Inhibition Of Avian Leukosis Virus Subgroup J Replication By RNA Interference Base On Targeting Gp37 Gene

Avian Leukosis (avian leukosis, AL), is defined as alpha-retroviral retrovirus avian leukosis virus in poultry, it is a transmissible neoplastic disease which can induce hematopoietic cells hyperplasia.ALV can be divided into A-J 10 subgroups, in which subgroup J avian leukosis (ALV-J) is endogenous recombinate exogenous ALV, since the group of poultry has been found to cause significant economic losses. ALV-J mainly caused myeloid leukemia in recent years appeared new clinical symptom, such as hemangioma tissue sarcomas, neural tumors, which leads to ALV-J-type of disease is more complicated. In order to alleviate the harm to the poultry industry and find a suitable antiviral therapy requiring urgent solution.According to antiretroviral infect cell us the mechanism of membrane fusion, this study used RNA interference (RNAi) technology, specifically block the key gene expression of membrane fusion in order to cut off the way of virus entering the host cell to achieve the purpose of anti-virus.After ALV-J binding to receptor site ,the conformational of gp37 gene-encoded transmembrane protein (TM) changes to implement the process of membrane fusion. Design six pairs of short double-stranded RNA (siRNA) according to gp37 gene, cloned into psilence2.1-U6 vector named pu6gp37-1, pu6gp37-2, pu6gp37-3, pu6 gp37-4, pu6gp37-5, pu6gp37-6, pu6gp (negative control). on CEF and 1-day-old SPF chicks do interference experiments to test the effects of viral replication . The results showed that siRNA played an inhibitory effect regardless in vivo or in vitro on the proliferation of ALV-J. This research laid a solid foundation for clinical application of anti-ALV-J siRNA drugs ,and provides a way to draw on for other virus disease of.pEGFP-N1 eukaryotic expression vector carrying the green fluorescent protein reportergene, the gp37 gene carrying pEGFP-gp37 fusion expression vector transfected into CEF cells, respectively, 24h, 48h, 72h to observe the changes in fluorescence. 24h after transfection found that you can see fluorescence, 48h strongest fluorescence, followed by gradually weakened on 48h when the highest transfection efficiency; PEGFP-N1 eukaryotic expression vector carrying the green fluorescent protein reporter gene, transfected the fusion expression vector pEGFP-gp37 carrying gp37 gene into CEF , respectively, 24h, 48h, 72h to observe the changes of fluorescence. after 24h fluorescence appearance, 48h strongest fluorescence and gradually weakened . pEGFP-gp37 and gp37 genes for different regions of the siRNA expression vector were cotransfected into CEF and observed after 48h, pu6gp37-2, pu6gp37-4 plasmid transfection group were reduced fluorecence and reduce the number of fluorescent cells . Preliminary confirmed pu6gp37-2, pu6gp37-4 inhibited the gp37 gene expression in the CEF.Will pu6gp37-2, pu6gp37-4, pu6gp transfected CEF, 8h after add ALV-J NX0101 strain, 5d after check infection cells with indirect immunefluorescence.Fluorescence can be seen under the microscope ,pu6gp37-2,the cell number and intensity of fluorescence is similar to pu6gp37-4 group but pu6gp37-4 group significantly diminished,indicating that pu6gp37-4 inhibite virus proliferation.Vivo experiment set up treatment group and prevention group,group means according to dose and inoculation was divided into high dose, low-dose and continuous injection of siRNA group , together with the positive control group and negative control group, the experimental test is divided into eight group.1 day after inoculation,autopsy 1 week,a totalof 4 times. By continuous detection of weight, immune organ weight, hematology indicators (antigens, antibodies, blood cells), thymus CD4+,CD8+ T lymphocyte changes, patho-gens in chicken to detect the role of RNAiin ALV-J proliferation.The analysis of 4 times test results can be seen that the low-dose prevention group can effectively alleviate growthinhibition on body weight, thymus, spleen, bursa, 5 weeks of age were positive for the weight control group 1.5 times, 6.7 times, 6.5 times, 1.9 times. Protective effects of the treatment group did not significantly.The results of T lymphocyte by flow cytometry showthat, CD4+T lymphocytes is higher than the positive control group and values close to thenegative control group shows no virus on the immune organ damage caused by too much.Blood cell analyzer for WBC, lymphocyte analysis also showed that the group of small doses siRNA has ascendancy on interference of viral replication with the advantages of WBC,lymphocytes were close to normal. Through in vitro and in vivo tests prove that theALV-J TM for the target protein, the RNAi approach to inhibit viral replication is very effective anti-viral approach. The completion of this experiment not only for prevention and treatment of retroviral provides a new way, but also other viral disease can followed.