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

摘要/Abstract

摘要：

为了揭示布鲁氏菌病的致病机制并研制其新型分子疫苗，试验利用酿酒酵母展示系统展示绵羊白细胞表面抗原DRB1基因，构建DRB1基因外显子2的酵母表面展示库。参照GenBank中绵羊MHC Ⅱ DRB1基因序列设计引物，以绵羊脾脏组织cDNA为模板，反转录扩增得到预期长度的产物，双酶切连接到克隆载体pEASY-T1，命名为pEASY-T1-DRB1。双酶切连接到表面展示载体pYD1上，成功构建了表面展示重组质粒pYD1-DRB1。以pEASY-T1-DRB1为模板，将DRB1基因外显子2两端进行点突变，产生新的酶切位点，然后对外显子2设计特异性引物。对500个绵羊样本进行DNA池化，扩增DRB1基因外显子2序列，双酶切后连接到经相同酶双酶切的表面展示重组突变载体pYD1-DRB1-TB上，构建酿酒酵母表面展示库。转化大肠杆菌DH5α感受态细胞，计算库容量。将其转化酿酒酵母EBY100感受态细胞，经半乳糖诱导，通过免疫荧光法在荧光显微镜下检测。结果显示，绵羊DRB1基因成功整合到酵母基因组中，酵母展示库的库容量在10⁵个以上，DRB1基因库成功展示在酵母细胞表面。由此说明，该库可用于下一步布鲁氏菌抗原肽的筛选。

关键词: 绵羊; DRB1基因; 外显子2; 点突变; 酵母展示库; 免疫荧光

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

中图分类号:

R392.11

王元元, 严国, 罗成, 齐江姣, 谭君, 周光普, 周永顺, 徐杰, 高剑峰. 绵羊DRB1基因外显子2酵母表面展示库的构建及鉴定[J]. 《中国畜牧兽医》, 2018, 45(4): 850-857.

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.

