[Cloning, expression and analysis of the heat shock protein of Cryptosporidium andersoni].
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.


CONCLUSIONS
The recombinant plasmid pET28a-CaHSP70 has been constructed. The purified rCaHSP70 exhibits high antigenicity and seems a potential candidate antigen for immunodiagnosis of cryptosporidiosis.
National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, MOH, WHO Collaborating Centre for Malaria, Schistosomiasis and Filariasis, Shanghai 200025, China).
Hai-peng
HP
Liu
Jian-ping
JP
Cao
Xiao-hong
XH
Li
Wei-yuan
WY
Lu
Yu-juan
YJ
Shen
Yu-xin
YX
Xu
Wei
W
Zang
Shu-xian
SX
Liu
D000818:Animals; D015153:Blotting, Western; D002417:