双峰驼天然重链抗体可变区酵母双杂交文库的构建与鉴定

doi:10.11843/j.issn.0366-6964.2015.06.025

畜牧兽医学报 ›› 2015, Vol. 46 ›› Issue (6): 1071-1076.doi: 10.11843/j.issn.0366-6964.2015.06.025

• 研究简报 • 上一篇

双峰驼天然重链抗体可变区酵母双杂交文库的构建与鉴定

胡湘云，高小龙，付向晶，刘丹丹，范文涛，王燕平，杜恩岐，王兴龙，党如意，杨增岐^*

(西北农林科技大学动物医学院，杨凌 712100)

收稿日期:2014-09-26 出版日期:2015-06-23 发布日期:2015-06-23
通讯作者: 杨增岐，教授，E-mail：yzq8162@163.com
作者简介:胡湘云（1990-），女，广西南宁人，硕士，主要从事动物传染病和基因工程抗体的研究，E-mail：821995274@163.com
基金资助:
国家自然科学基金(31272578)；西北农林科技大学引进人才科研启动基金(Z111021006)

Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody（VHH）Yeast Two-Hybrid Library

HU Xiang-yun ，GAO Xiao-long，FU Xiang-jing，LIU Dan-dan，FAN Wen-tao，WANG Yan-ping，DU En-qi，WANG Xing-long，DANG Ru-yi，YANG Zeng-qi^*

(College of Veterinary Medicine，Northwest A&F University，Yangling 712100，China)

Received:2014-09-26 Online:2015-06-23 Published:2015-06-23

摘要/Abstract

摘要：

构建双峰驼天然重链抗体可变区（VHH）酵母双杂交文库，并对文库质量进行鉴定。采集未经免疫的6月龄雌性双峰驼外周血、骨髓、脾和淋巴结，并从外周血中分离淋巴细胞。抽提总RNA，反转录为cDNA，用2对引物经过2轮PCR扩增得到400 bp左右的双峰驼VHH片段。根据Clontech公司Mate & Plate^TM library construction system使用手册，将带有同源臂的VHH片段与线性化的pGADT7-Rec质粒共转化Y187酵母感受态细胞，转化产物涂布SD/-Leu平板，30 ℃倒置培养3～5 d后，收集平板上的所有克隆，即为双峰驼天然重链抗体可变区酵母双杂交文库。对文库的滴度、重组效率及多样性进行鉴定。结果显示，本研究构建的双峰驼天然重链抗体可变区酵母双杂交文库库容为2.07×10⁷，滴度为7.6×10⁸ cfu•mL^-1。随机挑取47个独立克隆进行菌落PCR鉴定，43个含有VHH片段，重组率为91.5%；选取其中19个测序，均为独立克隆，且多样性好。该研究成功构建双峰驼天然重链抗体可变区酵母双杂交文库，且文库质量满足要求，为进一步获得特定抗原的VHH奠定了基础。

Abstract:

The aim of this study was to construct and characterize of a naive Camelus bactrianus variable domain of heavy chain of heavy-chain antibody（VHH）yeast two-hybrid library.The blood，bone marrow，spleen and lymph node were collected from a healthy non-immunized 6 month-old female Camelus Bactrianus. And peripheral lymphocytes were isolated from blood samples using Ficoll Paque PLUS reagent.Then total RNA was extracted from peripheral lymphocytes，bone marrow，spleen and Lymph nodes.Reverse-transcription was performed to generate the first-stand cDNA.VHH fragments were amplified through two rounds PCR using two pairs of specific primers.Naive Camelus Bactrianus VHH yeast two-hybrid library was successfully constructed according to Clontech Mate & Plate library construction system user manual.Briefly，5 μg VHHs fragment containing homologous arms in 20 μL were co-transformed with 6 μL linearized pGADT7-Rec plasmid into component Y187 yeast cells，and transformation products were plated on SD/-Leu plates.After 3-5 days incubation at 30 °C，the library was harvested and the library quality were evaluated by titer，recombination efficiency，diversity and complexity.The results showed that there were 2.07×10⁷ independent clones in the naive VHH yeast two-hybrid library and the library titer was 7.6×10⁸ cfu•mL^-1.PCR results showed that 43 independent clones inserted VHHs fragment in 47 randomly picked out clones，and the recombination efficiency was 91.5%.Nineteen independent clones were random picked out to sequence and sequence results indicate they all share unique sequences.In a conclusion，a naive Camelus Bactrianus variable domain of heavy chain of heavy-chain antibody yeast two-hybrid library was successfully constructed with large capacity，good diversity and complexity，which laid the foundation for preparation VHHs against interested antigens.

中图分类号:

Q789

胡湘云，高小龙，付向晶，刘丹丹，范文涛，王燕平，杜恩岐，王兴龙，党如意，杨增岐. 双峰驼天然重链抗体可变区酵母双杂交文库的构建与鉴定[J]. 畜牧兽医学报, 2015, 46(6): 1071-1076.

