Detection Of Clostridium Dfficile Toxins And Expression Of Toxin B Receptor-Binding Domain

Objective:(1) To investigate clostridium difficile infection of patients with diarrhea in Xiangya hospital by screening clostridium difficile in stool specimens and comparing detection rate in differert periods.(2) To construct a prokaryotic expresstion vector of Clostridium difficile toxin B receptor-binding domain and induce expression of the target protein.Methods:(1)106stool specimens of hospitalized patients with diarrhea during February to December in2009and during April to July in2011were collected, cultured in anaerobic environment and identified by API.Those positive culture isolates were detected toxin A、toxin B and binary toxin gene by PCR; Enzyme-linked fluorescence immunoassay was used for toxin A/B detection.(2) The genomic DNA of clostridium difficile was extracted, and then the gene fragments of clostridium difficile toxin B receptor-binding domain was amplified by PCR.The prokaryotic expression vector pGEX-4T-1-CDB3was constructed and the expression of target protein was induced by IPTG.The protein expression was verified by SDS-PAGE and Western blot.Results:(1) Of106specimens, the positive culture rate was15.09%(16/106) and all those positive culture strains were positive for toxin A and B by PCR and negative for binary toxin by PCR. The positive rate was12.26%(13/106) by directily detecting toxin A/B. The detection rates of clostridium difficile in two periods during2009and2011were compared. The positive culture rate were22.8%(13/57)、6.12%(3/49) respectivly, the difference was statistically significant; The amplification positive rates of toxins by PCR were22.8%(13/57)、6.12%(3/49) respectivly, the difference was statistically significant; The detection positive rates of toxin A、B were17.54%(10/57)、6.12%(3/49) respectivly, the difference was not statistically significant. From clinical datas of positive patients with clostridium difficile infection, we found that they used one or more antibiotics of cephalosporins、quinolones、 carbapenems、broad-spectrum penicillin and clindamycin during hospitalization.(2) The results of PCR、restriction enzyme digestion and gene sequencing showed that the prokaryotic expression vector pGEX-4T-1-CDB3was constructed successfully and the target protein about97KD was induced successfully,too.Conclution:(1) Clostridium diffilile-associated diarrhea is more serious in Xiangya hospital. The use of antibiotics is an important inducing factor to clostridium difficile infection.(2) The prokaryotic expression vector pGEX-4T-1-CDB3is constructed successfully and the target protein is expressed massly.