创伤弧菌溶细胞素特定氨基酸序列分析及其人源化单链抗体筛选

Characterization of specific amino acid sequences of Vibrio vulnificus VvhA and isolation of humanized single chain antibody fragments against VvhA peptides using phage display

本研究利用生物信息学方法分析创伤弧菌(vibrio vulnificus, V.vulnificus)溶细胞素的蓖麻毒素结构域内氨基酸序列并合成多肽,筛选人工合成的单链抗体噬菌体文库Tomlinson I+J,为创伤弧菌感染的抗体阻断治疗研究奠定基础。通过对未经处理的大容量Tomlinson I+J噬菌体文库进行3轮特异性亲和筛选,从中筛到特异性结合溶细胞素多肽的克隆,命名为scFv-D2,经氨基酸序列分析验证其含有完整的人源化抗体重链和轻链可变区。将scFv-D2克隆转化E.coli HB2151并诱导表达,用ELISA方法检测其可溶性scFv片段与溶细胞素毒性多肽的结合活性。结果显示从Tomlinson I+J文库成功筛选出一株具有结合活性的人源化噬菌体单链抗体,可用于创伤弧菌感染抗体阻断治疗策略的研究。

The bioinformatics was used to analyze the amino acid sequence of functional site of the Ricin B in Vibrio vulnificus VvhA,and the synthetic peptides were used to screen the naive human synthetic phagemid library Tomlinson I＋J for specific single chain antibodies,which can lay the foundation for antibody treatment with blocking infection of Vibrio vulnificus.Three rounds of specific panning were performed through naive phage displayed human antibody libraries.One of the specifically identified anti-peptides scFv was named scFv-D2,the coding sequences of which belongs to human nucleotide sequence giving containing a V_H fragment and a V_L fragment.The clone named scFv-D2 clone was transformed into E.coli HB2151 for induced expression,and the binding activity of soluble scFv fragments with cytotoxic polypeptides was detected by ELISA.Our study showed that the scFv-D2 was isolated implied successfully to be screened from the Tomlinson I＋J phagemid library and the soluble fragements had the meaningful affinity to VvhA peptides.Our work may lay the foundation for future studies involving blocking antibody therapy of the humanized antibody off against Vibrio vulnificus infection.