[Cloning, expression and analysis of the heat shock protein of Cryptosporidium andersoni].
Zhongguo Ji Sheng Chong Xue Yu Ji Sheng Chong Bing Za Zhi
; 25(3): 163-70, 2007 Jun.
Article
em Zh
| MEDLINE
| ID: mdl-18038769
ABSTRACT
OBJECTIVE:
To clone and express the partial encoding sequence of Mr 70,000 heat shock protein of Cryptosporidium andersoni (CaHSP70) in Escherichia coli and identify the recombinant protein.METHODS:
Total RNA was extracted from oocysts of C. andersoni isolated from Xuzhou, Jiangsu (XZ-BOV). The CaHSP70 gene was amplified by RT-PCR. The PCR product was cloned and then subcloned into pET28a vector, and the recombinant plasmids were transformed into E.coli BL21(DE3) subsequently. The expressed protein induced by IPTG was purified and identified by SDS-PAGE and Western blotting, and was further analyzed by relevant bioinformatics softwares. The specific IgG antibodies in mice immunized by rCaHSP70 were detected by Western blotting and ELISA respectively.RESULTS:
The deduced amino acid sequence showed to be identical with that of C. andersoni Mr 70,000 heat shock protein (HSP70). The recombinant protein expressed in the form of inclusion body was about Mr 43,000. It could be recognized by anti-His G labeled HRP antibodies and all the sera from mice infected with C. andersoni and children infected with C. parvum as well as sera from mice immunized with rCaHSP70 respectively. The rCaHSP70 possibly had multiple domains and potential antigenic determinants. Phylogenetic analysis showed that XZ-BOV and C. andersoni were in the same clade. ELISA showed that the level of specific antibodies against rCaHSP70 in immunized BALB/c and C57BL/6 mice was significantly higher than that of mice before immunization.CONCLUSION:
The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteínas de Protozoários
/
Proteínas de Choque Térmico HSP70
/
Cryptosporidium
Limite:
Animals
Idioma:
Zh
Ano de publicação:
2007
Tipo de documento:
Article