Clonging, Expression And Its Biological Characteristic Of The Domain Gene Of Clostridium Difficile Toxin B Mucosa Receptor Conjugate Region

Clostridium difficile was one major causative agent of pseudomembranous colitis (PMC) and many cases of antibiotic-associated diarrhea in humans. It was import mostly to diagnose quickly, identify C difficile, because these work could help to confirm originm, control its diffuse and treat patients.ELISA assay,which was high the sensitivity and its rapid turnaround time in 2 to 4 hours,had been as an in vitro diagnostic aid for C. difficile disease in developed countries,but the study of clostridium difficile Toxin B is very little in our country.In this paper,we perform cloning and expression of the C-terminal domain gene of Clostridium difficile Toxin B by gene work, purify of the recombinant proterin and preparation of multiple clone antibody of C-terminal domain C. difficile toxin B.The aim of study seeked after its biological characteristic in order to establish a method for development of an ELISA kit for Clostridium difficile toxinB. Methods:1. To perform cloning and expression of the C-terminal domain gene ofClostridium difficile Toxin B by gene techniques. C-terminal domain Clostridium difficile Toxin B gene was amplified from Clostridium difficile DNA by PCR techniques.The PCR product was inserted into the prokaryotic expression vector PET22b(+) after they are digested with EcoRI and Xhol for two hour.The recombinant plasmid was true through checking sequences,then the recombinant plasmid was transformed into the E.coli BL21(DE3),where the C-terminal domain Clostridium difficile Toxin B gene was expressed with FPTG.The expression products were analyzed using the SDS-PAGE method o2. Metal chromatography method for purification of the recombinant proterin and its antigenicity by western-blot assay. In this report, the prokaryotic expression vector PET22b(+) plasmid has secense of His.Tag,so it can combinece with Ni2+,at last, the recombinant proterin can be washed by imidazole. The western-blot assay masure its antigenicity.3. Preparation of multiple clone antibody of the C-terminal domain Clostridium difficile ToxinB and study its biological characteristic. The blood serum was gathered from new-zealand rabbits, which was immunized with purification of the recombinant proterin.Double immunodiffusion and ELSIA measure titre of the multiple clone antibody. Double immunodiffusion measure the antigen correlation between the recombinant protein and Clostridium difficile ToxinB from NOVagen incoprationAt last,we made a examination about Clostridium difficile ToxinB by indirect ELISA from the tested specimens including culture filtrates of 5 strains of toxigenic C. difficile, 1 strain of BL21 with PET-22b(+),l strain of E. coli, 1 strain of S. dysenteriae.Results1. The PCR product was shown consisting of about 1848 bp.lt was determined using restriction enzyme digestion PCR and sequencing methods, that the genesegment inserted into recombinant vector was identified 99% to the Gene Bank.It was shown the expression product of the C-terminal domain gene of Clostridium difficile Toxin B was with a molecular mass of 71 300 ?2. The method for purifying the recombinant protein was established.The purfied the recombinant protein was obtained. The purifying expression products, which can be degraded a little, was a molecular mass of 71 300 by the 10% SDS-PAGE method.Protein concentration is 0.762mg/ml by Coomassie brilliant blue G-250 chromatometry method. Western-blot shows it has strong antigenicity.3. The multiple clone antibody of the C-terminal domain Clostridium difficile ToxinB was obtained. The antigenicity of the recombintant protein was subdivision of the antigenicity of Clostridium difficile ToxinB. Western-blot shows not only the multiple clone antibody has strong specificity but also it could be combined with splitting protein of E.coli thallus.3 strains culture filtrates of toxigenic C. difficile and 1 strain of BL21 with PET-22b(+) gave a positive result, others strains culture filtrates were all negative.Conclusionsl.The recombinant vector constructed in this study with PET22b(+) and ToxinB3 could express the inserted gene at a high level, which might act as a necessary basis for the further research work on the clostridium difficile.2. This modified method for purification of toxin B of C. difficile was simple and convenient ,and offer a successful approach to produceing large volumes of the recombinant protein from C. difficile.3. This protein can act as gene engineering recombined antigen,and be used as for CD detection. The results indicated that the ELISA kit will be a beneficial tool to clinical detection of C. difficile toxin B.