| Subgroup J Avian leukosis virus(ALV-J)is widely prevalent in the world and can cause a variety of infectious benign and malignant neoplastic diseases in poultry,causing great economic losses.The host range of ALV-J in China has been expanded from commercial breeding chickens to local breeds of chickens.However,there is no effective vaccine or drug for prevention and control,so purifying breeding chicken farms is the only way to prevent and control.Therefore,the detection and monitoring of avian leukosis virus in progenitor chicken breeding farms and the establishment of a rapid,accurate and effective detection for avian leukosis virus are of great significance.In this study,998 samples of rooster semen were collected from a local progenitor chicken farm in east China.After detection by ALV ELISA antigen detection kit,isolated from DF-1 cells,a strain of ALV was finally isolated and named G4-4.The detection rate was 5.51 %(55/998)and the separation rate was 1.82 %(1/55).The isolate G4-4 was identified as ALV-J by specific primers.The p27 gene sequence of G4-4 is 91.8 % ~ 98.4 % similar to that of subgroup A,B,C,D,E,J and K,among which the similarity between G4-4 and JS09GY3 is as high as 98.4 %.Genetic and evolutionary analysis found that G4-4 and JS09GY3 were divided into the same branch,indicating that the two strains were closely related.The gp85 gene sequence of G4-4 was 46.7% ~ 85.6% similar to that of subgroup A,B,C,D,K and E,and 99.4% similar to that of subgroup J JS09GY3.Genetic and evolutionary analysis showed that the isolates G4-4 were in the same branch as the avian leukosis viruses of subgroups J and E,while the strains of subgroups A,B,C,D and K were in the other branch.In addition,the isolates G4-4 were in the same small branch as JS09GY3 and JS09GY6 strains,indicating a close genetic relationship.In order to prepare the high-immune serum of avian leukosis virus p27 protein,this study screen out several ALV-J p27 epitopes by biological software,then using serine and glycine in tandem,cloned them into P-particle expression vector,named pp354.After western-blot detection,the size of recombinant protein pp354 was about 73 kD.Recombinant protein pp354 in temperature is 25?,IPTG concentration is 0.4 mM,constant temperature oscillation expression 8 h express the highest amount,in this condition,the soluble of pp354 is about 50%.The results of ELISA ALV antigen detection kit showed that pp354 could effectively bind to the p27 antibody and had the antigenicityof p27 protein.Western-blot identification of pp354 with ALV-J positive serum showed obvious bands at 73 kD.The continuous popularity and diffusion of ALV-J require us to establish ALV-J negative progenitor breeding stock,and the development of ALV-J antigen detection kit is the basis for achieving this goal.In view of the defects and deficiencies of current commercial ALV-J detection kits and detection methods,such as expensive and high false positive rate,the establishment of a protein expression platform can express the specific antigen fragments of ALV-J is great help for the establishment of commercial ALV-J detection methods and the purification of ALV-J.In this study,ALV-J isolation and analysis were performed on samples collected from a progenitor chicken farm of a local variety in east China,and P-particle expression vector was used to construct the recombinant protein pp354 with p27 antigenicity,which laid a foundation for the production of specific antibody of ALV-J p27 and the development of domestic commercial ALV p27 antigen ELISA detection kit. |
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