英文文献汇报教学文案.ppt

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1、英文文献汇报 Avian leukosis viruses(ALVs),which belong to the family Retroviridae,are classified into A,B,C,D,E,and J subgroups1,2.The A,B and J subgroups of ALV are the most common ALVs in commercial poultry,whereas subgroups C and D have rarely been reported.Subgroup E is a ubiquitous endogenous leukosi

2、s virus of low pathogenicity 3.ALV predominantly causes lymphocytic leukemia,myeloid leukosis and other sarcomas and can also lead to immunosuppression effects,such as the abnormal development of immune organs,growth retardation,and decreased immune responses.4,5.ALV subgroup J(ALV-J)was first isola

3、ted in the UK from white meat-type chickens in 1988 2.In China,ALV-J was first reported in 1999.Since then,ALV-J isolates have been detected in layer chickens,broiler breeders and local chickens throughout most regions of China and have caused various tumors in chickens 68.In recent years,ALV-J has

4、become widespread and has resulted in severe economic losses to the poultry industry 6,8.1.Introduction Effective vaccines against ALV are not available.The control of ALV infection occurs primarily by establishing exogenous ALV-free poultry flocks by adopting eradication as the strategy of choice 9

5、.Because of substantial antigenic and genetic variation among ALV isolates and high levels of vertical and horizontal transmission,eradication has been difficult 10,11.Thus,effective methods for the accurate detection of ALV antigens in chickens are critical for the control of ALV infections.The gen

6、ome of ALV consists of 5-LTR-UTR-gag-polenv-UTR-LTR-3.The gag,pol and env genes are the viral structural genes 11.The P27 protein,which is encoded by the gag gene,is the capsid protein and acts as a group-specific antigen.The ALV P27 gene is highly conserved and exhibits 96%sequence identity among t

7、he exogenous subgroups(A,B,C,D and J).In addition,the P27 protein content accounts for more than 30%of the viral protein,and P27 has many viral antigen sites that are easy to detect.Therefore,the P27 protein is the first choice for preparing antibodies for detection.Epitopes of proteins are classifi

8、ed as either continuous or discontinuous depending on whether the amino acids included in the epitope are continuous in the peptide chain or not1214.Continuous epitopes sometimes do not represent the entire antigenic epitope in the viral protein but instead only a short cross-reactive portion of a l

9、arger,discontinuous epitope 12,13.B-cell epitopes are regions that are recognized by the binding sites or paratopes of antibody molecules when they are present in their free form in serum or as membrane-bound B-cell receptors 12.Correct identification of B-cell epitopes within an antigenic protein m

10、ay open the door for the design of molecules that mimic potentially protective epitopes and could be used to raise specific Abs or be used as prophylactic or therapeutic vaccines 1517.Identification of B-cell epitopes could promote protective immunity in the context of emerging and re-emerging infec

11、tious diseases and potential bioterrorist threats 15.The aim of this study was to generate a P27-specific antibody against ALV using purified recombinant P27 protein as the immunogen and subsequently to identify the B-cell epitope recognized by the antibody.The information described in this study wi

12、ll facilitate the development of ALV diagnostic tools and will further our understanding of the antigenic structure of the P27 protein.2.Materials and methods重组质粒的构建 构建重叠片段短肽免疫小鼠制备单抗IFA&Western blot检测单抗特异性鉴定最小抗原表位基序分析表位保守性3.ResultsFig.1 Characterization of the recombinant P27 protein and mAb 3A9.(A)

13、Expression and purification of the recombinant P27 protein.M,molecular marker(kDa);1,recombinant P27 protein before IPTG induction;2,recombinant P27 protein after IPTG induction;3,purified recombinant P27 protein.(B)Recognition of the purified ALV-J P27 protein by mAb 3A9 in western blot analysis.M,

