Isolation And Identification Of Sheep-origin Clostridium Perfringens And Establishment Of Indirect Elisa Detection Method In Xinjiang

Clostridium perfringens is a kind of gram-positive bacilli that parasites in many kinds of animals,soil,sewage and other environments,and it can infect humans and many kinds of animals.It produces a variety of toxin proteins in the process of growth and reproduction,those toxin proteins can cause a series of immune reactions,resulting in pathological changes in many organs and tissues of the body.Alp Ha toxins are secreted by all types of Clostridium perfringens,and it is the most important lethal toxin.The gene encoding alp Ha toxin protein is located on the chromosome,so the alp Ha gene is relatively conserved among all types.Clostridium perfringens type A secretes the most alp Ha toxin protein.Clostridium perfringens is prevalent in all countries in the world,but the prevalent bacterial types are very different.vaccines and antibiotics are widely used,the bacteria has a very serious drug resistance,so it is particularly important to establish different detection and epidemic prevention mechanisms in different regions.The main content of this experiment:(1)Before the experiment,the spleen,kidney,heart,jejunum,ileum and intestinal contents of sick and dead sheep were collected from a sheep farm in Xinjiang as samples of isolated strains.After the samples were treated in the laboratory,they were isolated and purified.When cultured in liquid medium,a large amount of gas is produced in the paraffin oil layer.The purified bacteria were examined by Gram staining,and the purplish red bacillus was found under the oil mirror.Obvious bands appeared during Bacteria 16S r RNA PCR amplification,and the sequence of the amplified products was found to be 99.4%similar to that of Clostridium perfringens after Blast in NCBI.The primers of?,?,?and permethrin were designed,and the?toxin bands were amplified from the genome of the isolated strain,indicating that the strain was Clostridium perfringens type A.(2)The recombinant plasmid p ET30a-?was constructed and transformed into Escherichia coli BL21(DE₃).The optimal induction conditions were selected and a large number of soluble recombinant proteins were obtained.The purity and reactivity of the proteins were analyzed by SDS-PAGE and Western immunoblotting after purification by Ni column.The results showed that the effect of 0.5 m M IPTG inducer was the best when induced at 37?for 5 h,and the protein size detected by SDS-PAGE was 58 KDa,which was consistent with the predicted value.(3)Using the purified protein coated with enzyme-labeled plate,the optimum coating concentration was 4.0?g/m L,the serum was diluted200 times,and the second antibody was diluted at 1:30 000 times.This method was used to detect870 serum in Xinjiang,and the positive rate was 90.11%.