利用酵母随机展示技术对猪肺炎支原体P97上B细胞表位的筛选和鉴定

doi:10.11843/j.issn.0366-6964.2018.07.019

Abstract

Abstract:

The experiment was conducted to construct a yeast random display library of P97 antigen from Mycoplasma hyopneumoniae(Mhp), and the specific B cell epitopes on P97 protein were screened and identified by using the library. The p97 gene was amplified from Mhp genome using specific primers. All TGA codons within the p97 gene were mutated into TGG by polymerase chain reaction, Mutated p97 gene was randomly digested into about 100-250 bp in the length with DNaseⅠ. After adding adenine at the 3' terminus, the digested fragments were ligated into yeast expression vector pCT-XcmⅠ treated with XcmⅠ. The ligation product was transformed into Saccharomyces cerevisiae EBY100 to obtain a random yeast display library of P97 protein.The positive yeast clones in the library that react with the sera from Mhp-infected porcine were screened by flow cytometry and verified the minimum peptide sequences of epitopes. Results:The size of the constructed library was 2.86×10⁶ clones. The inserted fragments were randomly distributed and covered the full length of p97 mutant gene. Upon induction and staining with the sera from Mhp-infected porcine, the positive clones were able to be detected from the library using flow cytometry. Then the sorting of the library was carried out using FACS and a positive peptide, named as 184, was identified. After one by one deletion from the C-terminal amino acid of 184, an epitope 184D(DEKTSSQKDPSTLRA) with 15 amino acids in length was determined. This epitope was able to react with many Mhp-positive porcine sera. These results indicate that the library can be used for the epitope screening from the P97 protein, and the 184D peptide on the P97 protein is a B-cell epitope which is capable of inducing antibody production in many hosts. These results will provide the base for the exploring epitope vaccine of Mhp and establishing a specific diagnostic method in the future.

CLC Number:

S852.62

LIU Lu, GUO Jie, ZHU Ke-meng, WANG Ning, XIN Jiu-qing, LIU Yu-fen, LIU Heng-gui. Screening and Identification of a B-cell Epitope on P97 Protein of Mycoplasma hyopneumoniae Using Yeast Random Display Library Technology[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2018, 49(7): 1492-1499.

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Screening and Identification of a B-cell Epitope on P97 Protein of Mycoplasma hyopneumoniae Using Yeast Random Display Library Technology

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