Zhao Yunsheng, Bu Youquan, Liao Fei. Rapidly cloning gene sequence of the uricase from Bacillus fastidious by degenerated primer. [J]. Journal of Chongqing Medical University 42(2):219-224(2017)
DOI:
Zhao Yunsheng, Bu Youquan, Liao Fei. Rapidly cloning gene sequence of the uricase from Bacillus fastidious by degenerated primer. [J]. Journal of Chongqing Medical University 42(2):219-224(2017) DOI: 10.13406/j.cnki.cyxb.001001.
Rapidly cloning gene sequence of the uricase from Bacillus fastidious by degenerated primer
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
Keywords
bacillus fastidious uricasedegenerated primer PCRgene cloning