Cloning And Construction Of The Expression Vector Of ALV-J Gp85 Gene From The Egg-type Chickens

Avian leucosis virus subgroup J (ALV-J) belonging to leucosis/sarcoma group of retrovirus was discovered in 1991 by Dr. Payne, and avian leucosis subgroup J produced by ALV-J caused considerable losses of the egg-type chickens industry in many countries. The nature disease of layer chikens has never been reported for more than ten years. In 2002, Xu Binrui, professor of China Agricultural university, first discovered the layer flocks were naturally infected by ALV-J. The study indicated that the morality of the egg-type cickens with avian leucosis subgroup J was as high as 10% and the production was 60%-70%, so the disease badly influenced the production. In the sudy, a pair of specific primers was designed according to the gene gp85 of the ALV-J HPRS-103 prototype stain. RNA was extracted from the illed material identified by ALV-J infection from the egg-type chickens. The complete gene gp85 of ALV-J was produced by RT-PCR and cloned into pGEM-T easy vector. After the gene was sequenced and compared with the gene gp85 of the HPRS-103 prototype stain and other strains of ALV-J, the nucleotide identidy to HPRS-103 was 93.7%, and the nucleotide identidy to other strains was between 92.1% and 94.3% (88.7% for the strain AF88); The identity of the deduced amino acid gp85 to HPRS-103 was 89.3%, and the identity of gp85 amino acid to other strains was between 87.3% and 90.9%. It indicated that the gp85 of ALV-J from the egg-type mutated, and the gp85 of ALV-J from the egg-type had near relationship with the strain HN0001.The subgroup specific of ALV-J was determined by the protein gp85 coded by ENV gene. To obtain the recombinant baculovirus with the gene gp85 of ALV-J, the pt-85 and the transfer plasmid pMelBac C were digested by Pst I and EcoR I . The gene gp85 of ALV-J was cloned into the digested thansfer plansmid pMelBac C, so the recombinant thansfer plasmid pMelBac-gp85 was obtained. After cotransfecting the cultured sf9 cell by pMelBac-gp85 and the linearized baculovirus gene DNA, the recombinant baculovirus identified by PCR was harvested. The study lay foundation for expressing from the sf9 cell the special diagnostic for the egg-type chickens, and has important potentially applied value.