大肠杆菌耐热性肠毒素Ⅰ融合基因的构建及其免疫原性研究

CONSTRUCTION OF FUSION GENE FOR ENTEROTOXIGENIC ESCHERICHIA COLI HEAT STABLE ENTEROTOXIN I AND ITS IMMUNIZATION

用限制性核酸内切酶ＢａｍＨＩ和Ｂｇ１Ⅱ双酶切质粒ｐＢＳＴ２－６，获得了大肠杆菌耐热性肠毒素Ⅰ（ＳＴ１）基因，再将含ＬａｃＺ基因（编码β－半乳糖苷酶）的载体ｐＵＣ１８用ＢａｍＨＩ酶切、碱性磷酸酶处理，然后与ＳＴ１基因通过Ｔ４ＤＮＡ连接酶连接，转化至受体菌ＤＨ５α中。通过菌落原位杂交筛选，共筛选出５３个ＳＴ１基因探针杂交阳性的重组子，对其中一个重组子ＤＨ５α（ｐＸＳＴ１）进行限制性核酸内切酶酶切分析和核苷酸序列分析，证明重组质粒ｐＸＳＴ１含有２个正向串连在一起的ＳＴ１基因，而且融合在ＬａｃＺ基因的上游，具有正确的阅读框架。又ＤＨ５α（ｐＸＳＴ１）菌株能在含Ｘ－Ｇａｌ的ＬＢ平板上长成蓝色菌落，而且ＥＬＩＳＡ也检测到ＳＴ１融合蛋白，这表明该菌株能表达具有β－半乳糖苷酶活性的大肠杆菌ＳＴ１—β－半乳糖苷酶融合蛋白。免疫实验结果表明，重组菌株ＤＨ５α（ｐＸＳＴ１）安全无毒，表达的ＳＴ１融合蛋白能够诱发ＢＡＬＢ／ｃ鼠产生抗体，该抗体具有中和天然ＳＴ１肠毒素的毒性作用，这表明ＤＨ５α（ｐＸＴ１）可以作为预防幼畜腹泻的菌苗候选株。

We used the restriction endonucleases Bam HI and BglII to cleave the 147bp ST1 gene fragment from the plasmid pBST2 6 containing ST1 gene,isolated by 3% agarose gel electrophoresis,recovered the ST1 DNA fragment,the 3terminus of the ST1 gene was genetica lly fused to the 5 terminus of the LacZ gene that encodes for the β galactosidase We constructed the recombinant plasmid pXST1 and studied in detail by restriction endonucleases analysis and DNA sequencing The results have shown that the plasmid pXST1 carried two ST1 genes and LacZ gene fragment, had positive reading frame By transformation of E coli DH5 α,we got DH5 α(pXST1) The strain could produce the STβ galactosidase fusion protein by ELISA More importantly,the fusion protein was nontoxic and immunogenic BALB/c mice immunized with crude preparation containing the fusion protein produced antibodies that were able to recognize ST1 enterotoxin in vitro Significantly,these sera antibodies were able to neutralize the biological activity of native ST1 enterotoxin in the suckling mouse assay Hence the recombinant strain DH5 α(pXST1) can be used candidate of vaccine strain