预防幼畜腹泻双价工程菌苗株的构建及表达

Construction and Expression of Divalent Vaccine Strain for the Prevention of Baby Animal Diarrhea

Bam HI部分酶切表达K99抗原的重组质粒pMGK99,将其与相同酶切的含有F41菌毛基因的片段相连接,构建了载有两种抗原基因的质粒pMG611。通过转化大肠杆菌K12HB101、RRI和C600,得到HB101(pMG611)、RR1(pMG611)和C600(pMG611)菌株。经甘露糖抗性血凝试验、玻片凝集试验和Western blot分析证明K99和F41两种菌毛抗原在每株菌中均获表达。SDS-PAGE结果表明所表达K99和F41菌毛亚单位的分子量与其各自野生株所产生的相同,分别是17200和29800Da,ELISA测定HB101(pMG611)菌株表达K99和K41菌毛抗原的水平与相应出发菌株相同,而高于其野生株。本实验对质粒pMG611表达K99和F41菌毛抗原的影响因素也进行了研究。

The F41 gene was isolated, and then inserted into K99 gene-carrying plasmid pMGK99 and was partially digested by the same enzyme BamHI. The transformants were screened by F41 gene probe that was labled with (α-^(32)P) dATP by random primer method. One of the positive transformants was tested by dot blot and Southern-blot assay. The results have shown that the recombinant plasmid carried K99 and F41 gene fragments. The recombinant plasmid was designated as pMG611. By transformation of E. coli K12 strains HB101, RRI and C600, we got three strains HB101 (pMG611), RRI(pMG611) and C600(pMG611).These strains can produce two kinds of antigens which were proved by using MRHA, slide agglutination and Western-blot assays. The molecular weight of K99 and F41 protein subunits were estimated by SDSPAGE and they are the same as wild fimbriae subunits, 17200 and 29800 Da, respectively.Double antibody sandwhich ELISA was established for detecting the expression levels of both K99 and F41 antigens. The levels of K99 and F41 fimbriae expressed by HB101 (pMG611) were higher than their corresponding parent strains. Expression conditions of both antigens were studied. The results of rabbit ileal loops test have indicated that HB101 (pMG611) and RRI(pMG611)have no enterotoxigenicity.We concluded that the recombinant vaccine strains can express both K99 and F41 antigens effectively and can be used for the vaccine candidate of the prevention of the neonatal animal diarrhea caused by ETEC,