| Bovine endemic leukemia(EBL)is a common neoplastic disease in cattle,caused by the Bovine leukemia virus(BLV).BLV belongs to the retrovirus of the retrovirus family Delta retrovirus,widely endemic in cattle countries around the world,which brings huge economic losses to the livestock industry,mainly including reduced reproductive performance,reduced milk production,prevention and control and monitoring of bovine leukemia is particularly important,the current main control measures for bovine leukemia is culling or isolating infected cattle.Therefore,the establishment of clinical diagnostic methods with strong specificity and high sensitivity is particularly necessary for the prevention and treatment of EBL.Among the known structural proteins of BLV,gp51 protein encoded by env gene and p24 protein encoded by gag gene have the strongest immunogenicity and good conservatism,which can induce strong immune response in animals,so gp51 protein and p24 protein have been the target proteins for the study of new vaccines for bovine leukemia virus.In this study,BLV-GP51 protein was truncated into four fragments,gp51-1(1aa-58aa),gp51-2(51aa-97aa),gp51-3(91aa-219aa),gp51-4(212aa-271aa),and BLV-P24 protein was truncated into two fragments,p24-1(1aa-124aa)and p24-2(119aa-196aa),and the above six fragments were constructed as prokaryotic expression plasmids The sizes of recombinant proteins obtained were27 k Da,23 k Da,31 k Da,27 k Da,26 k Da,and 24 k Da,respectively,and then the antigen reactivity of the above expressed recombinant proteins was detected by Western blot,and the results showed that the 1aa-58 aa,51aa-97 aa of BLV-GP51 protein,1aa-124 aa of BLV-P24 protein,119aa-196 aa has the ability to react positively with BLV infection.The above fragments were further truncated in the second round to obtain P24-1.1(1AA-36AA),P24-1.2(32AA-68AA),P24-1.3(64AA-97AA),P24-1.4(92AA-126AA),P24-2.1(119AA-160AA),P24-2.2(155AA-196AA),GP51-1.1(1AA-32AA),The recombinant proteins of gp51-1.2(26aa-58aaa),gp51-2.1(51aa-76aaa),gp51-2.2(71aa-97aa)were detected by Western blot to obtain the epitope dominance region of gp51 and p24,of which the epitope dominance region of gp51 was located in 26aa-76 aa,and the epitope dominance region of p24 was located in 1aa-68 aaa,119aa-160 aaa.The obtained three antigen dominant fragments were connected in series and connected with p ET-32 a vector,p ET-32a-gp-2p expression plasmid was constructed,and gp-2p tandem protein was induced,and the purified recombinant fusion protein was identified as a coating protein as a coating protein to establish an indirect ELISA method for the detection of bovine leukemia,and the conditions of the method were optimized,and the results showed that the optimal coating concentration of antigen was 1μg/m L and the optimal serum dilution concentration was 1 :40,the optimal working concentration of secondary antibodies is 1:20000,and the critical value of this detection method is 0.65.The compliance rate of this method with nucleic acid detection was92.86%,and the compliance rate with gp51 and p24 double antibody sandwich ELISA was 93.75%.In summary,the dominant antigenic regions of GP51 protein and P24 protein were identified,and an indirect ELISA detection method based on gp-2p tandem protein was established,which had good specificity and sensitivity.It has added new and effective technical means for the seroepidemiological investigation of BLV in China. |
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