Cloning,Expression And Application Of RgpA/B Gene In Porphyromonas Gingivalis Dominant Strain In Esophageal Cancer Patients

Objective The RgpA/B gene in patients with and without esophageal cancer was compared and analyzed,and the dominant strains in patients with esophageal cancer were screened for cloning and expression;Monoclonal antibodies were prepared using hybridoma technology to provide the basis for further development of a kit for clinical detection and diagnosis of P.Gingivalis infection.Methods(1)Differences in RgpA/B genotype between patients with and without esophageal cancer:Collect oral gingival samples from 74 esophageal cancer patients and 135 without esophageal cancer patients,RgpA/B gene was amplified by PCR and sequenced,the detection rate of RgpA/B gene in the two groups was calculated.Homology analysis was performed using Clustal X software to observe the aggregation and distribution of RgpA/B gene,find out the dominant strains of esophageal cancer patients.(2)Cloning and expression RgpA/B genes in the dominant strains of esophageal cancer patients:The dominant strains of esophageal cancer patients were used toconstruct prokaryotic expression plasmids PET-28a/RgpA and PET-28a/Rgp B,which were converted into E.coli BL21,constructed prokaryotic expression strains BL21/PET-28a/RgpA-P046 and BL21/PET-28a/Rgp B-P046.The recombinant target protein was induced by IPTG expression,and purified by His Trip HP nickel column affinity chromatography.(3)Preparation and application of RgpA/B protein monoclonal antibody:Immunize mice with the above-mentioned gene engineering target protein as antigen,prepare monoclonal antibody and identify its characteristics,prepare ELISA reagent and compare with PCR method to determine its clinical performance.Result(1)Differences in RgpA/B genotype between patients with and without esophageal cancer:PCR amplified RgpA/B genes,The positive rates of RgpA/B genes in esophageal cancer group and non-esophageal cancer group were 53(71.62%)/54(72.97%)and 59(43.70%)/58(42.96%).The detection rate in esophageal cancer group was significantly higher than that in non-esophageal cancer patients(P<0.01),and the difference was statistically significant.By homology analysis,RgpA/B genes in esophageal cancer patients had obvious clusters in multiple regions,which was defined as the dominant strain in esophageal cancer patients.(2)Cloning and expression of RgpA/B genes in dominant strains of esophageal cancer patients:In this experiment,the prokaryotic expression plasmid(p ET-28a/RgpA、p ET-28a/Rgp B)and prokaryotic expression strain(BL21/p ET-28a/RgpA-P046、BL21/p ET-28a/Rgp B-P046)were successfully constructed.The prokaryotic expression strain was cultured in the culture medium containing kanamycin,the IPTG was added to induce,when cultured to 24h,the target protein expression was the highest.The molecular weight size is consistent with the predicted results.After His Trip HP nickel column chromatography,the purity>90%was identified by PAGE electrophoresis.(3)Preparation and Preliminary Application of RgpA/B Protein Monoclonal Antibodies:The hybridoma cell line with Rgp-P046 protein and monoclonal antibodies were successfully prepared.The titer of ascites antibody was 1:10~6.Anti-RgpA-P046monoclonal antibody is Ig G1 type,which has good specificity and no cross reaction with other antigens.The prepared monoclonal antibody was used to detect the RgpA antigen of P.gingivalis in esophageal cancer patients by ELISA,compared with the RgpA gene amplified by PCR,the two methods was no statistically significant difference(P=0.46).Conclusion We successfully found the dominant strain of P.gingivalis,cloned its RgpA/B gene and expressed purified RgpA/B antigen as immunogen,Successfully prepared anti-RgpA-P046 monoclonal antibody,and the preliminary identification effect was good identification effect was good material basis for the development of gold standard strip or ELISA kit for rapid diagnosis of Porphyromonas gingivalis.