HU Xiang-yun,GAO Xiao-long，FU Xiang-jing，LIU Dan-dan，FAN Wen-tao，WANG Yan-ping，DU En-qi，WANG Xing-long，DANG Ru-yi，YANG Zeng-qi. Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody（VHH）Yeast Two-Hybrid Library[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2015, 46(6): 1071-1076.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.xmsyxb.com/CN/10.11843/j.issn.0366-6964.2015.06.025

https://www.xmsyxb.com/CN/Y2015/V46/I6/1071

参考文献

［1］VANLANDSCHOOT P，STORTELERS C，BEIRNAERT E，et al.Nanobodies^®：new ammunition to battle viruses ［J］.Antiviral Res，2011，92(3)：389-407.
［2］MONEGAL A，AMI D，MARTINELLI C，et al.Immunological applications of single-domain Ilama recombinant antibodies isolated from a naïve library ［J］.Protein Eng Des Sel，2009，22(4)：273-280.
［3］HAMERS-CASTERMAN C，ATARHOUCH T，MUYLDERMANS S，et al.Naturally occurring antibodies devoid of light chains ［J］.Nature，1993，363(6428)：446-448.
［4］GREENBERG A S，AVILA D，HUGHES M，et al.A new antigen receptor gene family that undergoes rearrangement and extensive somatic diversification in sharks ［J］.Nature，1996，374(6518)：168-173.
［5］HARMSEN M M，DE HAARD H J.Properties，production,and applications of camelid single-domain antibody fragments ［J］.Appl Microbiol Biotechnol，2007，77(1)：13-22.
［6］DUMOULIN M，CONRATH K，VAN MEIRHAEGHE A，et al.Single-domain antibody fragments with high conformational stability ［J］.Protein Sci，2002，11(3)：500-515.
［7］MUYLDERMANS S，BARAL T N，RETAMOZZO V C，et al.Camelid immunoglobulins and nanobody technology ［J］.Vet Immunol Immunopathol，2009，128(1-3)：178-183.
［8］RAHBARIZADEH F，AHMADVAND D，SHARIFZADEH Z.Nanobody：an old concept and new vehicle for immunotargeting ［J］. Immunol Invest，2011，40(3)：299-338.
［9］SABIR J S，ATEF A，EI-DOMYATI F M，et al.Construction of naive camelids VHH repertoire in phage display-based library ［J］.C R Biol，2014，337(4)：244-249.
［10］RYCKAERT S，PARDON E，STEYAERT J，et al.Isolation of antigen-binding camelid heavy chain antibody fragments (nanobodies) from an immune library displayed on the surface of Pichia pastoris ［J］.J Biotechnol，2010，145(2)：93-98.
［11］SILACCI M，BRACK S，SCHIRRU G，et al.Design，construction，and characterization of a large synthetic human antibody phage display library ［J］.Proteomics，2005，5(9)：2340-2350.
［12］YANG M，WU Z，FIELDS S.Protein-peptide interactions analyzed with the yeast two-hybrid system ［J］.Nucleic Acids Res，1995，23(7)：1152-1156.
［13］FU X，GAO X，HE S，et al.Design and selection of a camelid single-chain antibody yeast two-hybrid library produced de novo for the cap protein of porcine circovirus type 2 (PCV2) ［J］.PLoS One，2013，8(3)：e56222.
［14］ARBABI GHAHROUDI M，DESMYTER A，WYNS L，et al.Selection and identification of single domain antibody fragment from camel heavy-chain antibodies ［J］.FEBS Lett，1997，414(3)：521-526.
［15］KASTELIC D，FRKOVIC＇-GRAZIO S，BATY D，et al.A single-step procedure of recombinant library construction for the selection of efficiently produced Ilama VH binders directed against cancer markers ［J］.J Immunol Methods，2009，350(1-2)：54-62.
［16］VU K B，GHAHROUDI M A，WYNS L，et al.Comparison of Ilama VH sequences from conventional and heavy chain antibodies ［J］.Mol Immunol，1997，34(16-17)：1121-1131.

[1]	许倩茹, 张二芹, 郭军庆, 杨继飞, 刘运超, 王爱萍, 王丽, 万博, 汪磊, 蒋大伟, 邓瑞广, 张改平. 新城疫病毒F蛋白在水稻中的表达及检测[J]. 畜牧兽医学报, 2017, 48(6): 1167-1172.
[2]	王向鹏，魏蕊芳，肖书奇，周恩民. 慢病毒载体介导稳定表达CD163 的PAM细胞系的建立及对PRRSV感染的研究[J]. 畜牧兽医学报, 2013, 44(11): 1797-1804.
[3]	贺生芳，付向晶，刘阳坤，慕国辉，刘海金，王兴龙，杜恩岐，杨增岐. 1种高效纳米抗体生产方法的建立[J]. 畜牧兽医学报, 2013, 44(9): 1487-1493.

双峰驼天然重链抗体可变区酵母双杂交文库的构建与鉴定

Construction and Characterization of a Naive Camelus Bactrianus Variable Domain of Heavy Chain of Heavy-Chain Antibody（VHH）Yeast Two-Hybrid Library

RichHTML

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 3

编辑推荐

Metrics

本文评价