The Establishment Of Culture And Identification Of Clostridium Novyi,preparation Of Monoclonal Antibodies And Polyclonal Antibodies Against ? Toxin

Clostridium novyi was once known as Clostridium oedematiens,Edematobacter,Type II malignant edema bacilli,Clostridium megabacterium and Clostridium buffalo,it is the causative agent of black diease.It widely exists in nature,especially in soil.After sheep eat forage polluted by spores,spores reach the liver from gastrointestinal wall through an unknown pathway to cause disease.Sheep and cow can be infected.The bacteria can cause disease in sheep over one year old,while sheep aged 2 to 4 years old and well-nourished are more susceptible to the disease.Goats can also suffer from this disease,cattle are only occasionally infected.Black diease is acute,and death is fast.Livestock often die without showing obvious symptoms.Some cases can be delayed for 1-2 days,but no more than 3 days,which brings huge losses to the animal husbandry.Therefore,there is an urgent need to establish a serological detection technique for rapid identification of Clostridium novyi in sheep,such as ELISA diagnostic methods.Clostridium novyi is a strict anaerobic bacterium,and its growth and reproduction have high nutritional requirements and environmental requirements,and common media cannot meet its needs.Therefore,the objectives of this study are as follows: to synthesize new media and obtain high-density bacterial cultures to meet the needs of laboratory or vaccine production;to prepare natural toxins,monoclonal antibodies and polyclonal antibodies for the preparation of new diagnostic kits such as ELISA kits;to obtain monoclonal antibodies and polyclonal antibodies by using recombinant proteins instead of natural toxins when natural toxins are difficult to obtain;to prepare monoclonal antibodies and polyclonal antibodies against Clostridium novyi recombinant protein for lying a foundation for rapid diagnosis in sheep and construction of ELISA diagnostic methods.The main tasks of this study include the following four aspects: exploring a new culture medium for Clostridium novyi;designing suitable primers for PCR identification of Clostridium novyi;SDS-PAGE identification of natural toxins;and preparing monoclonal antibodies and polyclonal antibodies using recombinant Clostridium novyi toxin?toxin is the main lethal toxin produced by Clostridium novyi type A and B.Therefore,this study first selected the appropriate target fragment,and then synthesized the target fragment after codon optimization.After prokaryotic expression and purification with nickel column,a relatively pure recombinant Clostridium novyi ? toxin was obtained.The purity of the recombinant protein was detected by polyacrylamide gel electrophoresis(SDS-PAGE).The recombinant protein purified by this method plays an important role in the preparation of monoclonal antibodies and polyclonal antibodies.In this study,the purified recombinant ? toxin protein was used to immunize BALB/c mice aged 6-8 weeks.After 3 days of intensive immunization,spleen cells of immunized mice were harvested and fused with myeloma cell F0.The fusion cells were screened by indirect ELISA and two hybridoma cell lines secreting alpha toxin recombinant protein of Clostridium novyi were successfully obtained by limited dilution subcloning.In this study,positive hybridoma cells with good growth status were injected into the abdominal cavity of mice,and ascites were collected after abdominal natural enlargement of mice.The ascites collected were purified by octanoic acid-ammonium sulfate purification method to obtain monoclonal antibodies against Clostridium novyi ? recombinant protein.In this paper,two hybridoma cells targeting Clostridium novyi ? toxin recombinant protein were successfully prepared,named 1978CT716.15.65 and 1978CT142.38.7,respectively.The titers of antibodies secreted by the two hybridoma cells were determined by indirect ELISA.The purified ascites antibody titers could reach 1:512000.Western Blot analysis showed that both monoclonal antibodies could specifically bind to the recombinant protein.In view of the advantages of monoclonal antibodies,such as single biological activity,relatively high purity and strong antigen binding specificity,we can use monoclonal antibodies to rapidly detect and treat diseases.The monoclonal antibody against recombinant protein of Clostridium novyi ? toxin was prepared by polyacrylamide gel electrophoresis(SDS-PAGE)and Western blot analysis.The purity and reactivity of the monoclonal antibody were further verified.This study laid a foundation for the construction of rapid detectiontechnology and detection methods for Clostridium novyi.