Cloning, expression and characterization of HSP gene from Eimeria tenella.
- Author:
Yan YAN
1
;
Hongyu HAN
;
Bing HUANG
;
Qiping ZHAO
;
Hui DONG
;
Lianlian JIANG
;
Yujian LI
;
Yujuan FAN
;
Qian YAO
Author Information
1. Key Laboratory of Animal Parasitology of Ministry of Agriculture, Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai 200241, China.
- Publication Type:Journal Article
- MeSH:
Animals;
Chickens;
Eimeria tenella;
genetics;
metabolism;
Escherichia coli;
genetics;
metabolism;
Heat-Shock Proteins;
genetics;
immunology;
metabolism;
Inclusion Bodies;
metabolism;
Male;
Molecular Sequence Data;
Open Reading Frames;
genetics;
Rabbits;
Recombinant Fusion Proteins;
genetics;
immunology;
metabolism;
Sequence Analysis, Protein
- From:
Chinese Journal of Biotechnology
2009;25(8):1121-1129
- CountryChina
- Language:Chinese
-
Abstract:
In order to study the functions of the HSPs (Heat shock proteins) of Eimeria tenella, we cloned a novel gene (which designated EtHSP) coding HSP of Eimeria tenella by RT-PCR and RACE (Rapid-amplification of cDNA ends). The full-length cDNA sequence of EtHSP was 1802 bp, containing a 1455 bp ORF (Open reading frame) (GenBank Accession No. FJ911605) encoding a deduced protein of 484 amino acids. Real-time PCR revealed that the mRNA level of EtHSP was much higher in sporozoites of E. tenella than other developmental stages (unsporulated oocysts, sporulated oocysts and merozoites). We constructed the recombinant plasmids pET28a(+)-EtHSP, then transformed it into E. coli BL21(DE3) for expression. SDS-PAGE indicated that the fusion protein was expressed in included bodies, with peak expression 6 h after induction by IPTG Western blotting revealed that the protein was specifically recognized by polyclonal antibodies against E. tenella, showing that the fusion protein was native antigen.
