新疆双峰驼纳米抗体噬菌体展示文库多样性的生物信息学分析

Analysis of the Diversity of VHH Library from Bactrian Camel(Camelus bactrianus) with Bioinformatics

【目的】骆驼免疫系统中只存在重链抗体,其可变区VHH片段是最小的天然抗体片段。通过对双峰驼VHH基因进行序列分析,了解双峰驼VHH基因特征,分析VHH文库多样性组成。【方法】采用新疆双峰驼VHH特异引物扩增全部抗体基因片段,连接到M13噬菌粒载体上,与p III蛋白融合表达在噬菌体表面,构建噬菌体展示文库。基因测序和菌落PCR方法,分析文库大小和阳性克隆插入率。NCBI-Ig GBLAST工具对抗体序列进行IMGT编号命名,Web Logo工具分析抗体保守序列分布频率,MEGA6软件对VHH序列进行同源性比对和系统进化树构建。【结果】利用免疫的新疆双峰驼淋巴细胞,构建了库容量为1.4×109的VHH抗体噬菌体展示文库。对91个随机挑选的文库TG1单菌落进行PCR。结果表明该文库VHH阳性插入率为97%,DNA测序表明文库多样性为97.8%。对91个克隆DNA测序结果进一步分析表明,这些基因中除2个为常规VH抗体外,其余均为重链VHH抗体。根据抗体序列中的特征氨基酸分布,将91个抗体序列划分为7个亚群。【结论】构建的新疆双峰驼VHH文库多样性较好,生物信息学分析揭示出文库中多样性克隆的分布和特征,有助于新疆双峰驼纳米抗体噬菌体展示文库的进一步构建。

[ Objective] Heavy chain antibodies exist only in the immune system of the camel and the VHH fragment of the variable region is the smallest natural antibody fragment. Through the sequence analysis of VHH gene to understand the genetic characteristics of Bactrian camel, camel VHH, and analyze the diversity of VHH Library are the purpose of this project. [ Method] The VHH library was constructed by PCR amplification of the VHH sequences from Xinjiang Bactrian camel （ Camelus bactrianus） peripheral blood lymphocytes. Sanger DNA sequencing and colony PCR were used to determine the size and VHH inserts of the library. A comprehensive analysis of VHH gene including the sequence homologous, CDR3 length distribution, sequence clustering were conducted via bioinformatics. [ Result] The size of the VHH library was 1.4×10^9. The positive VHH inserts in the 91 randomly selected clones was 97% in colony PCR detection. The result of DNA sequencing revealed the diversity of the library was 97.8%. Bioinformatics analysis showed almost all sequences were VHH derived except that from two clones. According to the sequence similarity, 91 sequences were classified into 7 clusters. [ Conclusion ] The constructed library has relatively large diversity and it will be helpful for analyzing the diversity of library by using bioinformatics tools.