用简并引物快速克隆苛求芽孢杆菌尿酸酶的编码序列

发布时间：2018-03-04 07:33

本文选题：苛求芽孢杆菌尿酸酶　切入点：简并引物PCR　出处：《重庆医科大学学报》2017年02期 　论文类型：期刊论文

【摘要】：目的:从头克隆苛求芽孢杆菌尿酸酶基因的编码序列。方法:据Edman降解所得N端氨基酸序列(AERTMFYGKGDV)用BLASTp搜索N端相似性70%细菌尿酸酶;多序列联配比对发现靠近N端和C端的2个保守肽段。据N端已知序列和2个保守肽段设计简并引物进行PCR扩增;将所得片段连接到T载体,转化后筛选阳性克隆测序。结果:据密码子偏好性调整简并引物,获得该尿酸酶从第六位氨基酸到终止密码子之间的编码序列;此尿酸酶共含322个氨基酸残基,原核尿酸酶靠近N端的保守序列ATDSMKNFI从该尿酸酶第70位氨基酸残基左右开始,另一保守序列GKVYTEPR从该尿酸酶第290位氨基酸残基开始但变为GAVFTEPR。结论:用简并引物PCR快速克隆获得了苛求芽孢杆菌尿酸酶的编码序列。
[Abstract]:Objective: to clone the coding sequence of urease gene from Bacillus causticus. Methods: according to the N-terminal amino acid sequence of Edman degradation, BLASTp was used to search for the N-terminal similarity of 70% bacterial uric acid enzyme. Two conserved peptides close to N-terminal and C-terminal were found by multisequence matching pair. Degenerate primers were designed for PCR amplification according to N terminal known sequence and 2 conserved peptide segments, and the resulting fragments were ligated to T vector. Results: according to the preference of codon to adjust degenerate primers, the coding sequence from 6th amino acid to stop codon was obtained, and the urease contained 322 amino acid residues. The conserved sequence ATDSMKNFI of prokaryote near N-terminal starts from the 70th amino acid residues of the urease. Another conserved sequence, GKVYTEPR, started from the 290th amino acid residue of the uricase but became GAVFTEPR.Conclusion: the coding sequence of uricase from Bacillus causticus was rapidly cloned by degenerate primer PCR.
【作者单位】：秦皇岛市第一医院检验科;重庆医科大学基础医学院生物化学与分子生物学教研室;重庆医科大学检验医学院临床检验诊断学教育部重点实验室;
【分类号】：R440

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