Sequence Analysis Of Avian Leukosis Virus In Guizhou And Screening Of Traditional Chinese Medicines Against Avian Leukosis

Avian Leukosis(AL)is a general term for a variety of neoplastic diseases characterized by malignant proliferation of hematopoietic cells caused by Avian Leukosis virus(ALV).Because it can cause tumors,resulting in decreased egg production,and can make the body produce severe immunosuppression,it has great harm to the poultry industry.The control of avian leukemia can only rely on population purification.Screening a specific drug that can resist avian leukosis virus is great significance for the prevention and treatment of the disease.Studies have shown that subtype A and J subtype Avian leukosis viruses are the major subtypes threatening the poultry industry in our province.In this project,the isolates of the subtype J avian leukosis virus in Guizhou Province were isolated and identified,and the sequence analysis was performed.The molecular biology characteristics were used to infer the prevalence of avian leukosis virus in Guizhou Province in recent years.At the same time,the subtype A standard strain was Subjects,from the 18 kinds of traditional Chinese medicines,screened out the traditional Chinese medicines that had a significant inhibitory effect on their replication,and further explored the best mode of action of traditional Chinese medicines.The results laid the foundation for the prevention and treatment of avian leukemia.1.ALV-J Clinical Case Detection and Sequence Analysis of gp85 in Guizhou Province: In this study,we designed a pair of J subtype avian leukosis virus-specific primers,25 cases of clinical cases were amplified by PCR and 17 cases of ALV-J positive cases were identified.The positive rate was 68%(17/25).Three cases of ALV-J subtypes were selected and the gp85 gene was cloned,sequenced and analyzed by biological software.The results showed that:(1)The gp85 gene of 3 strains(GZ161219,GZ170813 and GZ180116)were all 924 bp in size,and the nucleotide similarity was between 98.3% and 99.5%.The epidemic strain and subtype J reference strain AHaq02(Gen Bank accession number: KF534753)with highest similarity of 98.2%-99.7%,similarity with other subtype strains of only 49.1%-51.2%;(2)derivation of gp85 gene of 3 strains with DNAStar software,3 All of them can encode 308 amino acids.The amino acid sequence similarity analysis results are consistent with the nucleotides.Compared with the J subtype strains HPRS-103(Gen Bank accession number: Z45390)and GZN49(Gen Bank accession number:KX077613),the mutations The rate is lower than 3%;(3)The prediction of secondary structure of amino acids deduced from three epidemic strains gp85 shows that the content of random coils is the highest,followed by the extension chain,and the content of ?-helix and ?-turn is the lowest.The parameters of the B cell epitopes of the gp85 gene-encoded protein were predicted.The results showed that the gp85gene-encoded protein has good hydrophilicity,the amino acid flexible region is more evenly distributed,the antigen index and surface accessibility are high,and various parameters are integrated.The possible dominant epitope of gp85 protein was obtained.The gp85 proteins of the three epidemic strains mainly contained conserved domains of the gp85 protein family,and no transmembrane structure or signal peptide was present.The research results provide reference data for the biological characteristics and epidemic variation of the subtype J avian leukosis virus in Guizhou Province.2.Prokaryotic expression of ALV p27 and preparation of its hyperimmune serum:(1)Design a pair of p27 gene-specific primers,clone the target gene into the p Cold I plasmid,transfer it to E.coli BL21,induce expression by IPTG,lyse by ultrasound,and detect by SDS-PAGE.As a result,a specific band of 26 k D was seen,and the recombinant protein His-p27 mainly existed in the form of inclusion bodies.(2)After purification by recombinant protein nickel column,immunize healthy mice,collect serum,measure titer by ELISA,and identify by Western Blotting.The results showed that this study successfully obtained a hyperimmune serum against p27 protein with a serum titer of 1:3200.The results laid the foundation for the detection of ALV and the study of the function of p27 protein.3.Determination of the maximum safe concentration of traditional Chinese medicines: Select 18 kinds of traditional Chinese medicines(Astragalus,Guanzhong,Millettia,Eclipta,Astragalus,Astragalus,Agrimonia,Salvia miltiorrhiza,Dandelion,Motherwort,Hedyotis diffusa,Epimedium,paeonol,Yujin,Prunella,Bandits,Gardenia,Houttuynia),prepared those medicine by decoction,and the maximum safe concentration(TC0)of Chinese herbs was determined by cytopathic and MTT assays.The results showed that different Chinese herbs were cytotoxic.Differently,among 18 Chinese herbs,the least cytotoxicity was Astragalus membranaceus,with a TC0 of62.5 mg/m L;the most toxic was Guanzhong,with a TC0 of 0.061 mg/m L,and the toxicity of some Chinese herbs to cells increased gradually with the increase of the action time.Big.The MTT method is more sensitive than the two detection methods.The measurement results can be used as the basis for the next experiment.4.Effects of different Chinese herbs on the transcription level of ALV: The TCID50 of the ALV strain was determined using the ALV p27 antigen detection kit,and the results were calculated using the Reed-Muench method.The TCID50 of the virus was determined to be 10-3.8/0.1 m L diluted virus solution.The fluorescent primers were designed with reference to the more conserved gp37 and 18 sr RNA gene sequences,and standard plasmids were constructed.Standard strains with high specificity and high amplification efficiency were successfully established.[Establishment of ALV fluorescence quantification method] Established direct inactivated group,prevention group and treatment group 3 groups,Chinese medicine and virus were inoculated separately or simultaneously in DF-1 cells,and cultured at37°C for 72 hours,each was extracted.The total RNA of the wells was reverse-transcribed into c DNA and the 18 sr RNA was used as the reference gene for q PCR amplification to obtain the Ct value.The difference was calculated using the2-??Ct method.The results showed that Astragalus had the strongest inhibitory effect on ALV,Yujin,Motherwort,and Sophora japonica had a certain inhibitory effect on ALV,while Millettia,Dandelion and Guanzhong had little effect on the replication of ALV.