Isolation And Characterization Of JL-2 Strain Of Avian Leukosis Virus Subgroup J And Its Gp 85 Gene Cloning And Expression In Escherichia Coli

Avian leukosis virus Subgroup J (ALV-J) is a retrovirus and was identified in 1990s', which infects meat-type chickens and causes serious economic losses. Envelope glycoprotein of ALV-J determines the host range of virus infection and cross-neutralization patterns. The SU, which is encoded by gp85 of env gene, determines the subgroup of the ALV-J.In this experiment, using a pair of primers reported before, a strain of new virus was detected by RT-PCR in a flock of commercial broilers in Jin Lin province and designated JL-2. CEF cells were used for the replication and isolation of the virus. According to the reported complete nucleotide sequences of ALV-J in GenBank, a pair of primers was designed with Oligo 6.0 to amplify a segment about 1700bp using the template extracted from the CEF infected with this isolate. This segment was cloned into pMD18-T vector and sequenced .The results of sequencing showed that the complete gp 85 gene of JL-2 isolate was 924bp, which can be translated into 308 amino acid. And the molecular weight is 34Ku. Computer analysis results showed that, in comparison with the gp85 gene of HPRS-103, the homology of JL-2 isolate to HPRS-103 strains is about 91.8%, and the deduced amino acid sequences were 86%. According to the nucleotide sequences of vector pGEX-6P-1, we designed another pair of primers to amplify the gp85 gene from CEF .The target gene was inserted into the vector and constructed the recombinant expression vector, pGEX-6P-1/gp85. This recombinant plasmid was transformed into E.coli BL21 (DE3) and induced with IPTG. The result showed that the gp85 gene had been well expressed, the target protein is 31.8% compared to the total bacterial protein of the BL21. The expressed product was analyzed by western-blot. The result showed that gp85 fusion proteins have a positive reaction with anti-JL-2 serum.In this experiment, the JL-2 strain was first identified. Then the gp85 gene of JL-2 was cloned and compared with other strains isolated around the world. Based on this, the SU protein was well expressed in prokaryotic system. This study would be worth in ALV-J identification, molecular epidemiology investigation, research of diagnostic reagent and genetic engineering vaccine.Candidate: Song Suquan Speciality: Preventive Veterinary Science Supervisor: Prof.Weiping Prof.Wang Xiaomei...