论文部分内容阅读
通过易错PCR方法建立了一个鼠肺不同长度的nGLP-1R(从第21个氨基酸开始到第145个氨基酸)的噬菌体随机突变展示肽库,通过噬菌体表面展示技术检测胰高血糖素样肽1受体N端片段(nGLP-1R)在缺失一段或两段基因后是否还具有结合Exendin-4的活性。经ELISA分析发现了一株无结合活性的突变株,命名为EP16。经测序比对,发现EP16缺失了前20个和后10个氨基酸,且第52位色氨酸突变为精氨酸。为确定EP16与Exendin-4无结合活性的原因,重新构建了无前20个和后10个氨基酸的EP16野生型及第52位色氨酸变为精氨酸的全长nGLP-1RW52R与EP16进行对比分析。结果表明,EP16的活性丧失是由保守的第52位色氨酸突变为精氨酸引起的,缺失的前20个和后10个氨基酸没有影响其生物学活性。关键位点单个氨基酸残基的突变可以改变胰高血糖素样肽1受体N端片段整个蛋白质的生物学活性。
A randomized phage display of nBLP-1R (from the 21st amino acid to the 145th amino acid) different lengths of murine lung was performed by error-prone PCR to display the peptide library. The expression of glucagon-like peptide 1 The receptor N-terminal fragment (nGLP-1R) also has the activity of binding to Exendin-4 after deletion of one or two genes. A non-binding mutant was identified by ELISA and named as EP16. After sequencing comparison, it was found that the first 20 and the last 10 amino acids were deleted in EP16, and the tryptophan in position 52 was changed to arginine. To determine the non-binding activity of EP16 with Exendin-4, full-length nGLP-1 WR52R was reconstructed with EP16 wild-type without EGF before and after 10 amino acids and arginine with tryptophan at position 52 with EP16 Comparative analysis. The results showed that the loss of activity of EP16 was caused by the mutation of the conservative tryptophan at position 52 to arginine, and the first 20 and last 10 amino acids of the deletion did not affect its biological activity. Mutation of a single amino acid residue at the key site can alter the biological activity of the entire protein at the N-terminal fragment of the glucagon-like peptide 1 receptor.