Sequence Analysis For The Complete Provial Genome Of Endogenous ALV/SD0501 From SPF Chicken And Construction Of Infectious Clone Of PSD0501

Avian Leukosis (AL) is caused by the avian leukosis virus in poultry in some of hematopoietic cell hyperplasia composition consisting mainly of various infectious diseases and cancer, with poultry broiler lymphocytic leukemia and the myeloid Leukemia more common. So far, A～J, ten subgroups ALVs have been isolated and identified. Thereinto, subgroup A, B, C, D, E and J were isolated from chicken. Besides subgroup E, endogenous virus contain endogenous virus site, medium repeart sequence EAV(endogenous avian virus), ART-CH(Avian retrotransposen which rooted from chicken genome), and high repeat sequence CR1(Chicken Repeat 1). Chicken in the endogenous retrovirus-like elements are eukaryotes there are a lot of anti-retroviral elements typical example, generally believe that E subsets ALV representatives from the cells mobile genetic elements (transposon) anti-retroviral. The evolution stage, while other components were considered to be of exogenous anti-retroviral degradation of the virus before the body, the loss due to mutation of the virus infection have the ability. The existence of these components is a lot of practical significance on the subject.ALV to human health there may be a potential threat. In recent years, ALV with other immunosuppressive virus infection become increasingly serious, resulting in flocks of immune suppression and cancer incidence rate increased year by year, because in general ALV widespread in chickens, caused by immunosuppression to the poultry industry losses Incalculable.and ALV direct impact on the poultry eggs (embryos) source or sources of chicken cells in medical research and the development of biological products. Therefore, control and eradication of the disease is an important and urgent task.The reference to endogenous ALV-related sequence, synthesized the four pairs of detection primer for the SPF from poultry flocks in the detection of endogenous leukemia virus gene fragments. Will collect the different batches of different manufacturers SPF 9-11 day-old chick embryos to hatch, the conventional preparation of CEF, 5% CO2, 37℃temperature, CEF cells extracted DNA, as a template of PCR amplification. PCR test products results with Dot-blot for validation. The results showed that PCR amplification are specific amplification. SPF chickens from different regions of the positive rate of statistical results, the endogenous ALV carrying a higher rate(gag,29/46;pol,27/46;env,24/46;LTR,31/46), of which the LTR fragment are carrying the highest rate, and different chicken farm show larger difference.Eight continuous and overlapping fragments were amplified from one DNA sample with 8 pairs of primers according to published sequences, then cloned into the TA vector and sequenced. The complete sequence of the whole genome of ALV strain SD0501 was established and analyzed with DNAstar software. Comparisons of SD0501 sequence with that of other representative endogenous avian virus strains demonstrated that the genomes of ALV were relatively conservative, the nucleotide identity of all the strains was over 99.1%, and env- gene was over 98.5%.However, a low identity was demonstrated among the representative strains of different subgroups, especially, env- gene showed obvious difference, the corresponding identity was as low as 56.3%～91.5%.The completed the sequencing of the ALV/SD0501 env gene sequences as a template, the design of primers for PCR amplification, digestion, and ligates into the pGEX-6P-1 vector, and transform into BL21 strain. After the preliminary identification, verification by the correct sequence, IPTG induced expression; SDS-PAGE analysis showed that gene has been relatively efficient expression. Cut plastic recycling the original purpose of protein expression, injact mice. In mice, the preparation of anti-serum. For the future more in-depth research work has laid a necessary foundation.The completed the sequencing of the endogenous ALV/SD0501 genome-wide cDNA template for using PCR method sub-paragraph 5 of the amplified ALV/SD0501 provial virus genome, PCR product of the cloning after the turn, ligate into the vector pUC19, was a virus that is the complete genome ALV/SD0501 before the recombinant, named ALV/pSD0501. Purified plasmid transfected CEF, IFA test showed that CEF ALV/SD0501env are against the gene expression that was infected with the cloning of the virus.