Construction and identification of prokaryotic expression system for galactose/N-acetyl galactosamine-specifric lectin p30 gene from Cryptosporidium
10.3969/j.issn.1002-2694.2010.02.010
- VernacularTitle:隐孢子虫Gal/GalNAc特异性凝集素p30原核表达系统的构建及鉴定
- Author:
Huicong HUANG
;
Qiaolian ZHAO
;
Feng TAN
;
Changwang PAN
- Publication Type:Journal Article
- Keywords:
Cryptosporidium;
p30 gene;
cloning;
prokaryotic expression
- From:
Chinese Journal of Zoonoses
2010;(2):134-139
- CountryChina
- Language:Chinese
-
Abstract:
To clone and construct a prokaryotic expression system containing the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium, the p30 gene was amplified from genomic DNA of Cryptosporidium parvum by PCR and cloned into vector pMD18-T directly. The positive clones were identified by EcoR I, Xho I digestion and sequenced. The gene structure and its possible function were analyzed and predicted by using related bioinformatics softwares. The P30 gene was recombined with plamid pET-28 a (+) to construct the prokaryotic expression vector and was expressed in E. Coli with IPTG induction. Ni-NTA affinity chromatography was used to extract P30 protein and the expression effect and purification of P30 protein were determined by SDS-PAGE and Western blotting. It was demonstrated that the galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum was specifically amplified, and its sequence homology of nucleotide and the deduced amino acid sequence of P30 gene with relevant sequences in GenBank were 98%-100% and 99%-100% respectively. Its theoretical iso-electric point and molecular weight were found to be 6.4854 and 31842 dalton.It was predicted to contain 9 potential epitopes. The expressed plasmid was identified by EcoR I/ Xho I digestion and sequenced and the recombinant P30 protein could be identified by SDS-PAGE and Western blotting assay. It is evident that the prokaryotic expression system for galactose/N-acetyl galactosamine-specific lectin p30 gene of Cryptosporidium parvum has been constructed successfully.
