用简并引物快速克隆苛求芽孢杆菌尿酸酶的编码序列  

Rapidly cloning gene sequence of the uricase from Bacillus fastidious by degenerated primer

作　　者：赵运胜[1] 卜友泉[2] 廖飞[3] 

机构地区：[1]秦皇岛市第一医院检验科,秦皇岛066000 [2]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400016 [3]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,重庆400016

出　　处：《重庆医科大学学报》2017年第2期219-224,共6页Journal of Chongqing Medical University

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。Objective：To clone the whole encoding sequence of the uricase from Bacillus fastidious. Methods：According to the N-terminus amino sequence AERTMFYGKGDV from Edman degradation,bacterial uricases bearing similar N-terminus were sought in NCBI database by BLASTp;through multiple sequence alignment,two short conserved peptide fragments were found,one close to the N-terminus while the other close to the C-terminus. Using the known N-terminus and two conserved peptides,degenerated primers were designed for cloning the encoding sequence of the uricase by PCR;the resulting fragments were inserted into T vector for sequencing. Results：After the adjustment of degeneration of the primers according to the code preference,the encoding sequence of the uricase was obtained from the sixth amino acid residue to the termination code. The translated enzyme contained 322 amino. The conserved peptide fragment ATDSMKNFI started from 70 th amino acid residue while the other conserved peptide fragment GKVYTEPR started from 290 th amino acid residue with the mutated sequence of GAVFTEPR. Conclusion：The encoding sequence of the uricase is cloned rapidly by degenerated primer PCR.

关 键 词：苛求芽孢杆菌尿酸酶 简并引物PCR 基因测序 

分 类 号：Q523[生物学—生物化学]