参考文献

[1] ZHAO Y X,YANG J,LV F H,et al.Genomic reconstruction of the history of native sheep reveals the peopling patterns of nomads and the expansion of early pastoralism in East Asia[J].Molecular Biology and Evolution,2017,34(9):2380-2395.
[2] KETEVAN S,JUN H,YU Y,et al.Genome sequences of human and livestock isolates of Brucella melitensis and Brucella abortus from the country of georgia[J].Genome Announcement,2017,5(6):e01518.
[3] DUCROTOY M J,CONDE-ÁLVARZE R,BLASCO J M.A review of the basis of the immunological diagnosis of ruminant brucellosis[J].Veterinary Immunology and Immunopathology,2016,171:81-102.
[4] FENG Y,PENG X,JIANG H,et al.Rough Brucella strain RM57 is attenuated and confers protection against Brucella melitensis[J].Microbial Pathogenesis,2017,107:270-275.
[5] GHAZAEI C.Advances in brucellosis and Brucella infection biology[J].Reviews in Medical Microbiology,2016,27(4):153-158.
[6] ÇIFTICI A,KCA T,SAVASAN S,et al.Evaluation of PCR methods for detection of Brucella strains from culture and tissues[J].Tropical Animal Health and Production,2017,49(4):755-763.
[7] PERPⅡÑÁ C,VINAIXA J,ANDREU C,et al.Development of new tolerant strains to hydrophilic and hydrophobic organic solvents by the yeast surface display methodology[J].Applied Microbiology and Biotechnology,2015,99(2):775-789.
[8] TILLOTSON B J, CHO Y K, SHUSTA E V.Cells and cell lysates:A direct approach for engineering antibodies against membrane proteins using yeast surface display[J].Methods,2013,60(1):27-37.
[9] ERIC T B,JEROME R B,ANDREW W N,et al.Yeast surface display of a noncovalent MHC class Ⅱ het-erodimer complexed with antigenic peptide[J].Biotechnology and Bioengineering,2005,92(4):485-491.
[10] MEI M,ZHOU Y,PENG W,et al.Application of modified yeast surface display technologies for non-antibody protein engineering[J].Microbiological Research,2017,196:118-128.
[11] ZHAO J Z,XU L M,LIU M,et al.An efficient and simple method to increase the level of displayed protein on the yeast cell surface[J].Journal of Microbiological Methods,2017,135:41-47.
[12] FORSYTH K S,EISENLOHR L C.Giving CD⁴⁺ T cells the slip:Viral interference with MHC class Ⅱ-restricted antigen processing and presentation[J].Current Opinion in Immunology,2016,40:123-129.
[13] EMMA R,EDWARD J.Antigen processing and immune regulation in the response to tumours[J].Immun-ology,2017,150(1):16-24.
[14] AFRIDI S,HOESSLI D C,HAMEED M W.Mechanistic understanding and significance of small peptides interaction with MHC class Ⅱ molecules for therapeutic applications[J].Immunological Reviews,2016,272(1):151-168.
[15] BALLINGALL K T,ROCCHI M S,MCKEEVER D J,et al.Trans-species polymorphism and selection in the MHC class Ⅱ DRA genes of domestic sheep[J].PLoS One,2010,5(6):e11402.
[16] VALILOU R H,RAFAT S A,DAVID R,et al.Fecal egg counts for gastrointestinal nematodes are associated with a polymorphism in the MHC-DRB1 gene in the Iranian Ghezel sheep breed[J].Frontiers in Genetics,2015,6:1-11.
[17] 陈月娥,苟亚峰,高剑峰,等.中国美利奴羊MHC-DRB1基因exon 2 SNPs及其与布鲁氏菌病易感性相关性[J].中国畜牧杂志,2014,50(1):15-20. CHEN Y E,GOU Y F,GAO J F,et al.China Merino sheep MHC-DRB1 gene exon 2 SNPs and its correlation with the susceptibility of brucellosis[J].Chinese Journal of Animal Science,2014,50(1):15-20.(in Chinese)
[18] CINARAB M U, MOUSELC M R,WHITEACE S N,et al.Ovar-DRB1 haplotypes *0404 and *0141 are associated with sheep growth and ewe lifetime prolificacy[J].Gene,2016,595(2):187-192.
[19] STAVROS S,PANAGIOTA K,GEORGIOS T,et al.Allelic polymorphism of Ovar-DRB1 exon 2 gene and par-asite resistance in two dairy sheep breeds[J].International Journal of Health,Animal Science and Food Safety January,2016,3(1):1-19.
[20] MINIAS P,WHITTINGHAM L A,DUNN P O.Coloniality and migration are related to selection on MHC genes in birds[J].Evolution,2017,71(1):432-441.
[21] PIEROBIO P,LENNON-DUMENIL A M.To use or not to use the force:How B lymphocytes extract surface-tethered antigens[J].The Journal of Cell Biology,2017,216(1):17.
[22] 戴银,胡晓苗,赵瑞宏,等.鸡MHCⅠα和β2m链基因真核表达载体的构建及其在293T细胞中的表达[J].中国畜牧兽医,2016,43(8):1938-1943. DAI Y,HU X M,ZHAO R H,et al.Construction of eukaryotic expression vector of chicken MHCⅠ α and β2m genes and its exression in 293T cells[J].Chinese Animal Husbandry & Veterinary Medicine,2016,43(8):1938-1943.(in Chinese)
[23] PASWAN C,KUMAR R,SINGH D,et al.Molecular characterization of Ovar-DRB1 exon 2 gene in Garole sheep resilient to gastrointestinal nematodes[J]. Indian Journal of Animal Research,2016,50(2):143-147.
[24] KOUTSOGIANNOULI E A,MOUTOU K A,STAMATIS C,et al.MHC class Ⅱ DRB1 and DQA2 gene polymorphisms in four indigenous breeds of sheep (Ovis aries)[J].Mammalian Biology-Zeitschriftfür Säugeti-erkunde,2016,81(6):628-636.
[25] POUYA Z,HOSEIN S D,ABBAS D,et al.Ovine DRB1 polymorphism and its associations with body weight,milk contents and immunological parame-ters[J].Annals of Animal Science,2016,16(2):425-438.
[26] FALORNI A,BROZZETTI A,PERNIOLA R.From genetic predisposition to molecular mechanisms of auto-immune primary adrenal insufficiency[J].Rontiers of Hormone Research,2016,46(2):115-132.
[27] KROGMAN A,TILAHUN A,DAVID C S,et al.HLA-DR polymorphisms influence in vivo responses to sta-phylococcal toxic shock syndrome toxin-1 in a transgenic mouse model[J].HLA,2017,89(1):20-28.
[28] 李振军,侯雪新,田国忠,等.布鲁氏菌种及种内部分生物型快速PCR鉴定方法的建立及应用研究[J].中华微生物学和免疫学杂志,2013,33(2):133-137. LI Z J,HOU X X,TIAN G Z,et al.Development and application of one-step polymerase chain reaction (PCR) for rapid identification of Brucella species and some biovars[J].Chinese Journal of Microbiology and Immunology,2013,33(2):133-137.(in Chinese)
[29] MILLER D,TALLMADGE R L,BINNS M,et al.Polymorphism at expressed DQ and DR loci in five common equine MHC haplotypes[J].Immunogenetics,2017,69(3):145-156.
[30] PIEHL F,OLSSON T,HOLMDAHL R,et al.Effects of C2ta genetic polymorphisms on MHC class Ⅱ ex-pression and autoimmune diseases[J].Immunology,2017,15(4):408-417.