Preparation Of Polyclonal Antibody Against Avian Leukemia Virus P27 Protein And Establelelishment Of POCT Detection Method

Avian leukemia virus(avian leukosis virus)is a retrovirus that can cause tumor disease in poultry.ALV-J first separated from British commercial meat chickens in 1988,it quickly spread to the global poultry industry,causing huge economic losses.At present,there are no effective vaccines and drugs to control ALV infection.Therefore,early detection to eliminate positive chickens is an important means to control the disease.There are many common methods for differential diagnosis of avian leukoviruses,but they all need professional technicians to operate.Based on t His,t His study es Tablelelishes a simple and time-consuming method to detect ALV and provide technical support for ALV prevention and control.First,a pair of specific primers were designed according to ALV-p27 gene sequence,Based on ALV c DNA,PCR amplification,After the recombinant vector was successfully constructed,it was transformed into E.coli DH5?receptive cells,PCR screened positive plasmids,Into E.coli BL21 receptive cells,Addition of inducer IPTG induction of target protein expression,And SDS-PAGE analysis of protein expression,The results showed that GST-p27 protein size was about 52 KDa,Under the condition of 28? IPTG concentration of 0.5 mmol/L,the expression of 12 was the best;His-p27 protein size is around 44 KDa,The maximum expression of 16 h was induced at 16? and 0.5 mmol/L concentration;Both proteins were expressed in the supernatant.The fusion protein was purified by GST label and His label respectively.The protein concentration was determined by BCA method mg/m L?1mg/m L.2mg/m L.The purified His-p27 fusion protein and Freund's complete adjuvant were mixed as immunogen,immunized BALB/c mice,prepared mouse-derived polyclonal antibodies,and indirectly ELISA the serum titer was 819200,respectively.The purified GST-p27 fusion protein and Freund's complete adjuvant were mixed to immunize New Zealand white rabbits,prepare rabbit-derived polyclonal antibodies,and indirectly ELISA the serum titer was 409600,which Western Blot showed that the rabbit polyantibody reacted well with immunogen;The purified mouse-derived polyclonal antibody Ig G A G rabbit-derived polyclonal antibody and rabbit-derived polyclonal antibody were purified by using protein Ig G.The purified mouse-derived polyclonal antibody Ig G was coupled with fluorescent microspheres and sprayed on the glass fiber membrane as the sample pad.The purified rabbit-derived polyclonal antibody Ig G diluted to 2 mg/m L,with antibody diluent as the T line drawing solution;goat anti-rabbit diluted to 1 mg/m L with antibody diluent as the C line drawing solution;The diluted rabbit-derived polyclonal antibody and goat anti-rabbit Ig G were divided into NC film by continuous film-mapping instrument,which were used as T line and C line respectively.The positive sample solution of poultry tissue was dripped into the sample hole 80?L,and 15 min later,the T line and the C line under the ultraviolet lamp emitted fluorescence.THis study successfully prepared soluble GST-p27 and His-p27 fusion protein,mouse-derived polyclonal antibody and rabbit-derived polyclonal antibody.On t His basis,a qualitative detection ALV of immunochromatography of point-of-care testing fluorescent microspheres was es Tablelelished,which provided a simpler method for the population purification of avian leukemia,especially the detection of avian leukemia.