Cloning And Expression Of FimA From Uropathogenic Escherichia Coli

ObjectiveUrinary tract infection is a kind of commonly encourted disease, the pathogen of which is called uropathogenic Escherichia coli (UPEC). Type I fimbriae is a chief toxin factor of UPEC.fimA, a structural gene of UPEC 132, was cloned and expressed in this study, after which FimA recombination protein was used for antiserum preparation which would be used later projects.Method1. The phenotype of type I fimbriae on UPEC 132 surface was detected by MSHA test, while fimA was detected by PCR.2. fimA gene of type I fimbriae within UPEC 132 was cloned by PCR, that was linked with T vector later. And then it was treated with restricted enzyme BamHâ… and Hindâ…¢, as well as pET32a. After conjuncting with pET32a vector, recombinated phage pET32a-fimA was constructed. The recombinated phage was identified by PCR and sequence analysis.3. pET32a-fimA was transformated into E. coli BL21 (DE3), as well as to be inducted by IPTG.. Recombinated bacterium was analysised via SDS-PAGE after ultrasoni cation.4. FimA recombinated protein was purified by Ni-affinity chromatography. The target protein was renatured through dialysis after purification, which was used for detection of concentration and purity, as well as its molecular weight.5. Purified FimA recombinated protein was used for antiserum preparation through immunizing BALB/c mice. The titer of antiserum was detected via ELISA, while the specificity of which was identified with Western blot.6. UPEC 132 was used as antigen, while FimA antiserum was used as antibody for whole bateria ELISA finally.Result1. It was showed that UPEC 132 does not have type I fimbriae through MSHA. It was identified that fimA exist within genome of UPEC132. 2. Recombinated phage pET32a-fimA was constructed, which DNA sequence was the same as fimA gene (ID 1037247) from GenBank.(similitude 100%) that was identified by PCR and restricted enzyme.3. Recombinated bacterium E. coli BL21 (DE3)/pET32a-fimA expressed with the inducement of IPTG. Recombinated protein FimA did not exist in supertanant identified by SDS-PAGE after centifugation, while it was found within deposit as inclusion body. It was measured that FimA is 37% of deposit expressed protein.4. Purified FimA recombinated protein was acquired after Ni-affinity chromatography, whose purity is 95.6%. The moleculare weight of FimA recombinated protein is 39.93kD, which concentration is 5.87mg/mL analysised from SDS-PAGE.5. The tiiter of FimA antiserum was mor than 1:51200, which suggested that FimA had good immunogenicity. It was showed that a single band displayed by Western blot.6. It was showed by whole bacteria ELIS A that the S/N values of reaction between UPEC132 strain and 1:400 of antiserum was 1.34, while the negative control E.coli k12 p678-54 was 1.12, as well as positive control E.coli 136 was 6.27. The result of whole bacteria ELISA of UPEC132 was negative, which confirmed the result of MSHA.ColusionThe whole gene sequence of fimA from UPEC132 was cloned succesfully, which was used to constructured the recombinated bacterium E. coli BL21 (DE3)/pET32a-fimA. The FimA recombinated protein presented 37% of deposit expressed protein. The purified FimA recombinated protein was acquired used for polyclone antibody preparation, which has good specificity. Our study lay a foundation for the in depth investigation on UPEC.