MOLECULAR BIOLOGY OF VIRULENCE FACTORS

毒力因子的分子生物学

基本信息

批准号：
3087008
负责人：
ROBERT M LAWRENCE
金额：
$ 7.57万
依托单位：
NEW YORK UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
1987
资助国家：
美国
起止时间：
1987-09-30 至 1992-08-31
项目状态：
已结题

项目摘要

The gene coding for the STb enterotoxin will be inserted into a plasmid vector and cloned into a K12 strain of E coli. The clones will be screened by DNA hybridization, immunoassay and bioassay to select strains which hyperproduce the STb toxin. Purification of the toxin will be done on culture supernatants using standard chromatography, an FPLC unit and antigen-antibody affinity columns. The purified toxin will then be biochemically analyzed. Antibodies to the STb toxin will be made by coupling purified or synthetic toxin to a large molecule and injecting the coupled protein into rabbits. The antibodies will be used in an immunoassay, and antigen-antibody affinity column and neutralization an receptor site studies. The gene for CJT toxin production will be localized on plasmid or chromosomal DNA. A genomic library will be created in pUC or lambda phage and screened for toxin production byh bioassay, Y1 cell assay and immunoassay. Once the gene for CJT production is identified it will be sequenced and then cloned into an expression vector. The cjt pUC clones will be transferred into K12 E. coli and selected for hyperproduction of C. jejuni enterotoxin. The toxin will be purified by standard chromatography methods for use in future studies.

STB肠毒素编码的基因将插入质粒载体并将其克隆成E大肠杆菌的K12菌株。克隆将通过DNA杂交，免疫测定和生物测定筛选选择过度生产STB毒素的菌株。纯化毒素的含量将使用标准进行培养上清液色谱法，FPLC单元和抗原抗体亲和力列。然后将对纯化的毒素进行生化分析。耦合纯化或合成毒素到大分子并注入耦合蛋白质成兔子。抗体将用于免疫测定和抗原抗体亲和力柱和中和受体位点研究。 CJT毒素产生的基因将定位于质粒或染色体DNA。基因组库将在PUC或 lambda噬菌体并筛选毒素生产Byh Bioassay，Y1 细胞测定和免疫测定。一旦CJT生产基因是确定它将被测序，然后克隆到表达式中向量。 CJT PUC克隆将被转移到K12大肠杆菌中并选择用于过多生产空肠梭菌肠毒素。这毒素将通过标准色谱法纯化在未来的研究中。