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

畜牧兽医学报 ›› 2018, Vol. 49 ›› Issue (7): 1492-1499.doi: 10.11843/j.issn.0366-6964.2018.07.019

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

刘璐¹, 郭杰², 朱可蒙², 王宁², 辛九庆², 刘玉芬^1*, 刘恒贵^2*

1. 哈尔滨师范大学生命科学与技术学院, 哈尔滨 150025;
2. 中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室/动物支原体团队, 哈尔滨 150069

收稿日期:2018-01-15 出版日期:2018-07-23 发布日期:2018-07-23
通讯作者: 刘玉芬(1972-),E-mail:liuyufen0825@126.com;刘恒贵(1975-),E-mail:LHG7519@163.com
作者简介:刘璐(1991-),女,黑龙江哈尔滨人,硕士生,主要从事分子免疫学研究,E-mail:coffeell1991@163.com
基金资助:
中央级公益性科研院所基本科研业务费专项（1610302016005；1610302017014）

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

LIU Lu¹, GUO Jie², ZHU Ke-meng², WANG Ning², XIN Jiu-qing², LIU Yu-fen^1*, LIU Heng-gui^2*

1. College of Life Science and Technology, Harbin Normal University, Harbin 150025, China;
2. Division of Bacterial Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin 150069, China

Received:2018-01-15 Online:2018-07-23 Published:2018-07-23

摘要/Abstract

摘要：

构建猪肺炎支原体P97蛋白的酵母随机展示文库，并利用该文库筛选和鉴定P97蛋白上的特异性B细胞表位。设计特异性引物从Mhp基因组中扩增p97基因，通过PCR方法将基因内部的TGA密码子突变为TGG，用DNaseⅠ将p97突变基因随机消化成长度100~250 bp的片段，回收产物经3'端加A处理后与用XcmⅠ酶切的酵母表达载体pCT-XcmⅠ连接，将连接产物转化酿酒酵母EBY100获得P97蛋白酵母随机展示文库。利用流式细胞分选技术筛选出文库中能与Mhp天然感染的猪阳性血清结合的阳性酵母克隆，并进一步鉴定其最小抗原表位。结果：1）克隆计数显示构建的随机文库库容量为2.86×10⁶克隆，所有插入的片段随机分布，覆盖了全长的p97突变基因，文库经诱导后可以利用流式细胞仪检测到与Mhp天然感染的猪阳性血清反应的阳性克隆；2）随后对文库进行流式细胞分选，经鉴定选出184号一个抗原多肽。从短肽C端氨基酸逐个缺失后发现长度为15个氨基酸的184D（DEKTSSQKDPSTLRA）多肽为一个B细胞表位，该表位能与多份Mhp阳性猪血清反应。P97蛋白酵母随机展示文库可以用于抗原表位的筛选鉴定，184D多肽是P97上一个在宿主体内能普遍诱导抗体免疫应答的B细胞表位，本研究对于研发Mhp的表位疫苗和建立特异性诊断方法具有重要意义。

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.

中图分类号:

S852.62

刘璐, 郭杰, 朱可蒙, 王宁, 辛九庆, 刘玉芬, 刘恒贵. 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[J]. 畜牧兽医学报, 2018, 49(7): 1492-1499.

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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参考文献

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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

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

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