Cloning,Expression And Application Of FimA Gene In Porphyromonas Pingivalis Dominant Strain In Esophageal Cancer Patients

Objective It has found dominant strain in esophageal cancer patients through homology analysis of fimA gene both in periodontal healthy population and esophageal cancer patients.In order to establish material foundation for the rapid diagnostic kit of Porphyromonas gingivalis(P.gingivalis)producing,the monoclonal antibody of Fim A protein was prepared by using techniques of genetic engineering and hybridoma.Methods(1)The difference of fimA gene expression in periodontal healthy population and esophageal cancer patients:Oral gingiva samples that conclude 74esophageal cancer patients and 135 periodontal healthy population were collected.Fim A gene were amplified by PCR and sequenced.The sequencing result of fimA was used to compare with the standard strain of six types of fimA gene.And calculating the detection rate in the two groups and the composition of different genotypes of fimA gene.Clustal X software was used for homology analysis of fimA gene to observe its distribution in the two groups(2)Clone and expression of fimA gene in the dominant strain in esophageal cancer patients:The plasmid p ET-28a/fimA prokaryotic expressed was constructed based on the fimA gene in the dominant strain in esophageal cancer patients,and was transformed into E.coli BL21,which aims to construct the BL21/p ET-28a/fimA engineering bacteria.And the recombinant protein was obtained by IPTG inducing and purified by His Trap HP nickel column.(3)Preparation and application of monoclonal antibody of Fim A protein:Hybridoma cell line was made from mice immunized with Fim A protein forementioned to produce monoclonal antibody,and its specificity identification was followed.By comparison between ELISA and PCR,discussed the clinical performance of monoclonal antibody.Result 1.The difference of fimA gene detection in periodontal healthy population and esophageal cancer patients:The positive rates of fimA gene amplified by PCR in esophageal cancer group and periodontal healthy group were 70.27%and41.48%respectively,with statistically significant difference(P<0.01);Fim A genotyping results demonstrated that the highest proportion of fimA II genetype in both groups,and no significant statistically difference in all genetypes(P=0.59);Homology analysis indicated that the fimA gene of esophageal cancer patients has obvious clusters in multiple areas,which was defined as dominant strain for esophageal cancer patients.2.Clone and expression of fimA gene in esophageal cancer patients dominant strain:The plasmid(p ET-28a/fimA-P008)prokaryotic expressed and the prokaryotic strain(BL21/p ET-28a/fimA-P008)were successfully constructed.The prokaryotic strain was cultured in containing kanamycin medium.When the A600 reached 0.6,it was induced with 1 m M IPTG.After cultured for 12h,the content of target protein expression was at the peak.The recombinant protein was categorized bacterial inclusion body protein,and the molecular weight detected was consistent with the predicted.And its purity was more than 90%by PAGE electrophoresis identification after His Trap HP nickel column chromatography purified.3.Preparation and application of monoclonal antibody of Fim A protein:The hybridoma cell line and monoclonal antibody against Fim A-P008 protein were successfully prepared.The titer of mouse ascites antibody is 1:10~6after detection.The anti-Fim A-P008 monoclonal antibody subtype was Ig G1 with high specificity that performed no cross-reactivity with other antigens.The prepared monoclonal antibody was used to detect Fim A antigen of P.gingivalis by ELISA.Compared with the fimA gene amplified by PCR,the difference of the two methods results were not statistically significance(P=0.20).Conclusion It is successfully that the dominant strain of P.gingivalis in esophageal cancer patients was found.The fimA gene was cloned and expressed.The Fim A protein was purified to use as antigen to prepare anti-Fim A-P008 monoclonal antibody,which has provided the material basis for the development of the rapid diagnosis kit of P.gingivalis.