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

作　　者：胡湘云[1] 高小龙[1] 付向晶[1] 刘丹丹[1] 范文涛[1] 王燕平[1] 杜恩岐[1] 王兴龙[1] 党如意[1] 杨增岐[1] 

机构地区：[1]西北农林科技大学动物医学院,杨凌712100

出　　处：《畜牧兽医学报》2015年第6期1071-1076,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：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)

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400bp左右的双峰驼VHH片段。根据Clontech公司Mate&PlateTMlibrary 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3~5d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7.6×108cfu·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。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 6month-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,5μg VHHs fragment containing homologous arms in 20μL were co-transformed with 6μL linearized pGADT7-Rec plasmid into component Y187 yeast cells,and transformation products were plated on SD/-Leu plates.After 3-5days incubation at 30°C,the library was harvested and the library quality were evaluated by titer,recombination efficiency,diversity and complexity.Theresults showed that there were 2.07×107 independent clones in the naive VHH yeast two-hybrid library and the library titer was 7.6×108cfu·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.

关 键 词：双峰驼 重链抗体可变区 酵母双杂交 

分 类 号：Q78[生物学—分子生物学]