| Since the late1980s,avian leucosis virus subgroup J (ALV-J) has become a kind of important diseases, threating the development of the meat-type poultry industry wordwide.The significant economic loss caused in the broiler industry by ALV-J is often due to mortality of meat-type chickens, tumor production, weight gain decrease and immunosuppression. In recent years, this disease showed development in our country and spreaded its tumorigenesis host range from meat-type chickens to commercial layer chickens and Chinese local flocks. Because of the lacking of valid vaccine and drugs, ALV-J can't be controlled effectively. The only means to control the disease is to eliminate the ALV-J antibody positive chickens from flocks. Therefore, it is very important to develop some economic and effective detection method for detecting ALV-J antibodies.In this study, ALV-J gp85 was amplified and expressed by the prokaryotic expression system, and one ELISA method based on the purified GP85 protein was developed and applied in field test. The process briefly described as follows.Firstly, according to the referenced sequence of ALV-J (HPRS103 strain), a pair of primer was designed to amplify a segment about 924bp of gp85, then the gene was cloned into pET-30a vactor (named pET-gp85), and transformed into E.coli BL21(DE3) for GP85 expression. The results of SDS-PAGE and western blot showed that gp85 gene was efficiently expressed and had well immunogenicity. FinAlly, a convenient ELISA method was developed based on the recombinant protein GP85, and the specific antibody against ALV-J could be detected easily from the infected chickens. The results of field test showed that this method had the following advantages,for example, specific good, convenience, repeatability and inexpensive et al. So it is suitable for large scale application for chicken farms.To elucidate the molecular mechanism of vertical transmission of ALV-J and lay the foundation for developing valid ALV-J vaccine, we concern study on the structure and function of the long terminal reapeat region (LTR) of ALV-J. Firstly, the LTR of ALV-J was amplified and cloned into pGL3-Basic vector(named pGL3-LTR), and a series of deletion mutants plasmids were constructed. Then, these plasmids were transfected into the DF-1 cells, respectively.48h later, the expression of the luminescence was detected and analyzed.The results showed that the LTR of ALV-J has well function of driving the transcription of the following reporter gene, U3 zone responsible for the function of LTR, if it was deleted, LTR would almost lose its function. On the contrary,if U5 zone was moved, the function of LTR would be improved, indicated that may be there are some negative control elements in the zone.In a word, these results for future exploring viral replication, transcription mechanism and control of ALV-J will have the important significance. |
