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.
ABSTRACT
To provide the basis for preparation of diagnostic kits and vaccines in Toxoplasma gondii infection, the gene coding for the qualified recombinant p30 protein (SAG1) of this parasite was amplified by PCR, and the amplified gene was cloned into prokaryotic expression vector pET-30a(+) to construct the recombinant plasmid, and then transformed to E.coli DH5α. The positive recombinant plasmid was screened by PCR and double enzymes digestion, and the nucleotide sequence of p30 gene was determined by automated DNA sequencing. Meanwhile, the identified recombinant plasmid was transformed to E.coli BL21(DE3) with the expression of p30 on bacteria induced by IPTG and the expressed protein was identified by SDS-PAGE. The protein obtained was then further purified and refolded, and its biological activity was checked by Western blotting. It was shown that the size of the amplified gene was 750 bp with molecular weight of 30 ku, and this protein could specifically react with monoclonal antibody against p30 protein.