Development And Characterization Of Monoclonal Antibodies To Subgroup A Avian Leukosis Virus And The Study Of Interaction In Chickens Co-infected With Different Subgroup Of Avian Leukosis Virus

Avian leukosis is a variety of tumor diseases caused by avian leukosis virus and avian sarcoma virus. In last ten years, leukemia / hemangioma cases increased significantly in layers breeder industry again. avian leukemia become a serious disease,which does severely harm to layer industry in China. Isolation and identification of virus showed that the vascular tumors was causednot only by the subgroup J avian leukemia virusbut also by the subgroup A and B. In order to greatly enhance the diagnosis and treatment of clinical diseases, the monoclonal antibodies (Mabs) to ALV–A were prepared and characterization of monoclonal antibodies to subgroup A avian leukosis virus were tudied in this study. To understand the dynamics of viremia, virus shedding and antibody responses in chickens inoculated with ALV-A and ALV-J and provide essential epidemiological data for prevention and eradicationo of ALV-A and ALV-J infection.1. Cloning and expression of gp85 gene of ALV-A-SDAU09C1The env-gp85 gene of ALV-A-SDAU09C1(GenBank:HM452339)was amplified by PCR and then subcloned into the prokaryotic expressing vector pET32a and recombinant vector pET32a-SDAU09C1-gp85 was further transformed into Escherichia coli strain BL21 for expression under the induction of IPTG.. After IPTG induction, there was a new protein band about Mr 53kD on SDS-PAGE. Western blot analysis also showed that a protein band with a molecular weight of about 53 kD was detected. Then the protein were purified and stored.2. Development and characterization of monoclonal antibodies to subgroup A avian leukosis virus2.1 Establishment of the Indirect ELISAAn alternative indirect enzyme- linked immunosorbent assay (ELISA) for detection of ALV antibody was developed using a truncated envelop glycoprotein(env)-gp85 protein of ALV produced in Escherichia coli.The results show that the appropriate testing condition is as follows: 2μg/ml antigen used to coat ELISA plate with PBS Buffe(rpH7.4), 1:1000 of sera as detecting samples, The working concentration of HRP-labelled sheep antichicken IgG was 1:2000 and the reaction time was 10 min. Cross-reactivity assay showed that this assay was not cross-react with REV,NDV,IBDVet al, this demonstrated it has good specifity. It showed that the ELISA had good repeativity, high specifity and high sensitivity.2.2 Development of monoclonal antibodies to subgroup A avian leukosis virusSix adult female Balb/c mice, 8–10 weeks old, were subcutaneously injected with purified recombinant myocilinemulsified in a 1:1 ratio with complete Freund's adjuvant. Two booster doses, again in 50% emulsion with Freund's incomplete adjuvant, were given at 2 weeks intervals. The mouse with the highest antibody titer tested by ELISA was boosted intraperitoneally with 100μg protein without adjuvant 3 days before the cell fusion. Feeder layer cells were prepared 1 day prior to fusion. Splenocytes from mice with the highest ELISA antibody titers were fused with murine myeloma cells Sp2/0 following standard procedures. Three strains (A6D1,A5C1,A4C8) were selected for subcloning. For the production of MAbs, BALB/c mice (4-6 weeks old) were injected intraperitoneally with 106 hybridoma cells per mouse. Ten days later, the ascitic fluid was further purified by using protein A agarose columns (Bio-Rad Laboratories). The concentration of three Mabs was : A6D1,1.575mg/ml;A5C1,0.903 mg/ml;A4C8,0.758 mg/ml. The purified ascitic fluids were used for further characterization.2.3 Characterization of monoclonal antibodies to subgroup A avian leukosis virus2.3.1 Titera of obtained McAbsThe titera of obtained McAbs from the mouse ascetic fluid were 210 (A6D1),28 (A5C1),27(A4C8), respectively, in indirect ELISA.2.3.2 Antibody secreting stabilities of hybridoma cell strainsThree primary hybridoma cell strains secreting McAb were resuscitated and cloned. After the cloning positive ratereached 100%, the 3 strains were passaged for 3, 6,9,12 months, and the antibody secreting stabilities of them were monitored by indirect ELISA. The results showed that the property of three strains secreting McAb was stable after long culture