14、protein marker(kDa);1,purified recombinant P27 protein;2,negative control(prokaryotic expression vector pET-30a).(C)IFA analysis of mAb 3A9 using different viral strains.mAb 3A9 on DF1 cells infected with(A)RAV-1;(B)RAV-2;(C)HPRS103;(D)NX0101;(E)ADOL-Hc1;(F)ADOL-7501;(G)HuB09JY03;(H)JL08CH3-1;(I)LN0

15、8SY10;(J)SD09DP04;(K)HLJ09SH01;(L)negative controlFig.2 Identification of the minimal linear epitope recognized by mAb 3A9.(A)Strategy for the identification of the epitope.Thirteen overlapping peptides were expressed and subjected to western blot analysis.The red peptides are the immunodominant reg

16、ions,and the blue ones are the non-immunodominant regions.(B)Western blot analysis of the reactivity of the GST fusions using mAb 3A9.GST fusions covering amino acids 181 to 197 were designed and expressed.GST was encoded by the prokaryotic expression vector pGEX6p-1,which served as a negative contr

17、ol.M,molecular marker(kDa);1,GST-S;2,GST-DCFRQKS;3,GST-IIDCFRQKS;4,GSTVIIDCFRQKS;5,GST-PVIIDCFRQKS;6,GST-APVIIDCFRQKS;7,GST RAPVIIDCFRQKS;8,GST-ARAPVIIDCFRQKS;9,GST.(C)Amino acid sequence,position in the P27 protein and reaction with 3A9 for each of the GST fusions.*represents the reaction between t

18、he GST fusions and mAb 3A9;?indicates that 3A9 could identify the GST fusion;indicates that 3A9 could not identify the GST fusionFig.3 ELISA analysis and amino acid sequence alignment analysis of the identified minimal linear epitope.(A)ELISA analysis for reactivity of the identified epitope with mo

19、use anti-ALV-J P27 sera.Three expressed proteins(GST,the minimal linear epitope,and the recombinant pET-30a-p27 protein)were used to coat the ELISA plates.(B)ELISA analysis for reactivity of the identified epitope with chicken anti-ALV sera.Error bars reflect the results of three experimental repeat

20、s.(C)Amino acid alignment analysis of the identified epitope in different ALV-A,ALV-B and ALV-J strains.Residues that match are represented by 4.Discussion Since the emergence of ALV,infection with this virus has become widely prevalent and has been identified as the cause of various tumors in many

21、chicken species,including meat-type chickens,layer chickens,and local breeder flocks68,25.Although western countries have successfully eradicated ALV from breeding chickens,the virus has expanded robustly in China since 2008 26.An effective vaccine against ALV is not available.Early detection and er

22、adication of virus-infected birds to reduce the spread of infection to other birds are very important for the control of ALV infection.Thus,effective methods for the rapid and accurate detection of ALV infection are essential for effective control of ALV spread.Traditional methods used to diagnose A

23、LV infection,such as reverse transcription PCR,virus isolation,and IFA,are slow and might yield inconclusive results 2729.In contrast,a specific mAb could be applied to develop a sensitive,rapid,and reliable method to detect virus infection.In this study,we produced and characterized the P27-specifi

24、c mAb 3A9 and defined the linear epitope.MAb 3A9 could react with the three(A,B and J)most common ALVs in commercial poultry.We used DNASTAR software to analyze the antigenic index and the surface probability of the ALV P27 protein.The identified linear epitope has a high antigenic index and a posit

25、ive surface probability,indicating that it is likely to be exposed on the surface of the P27 tertiary structure(data not shown).Thus,the specific mAbs and defined linear epitope may provide valuable tools for the development of new immunodiagnostic approaches for ALV detection and may also provide important information to further our understanding of the antigenic structure of the P27 protein.此课件下载可自行编辑修改，仅供参考！此课件下载可自行编辑修改，仅供参考！感谢您的支持，我们努力做得更好！谢谢感谢您的支持，我们努力做得更好！谢谢