Prokaryotic Expression Of Killer Yeast(Killer Yeast) And Its Effect On The Treatment Of Chinese Mitten Crab Milk Disease

Chinese mitten crab(Eriocheir sinensis)is a crustacean with a wide distribution,high nutritional value and significant economic benefits.It is a popular aquatic product and one of the important economically farmed aquatic animals.In 2021,a large-scale epidemic appeared in Tonghua Aquaculture Base of Jilin Province,with a mortality rate of more than 80%.The pathogen was isolated and identified as Metschnikowia bicuspidata(Metschnikowia bicuspidata for short: WCY).Because the main diseased organ,the liver and pancreas,was milky white,it was also known as "milk disease",seriously endangers the development of river crab aquaculture.At present,there is no specific drug for the prevention and treatment of this disease,so it is an urgent task to explore new convenient and reliable treatment methods at this stage.In this experiment,Eriocheir sinensis was taken as the research object.Disease materials were collected,and appropriate medium was selected for isolation,culture,microscopic observation,and biochemical test identification.At the same time,the length of the ITS gene amplification sequence of the pathogenic yeast strain was407 bp.The results of the amplified sequence were submitted to the gene bank for comparison to construct a developmental tree with a similarity of 96%.The results showed that the pathogen was successfully isolated as Metschnikowia bicuspidata.Studies have shown that the marine killing yeast YF07B has a good killing effect on Metschnikowia bicuspidata.The main functional protein is the marine killing yeast YF07B protein.According to the designed primers,gene cloning and identification are carried out,and prokaryotic recombinant expression plasmids are constructed for bioinformatics analysis.The BL21 expression strain was selected to explore the optimal expression induced by different time,temperature and IPTG.The protein was purified by His-tagged Ni ion chromatography column and refolded by urea gradient dialysis.The results showed that the CDS region of YF07B successfully cloned was1297bp;Prokaryotic expression showed that when the BL21 strain was induced at 37℃ for 8 h and the induction concentration of IPTG was 0.75 m M,the molecular weight of the fusion protein was 49.7 k Da,and the optimal elution concentration after protein purification was 250 m M imidazole.Refolding by urea denaturation followed by gradient dialysis has a native conformation.After the recombinant protein was purified and its concentration was determined,four sets of gradients were designed to be 200 μg/ml,400 μg/ml,700 μg/ml,and 1000μg/ml.The in vitro antibacterial zone test of Metschnikowia bicuspidata showed that700μg/ml had the best antibacterial effect.The best inhibitory concentration was used to conduct the experiment of injecting Chinese mitten crabs(adult crabs and buckling crabs)to treat the disease(has been challenged),respectively PBS group(negative),challenge group WCY,YF07B-700μg/ml+WCY group The results showed that the YF07B-700μg/ml+WCY group had the best tissue protection after collecting gills and hepatopancreas pathological sections after 7 days.In summary,this experiment successfully isolated the pathogenic strain,and constructed the prokaryotic expression plasmid of the marine killing yeast YF07 B,and determined the optimal induction expression conditions and the best antibacterial in vitro protein concentration of the marine killing yeast YF07B;After attacking the virus,100% of the disease occurs,and the recombinant protein is injected for treatment.The survival rate test shows that the survival rate of buckling crabs is 60%,and the survival rate of adult crabs is 66%.The optimum dosage of recombinant protein for treating this disease is 700μg/ml.