Cloning, sequencing of CPI gene from periodic Brugia malayi and prediction of B cell epitopes in its amino acid sequence
10.3760/cma.j.issn.1000-4955.2010.05.013
- VernacularTitle:周期型马来丝虫CPI基因克隆和序列分析及编码产物B细胞表位预测
- Author:
Hai-yan, TONG
;
Zheng, FANG
;
Sai-nan, ZHANG
;
Bang-sheng, XU
;
Hao, FANG
;
Wei-qun, HUANG
;
Dong-fang, XIE
;
You-qin, SHI
- Publication Type:Journal Article
- Keywords:
Cysteine protease inhibitors;
Genes;
Sequence analysis;
Filariasis;
B-cell epitope
- From:Chinese Journal of Endemiology
2010;29(5):515-518
- CountryChina
- Language:Chinese
-
Abstract:
Objective To clone and sequence the cysteine protease inhibitor gene of periodic Brugia malayi(BmCPI) and predict B-cell epitopes in amino acide sequence of BmCPI in order to provide basis for further study the expression of BmCPI and its function. Methods Total RNA was extracted from periodic Brugia malayi.A couple of specific primers were designed on the basis of known sequences of cysteine protease inhibitor gene from BmCPI. The desired gene was amplified by PCR technique from cDNA. The PCR products were purified and cloned into plasmid pGEM-T by T-A cloning method, transformed into Escherichia coli(E, coli) strain DH5α. The recombinant plasmids were screened and identified by digestion with restriction enzyme and PCR amplification. Five parameters and methods were used to predict B-cell epitopes in amino acide sequence of BmCPI. Results For RT-PCR, a specific band of around 621 bp was amplified. The same band was obtained by double restriction of recombinant plasmids or PCR using recombinant plasmid as template. The result of DNA sequencing showed that BmCPI shares 99% nucleotide sequence identity with that of published sequence. It showed that B-cell epitopes were probably at or adjacent to 23 - 32, 50 - 79 and 117 - 126 in its amino acide sequence. Conclusions pGEM-BmCPI is successfully constructed and sequenced, anticipated objective is reached and conditions is provided for further study of BmCPI expression and its function.
