The Expression Of Avian Leulosis Virus P27 Gene In Pichia Pastoris And The Establishment And Application Of Indirect Elisa To Avian Leulosis

Avian leukosis is a contagious neoplastic disease caused by avian leukosis virus(ALV).Avian leukosis could be horizontal and vertical transmitted,but there are still no available vaccines and effective drugs,and it has been spread widely in countries all over the world and has brought serious harm.Avian leukosis is one of the priority disease prevention and control diseases list in the national medium and long-term animal disease prevention and control plan among 2012 to 2020.The main ways to control the spread of the virus are population purification to reduce the chance of infection.Therefore,it is very necessary to establish a fast and effective detection method for avian leukosis,that will be good for clinical diagnosis,purification and monitoring.The p27 protein is a group-specific antigen encoded by the avian leukosis genome,and it is the main component of the ALV core capsid protein.There is a lot of viral antigenic sites on p27 protein,which are often the first considering in detecting antibodies.In this study,we use yeast expression system to express p27 gene in avian leukosis,and the ELISA detection method was established used the p27 protein as package antigen,it will provid a theoretical basis for the large-scale production of p27 protein and the further development of the detection kit to avian leukosis.Expression of avian leukosis virus p27 gene in Pichia pastoris According to the published sequence of p27 gene in the full sequence of avian leukosis gene published in Genebank,specific primers that containing EcoR I and Not I restriction sites were designed using Primer Premeir 5.0 software,using ALV RNA as template,The target gene was amplified by RT-PCR;the p27 gene was ligated into the pMD18-T vector to obtain the cloning plasmid pMD18-T-p27,then the yeast expression plasmid pPIC9K and pMD-p27 plasmid were digested,and ligated,the yeast expression plasmid pPIC9K-p27 was obtained by DNA recombination technology.The yeast expression plasmid pPIC9K-p27 was transfected into Pichia pastoris cell GS115 by electroporation,then was induced to expression.The best inducing conditions were that when the induction time was 24h,the methanol concentration was 1%,pH was 7.0 and the temperature was 28?,the p27 protein expression was best(26.2?g/mL).The establishment of an indirect ELISA method to avian leukosis based on p27 protein The reaction conditions were screened by the square matrix titration test,the optimum reaction conditions for the indirect ELISA method were as follows:the coating concentration of p27 protein is 2.5 ?g/mL.5%skim milk powder closure time is 1.5h,Serum dilution is 1:200 serum hatching time is 1.5 hours.HRP-labeled rabbit anti-chicken IgY dilution is 1:5 000,and 37?constant temperature culture 1h.Coloring time is 12 min.The critical value is 0.1979.The optimized ELISA detection method was also used to detect the infectious IBDV,MDV,NDV,IBV,fowl reticuloen dodermis virus,avian leukosis virus positive sera and negative sera,it shows good specificity.The optimized ELISA detection method was used to detect 80 suspected avian leukemic chicken serums and comparing with the commercialized ELISA kit,the coincidence rate was 96.92%,the positive rate was higher and the sensitivity was better.The intra batch and interbatch repeated tests were carried out,the coefficient of variation was less than 8%using the optimized ELISA detection method,it showed high repeatability.314 suspected ALV chicken serum samples were detected by the optimized ELISA detection method,the positive number was 236,the positive rate was 75.15%,so the optimized ELISA detection method is suitable for clinical application of avian leukosis.