| Avian leukosis（Avian Leukosis,AL）is a tumorous disease that is caused by avian leukosis virus（Avian leukosis virus,ALV）and is more harmful to poultry.It spreads vertically and horizontally.The incidence of the disease is 3%?5%,and the disease course is long,which seriously increases the losses of poultry breeding industry.There are currently no effective drugs and vaccines for the disease.Therefore,this study carried out an epidemiological investigation on AL in Hebei and the prokaryotic and eukaryotic expression of the gp85 gene in the main epidemic subgroup,and conducted a preliminary study on its immune effect.The research contents and results are as follows:1.Analysis on the carrying situation,epidemic subpopulation and molecular characteristics of ALV p27 in breeders in Hebei ProvinceIn this study,the egg and anal swab samples were collected from the breeding farms in Hebei area（including 3496 samples from 7 chicken farms in 6 regions,including 2533 local breeds,159 introduced breeds and 804 cultivated breeds）,and the p27 antigen ELISA kit was used to detect the carrying status of ALV p27.The results showed that in 2018-2019,the positive rate of breeder farms in Hebei was85.71%,the average positive rate of individuals was 13.79%,the positive rate of local breeds was 18.91%,the positive rate of introduced breeds was 1.26%,and the positive rate of cultivated varieties was only 0.12%;PCR method was used to detect the main epidemic subgroups in 2018-2019,and the results showed that J subgroup was detected The detection rate was 13.79%,the E subgroup detection rate was13.70%,the A subgroup detection rate was 0.09%,and the K subgroup detection rate was 2.23%;sequencing analysis of the main epidemic subgroup ALV-J gp85 genes,The homology between the isolate and the reference strain was 98.50%100.00%,and it mainly mutated around HPRS-103.2.ALV-J gp85 prokaryotic expression and immune protection evaluationIn order to clone the ALV gp85 gene,design primers,construct pET-32a-gp85expression vector,transform the recombinant vector into BL21 E.coli engineering bacteria,successfully express the target protein,the protein molecular weight is about54 kDa,Western blot method proved the protein Has good immunogenicity.The prokaryotic expressed protein was immunized with 100μg,150μg,200μg/dose.Blood was collected on the 7th,14th,21st,and 28th day after immunization,and the specific gp85 antibody level was detected by ELISA.Both were significantly higher than the control group（P<0.05）.The antibody level in each phase of the 150μg immunization group was the highest,and it reached a significant level compared with the 100μg and 200μg immunization groups（P<0.05）.On the 35th day,he was challenged with 3 TCID50,and his ELISA p27 antigen kit was used to detect its antigen carrying status,and then his protection rate was analyzed.The results showed that on the 21st day of challenge,the 100μg immunization group had the lowest protection,and the 150μg immunization group had the second protection,The protective power of 200μg immunization group was 26.67%.3.Expression of ALV-J gp85 eukaryotic protein in 293T cells and evaluation of its immune effectIn order to obtain the gp85 protein expressed by 293T cells,in this study,the gp85 gene was introduced into the expression plasmid pEGFP-N1 to obtain the recombinant plasmid pEGFP-N1-gp85,transfected into 293T cells,and identified by inverted fluorescence microscope and Western blot to obtain gp85 eukaryotic protein,The molecular mass is about 60 kDa.The pEGFP-N1-gp85 eukaryotic plasmid was immunized at 100μg,150μg and 200μg/dose.Blood was collected on the 7th,14th,21st and 28th days after immunization.The specific gp85 antibody level was detected by ELISA and T lymphocytes were detected by MTT Conversion rate,challenge with3 TCID500 on day 35d,detect ALV p27 antigen carrying status,and then analyze its protection rate.The results showed that the serum gp85 antibody level and the peripheral blood T lymphocyte transformation rate of each stage of the immunized groups were significantly higher than the control group（P<0.05）.The antibody level of the 150μg immunized group was the highest,compared with the 100μg and 200μg immunized groups.All reached a significant level（P<0.05）.On the 21st day of the challenge test,the protection of the 100μg immunization group was the lowest,the protection of the 200μg immunization group was second,and the protection of the150μg immunization group was 53.33%.It shows that the suitable immunization dose of this eukaryotic plasmid is 150μg. |
