| 摘要: |
| 【目的】构建H9N2禽流感病毒（AIV）纳米抗体（Nbs）酵母双杂交文库，为后期筛选H9N2 AIV Nbs奠定基础。【方法】以H9亚型禽流感疫苗（F株）免疫双峰驼，当血凝抑制（HI）抗体滴度大于1∶24时，颈静脉采集血液，分离淋巴细胞并提取淋巴细胞总RNA，用反转录试剂盒合成第一链cDNA，再以此为模板，通过巢式PCR扩增Nbs基因片段。将Nbs基因与pGADT7-Rec载体共转至Y187酵母感受态细胞，转化产物涂布SD/-Leu平板，30 ℃倒置培养约4 d，收集平板上的全部菌落，构建H9N2 AIV Nbs酵母双杂交文库，并对文库库容、滴度、重组效率和多样性进行鉴定。【结果】经H9亚型禽流感疫苗连续免疫4次后，双峰驼HI抗体滴度可达1∶29；通过巢式PCR成功扩增出400 bp的Nbs基因。对构建的H9N2AIV Nbs酵母双杂交文库的鉴定表明，其库容为2.4×108，滴度为4.2×109 CFU/mL，插入重组率为95.6%；随机挑取10个克隆测序，结果显示均符合Nbs序列特征，且为独立克隆，表明文库多样性良好。【结论】成功构建了H9N2 AIV Nbs酵母双杂交文库，且文库各项指标均达到了要求。 |
| 关键词: H9N2禽流感病毒 纳米抗体 酵母双杂交 |
| DOI： |
| 分类号: |
| 基金项目:青海省科技厅自然科学基金青年项目（2018-ZJ-952Q） |
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| Construction of nanobody yeast-two hybrid library against H9N2 avian influenza virus |
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ZHANG Cheng,GUO Chengyi,ZHOU Lei,et al
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| Abstract: |
| 【Objective】This study constructed a nanobodies (Nbs) yeast-two hybrid library against H9N2 avian influenza virus (AIV) to provide foundation for screening Nbs against H9N2 AIV.【Method】A female Camelus bactrianus was immunized with inactivated H9 AIV vaccine (F strain).When the hemagglutination inhibition (HI) antibody titer was higher than 1∶24,100 mL blood was collected from jugular vein for peripheral lymphocytes isolation.Then,total RNA was extracted from the peripheral lymphocytes and reverse transcription was performed to synthesise the first-strand cDNA.Subsequently,400 bp gene fragments that encoded Nbs were amplified through a nested polymerase chain reaction (PCR) with two pairs of specific primers.The gel-purified Nbs gene was cotransformed with linearized pGADT7 Rec into Y187 yeast competent cells for recombination.The transformation products were plated on SD/-Leu plates.After 4 days incubation at 30 ℃,the library was harvested and the capacity,titer,recombination efficiency and diversity were evaluated.【Result】The HI antibody titer was 1∶29 post four times immunization and the Nbs gene was successfully amplified through a nested PCR.The capacity,titer and recombination efficiency of the constructed library were 2.4×108,4.2×109 CFU/mL and 95.6%,respectively.Ten independent clones were then random picked out for sequencing and sequence results showed they all shared the feature of Nbs as independent clones,indicating a good diversity.【Conclusion】This study successfully constructed a H9N2 AIV Nbs yeast-two hybrid library. |
| Key words: H9N2 avian influenza virus nanobody yeast-two hybrid |