and cry preservation.2.3.3 Western blot analysis The purified protein was analysed by means of SDS polyacrylamide gel electrophoresis (SDS PAGE) and followed by Western blotting. The purified protein with a band of molecular mass 53 kDa were got in SDS-PAGE, and it bind specifically to McAbs in Western-blot and ELISA assay. The Western blot analysis showed the 3 strains McAbs were all specific to ALV env gp 85 protein.2.3.4 IFA analysisThree strains of monoclonal antibodies (McAbs) against ALV-A were obtained by using hybridoma technique and their characteristics were studied by IFA . The results showed that three MAbs (A5C1 and A4C8) reacted with ALV-A but not with subgroups B, or J of ALV. MAb A6D1 reacted with subgroups A and B of ALV but not with ALV-J.3. Dynamics of viremia and antibody responses in chickens Co-infected with ALV-A and ALV-JIn order to discuss the interference of viremia and antibody caused by avian leucosis virus of subgroup A(ALV-A) and avian leucosis virus of subgroup J(ALV-J), 200 SPF chickens are divided into 4 groups randomly at 1d: one is injected by 0.2ml cell culture supernatant of ALV-A(containing 103.01 TCID50 of 0.1ml infected cell culture supernatant), the other is ALV-J, the third is ALV-A+ALV-J and the fourth is Hank's liquid as negative control at 2d separately; the chickens are immuned as usual; on 14d, 21d, 35d and 49d, 30 chickens are taken out from these groups and the serial numbers are given contemporarily. The fresh blood containing anticoagulant are collected without any bacterias from 6 of the 30 chickens to test the viremia; the other blood are collected in the normal way to test antibodies; the viremia are detected by IFA and the level of antibody are detected by ELISA.3.1 Dynamics of viremia in chickens Co-infected with ALV-A and ALV-JViremia was detected from the 2nd week after inoculation with ALV-A at 2-day-old and was increasing gradually from the 3nd week to 7nd week; The viremia was 3.72 PFU/ml at 7-week-old(reach to peak and was decreased at 8week-old (3.01 PFU/ml);Viremia was detected from the 1nd week after inoculation with ALV-A at 2-day-old and was increasing gradually from the 3nd week to 8nd week; The viremia was 3.71 PFU/ml at 8-week-old; The coinfection of ALV-A can interfere the dynamic viremia level of ALV-J obviously(P<0.05)on 2w, but the coinfection of ALV-J can't interfere the dynamic viremia level of ALV-A(P>0.05).3.2 Dynamics of antibody responses in chickens Co-infected with ALV-J3.2.1 Iinfluence of coinfection with ALV-J on antibody to ALV-AThe antibody is 0 in ALV-A group and ALV-A+ALV-J group on 2w; Only some chicken produced specific antibodies to ALV-A on 3w, the antibody positive rates of in ALV-A group was significantly lower than that in ALV-A+ALV-J group (P<0.05), increasing with age. The antibody positive rates of in ALV-A group and ALV-A+ALV-J group was basically the same on 5w and 7w. The results showed that coinfection with ALV-J has no significantly influence on ALV-A antibody produce.3.2.2 Iinfluence of coinfection with ALV-A on antibody to ALV-JThe antibody positive rates of was 0 in ALV-J group and ALV-A+ALV-J group on 2w and 3w ; The antibody also was 0 ALV-A+ALV-J group on 5w and 7w; the antibody positive rates of in ALV-J group only was 20.00% on on 5w and 13.33% on 7w. The results showed that the emerge of antibody of ALV-J was inhibitated because of coinfection of ALV-A, although the coinfection of ALV-J don't affect the emerge of antibody of ALV-A.3.2.3 Interaction of Virema and Antibody in Chickens Coinfected with ALV-A and ALV-JCompared the dynamic viremia level and the level of antibody at the same time, the rate of coexistance of ALV and its antibody positive rates of is 0 in ALV group and ALV-A+ALV-J group on 2w and there is coexistance of ALV and its antibody positive rates of in ALV-A(33.33%) group and ALV-A+ALV-J group on 3w ( 6.25%) ; the antibody positive rates of ALV-A +ALV-J group was 50.00% on 5w, this phenomena lasts until 7w; the antibody positive rates of ALV-A group was 66.67%% on 5w, and 50% on 7w.The antibody in ALV-J group ALV-A +ALV-J group was 6.25% on 5w and 33.33% on 7w. The results showed that there isn't coexistance of ALV-J and its antibody at all in the whole test processa nd the rate declines obviously after coinfected ALV-A .