绵羊DRB1基因外显子2酵母表面展示库的构建及鉴定

doi:10.16431/j.cnki.1671-7236.2018.04.002

Abstract

Abstract:

In order to reveal the pathogenesis of brucellosis and develop a new molecular vaccine of brucellosis,the ovine lymphocyte antigen DRB1 gene was designed to be displayed on yeast cell surface using yeast surface display system,explored a platform for screening antigen peptides and developing a vaccine against sheep brucellosis.A pair of primers were designed according to the published sequences of sheep MHC Ⅱ DRB1 gene in GenBank.DRB1 gene was cloned using sheep cDNA extracted from spleen tissue as template,the PCR product was inserted into the cloning vector pEASY-T1 by double enzyme digestion,then named pEASY-T1-DRB1.Ligated into the yeast surface display plasmid vector pYD1 by double enzyme digestion,the recombinant plasmid pYD1-DRB1 was successfully constructed on the surface of yeast.Point mutation was made at the end of DRB1 gene exon 2 in order to make restriction enzyme cutting site using pEASY-T1-DRB1 as a template and the specific primers of exon 2 was designed according to mutated sheep DRB1 gene sequence.DNA pooling of 500 sheep samples was made and amplified the sequence of DRB1 gene exon 2 which was digested by double enzyme,then connected to surface display restructuring mutation carriers pYD1-DRB1-TB by the same double enzyme digestion,the yeast surface display libraries were constructed.E.coli DH5α competent cells were transformed and library capacity was calculated.It was transformed into yeast EBY100 cell,after galactose induced,it was detected that DRB1 gene library on the yeast cell surface under the fluorescence microscope by immunofluorescence assay.The results showed that DRB1 gene was successfully integrated into yeast genome,the library capacity of librariy was more than 10⁵,and DRB1 gene library was successfully displayed on the surface of yeast cells.The results suggested that this library could be used for screening of Brucella antigen peptide.

Key words: sheep; DRB1 gene; exon 2; point mutation; yeast display library; immunofluorescence

CLC Number:

R392.11

WANG Yuanyuan, YAN Guo, LUO Cheng, QI Jiangjiao, TAN Jun, ZHOU Guangpu, ZHOU Yongshun, XU Jie, GAO Jianfeng. Construction and Identification of Yeast Surface Display Library of Sheep DRB1 Gene Exon 2[J]. , 2018, 45(4): 850-857.

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Construction and Identification of Yeast Surface Display Library of Sheep DRB1 Gene Exon 2

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