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

Abstract

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.

CLC Number:

Q789

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.

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

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