Cloning of acidic mammalian chitinase gene mutant and its sequence analysis
- VernacularTitle:小鼠AMCase基因突变体的cDNA克隆及序列分析
- Author:
Ling CHEN
;
Zhu SHEN
;
Yufeng LIU
- Publication Type:Journal Article
- Keywords:
acidic mammalian chitinase;
mutant;
sequence analysis,DNA
- From:
Medical Journal of Chinese People's Liberation Army
1982;0(03):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone acidic mammalian chitinase (AMCase) gene and analyze the sequences of the nucleotides and encoded amino acids. Methods Total RNA was isolated from the stomach of BALB/c mouse and the mRNA was reversely transcribed into cDNA. AMCase gene was amplified by polymerase chain reaction and inserted into recombinant clone vector of pMD18-T. Sequence alignment was performed with DNAMAN 4.0 software. Isoelectric point, amino acid composition, signal peptide and transmembrane helices were analyzed with SignalP 3.0 Server and TMHMM Server v.2.0. Results The cloned gene encodes a protein of 473 amino acids with molecular weight of 51.93 kDa and isoelectric point 4.73. Extensive amino acid sequence homology (up to 99.57%) was found when compared with AMCase. Both protein encoded by this cloned gene and AMCase belonged to family 18 chitinase, which was composed of signal peptide (amino acid position 1-21), the catalytic domain (amino acid position 22-391), hinge region (amino acid position 392-425) and chitin binding domain (amino acid position 426-473). There were two mutations of amino acid in the catalytic domain and hinge region with this AMCase mutant. The amino acid of Ala(position 292)was mutated into amino acid of Pro, and the amino acid of Thr(position 404)was mutated into amino acid of Ser. Conclusion This sequence is a mutant of AMCase gene. Identification of the biological characteristics will play important role in the further study of its biological function by molecular biology technique.
