 017 was studied.Methods(1)random cloning was used to construction a gene library of L interrogans serovar Lai strain 017;(2)Situ blotting was used to selection recombinant plasmid in the bank;(3)Southern blotting was seleted to research on the DNA homlogy;(4)Sanger double-dideoxy-mediated chaintermination was employed to check the sequence of the fragment insered and its alignment with other bactereal omp(outer membrane protein);(5)To induce the recombinant protein expressed,IPTG was adoped;(6)Antigenicity of the protein expressed was studied with immunoblotting.EIA and MAT;(7)MTT was taken to test the actions of IL-2 and IL-6 in guinea pigs immunized with the protein expressed;(8)Initiative immunization was carried out to identify the immuno/protection on the model of guinea pigs with the protein expressed.Results(1)A recombinant clone,designated as pDJH 2 (subclone was pDJt)in the bank was obtained.It contained 1 9kb DNA fragment from L Strain 017;(2)The 1 9kb had highly homlogy with pathogenic leptospires;(3)The sequence analysis showed that total numbers of nt for the inserted ORA were 1811bp.There were two open reading frames(ORF)of 565 nt and 662nt there are promoters,terminators and initiatin codons respectively,in ORFs.There were not similar sequence in the GenBank/EMBL;(4)The molecular weight of protein expressed of pDJH 2/pDJt was 68kD(p68);(5)p68 could elicit a strong and specific humoral immune response that T and B cell took part in.Antiserum titer of p68 was 1:524288(guinea pigs,EIA).The activity of IL-6 was 83 25 IU·ml,IL-2 was 75IU·ml,and p68 was idetified as a major antigen of OMP from L serovarLai;(6)Guinea pigs immunized with p68 could resist well challenge of the strong toxicity L serovar Lai strain 017 .Conclusion(1)Code gene of p68 was a protective antigen of pathogenic leptospires;(2)p68 was a good protective antigen for leptospirsis and candidate for gene engineering vaccine of leptospirosis.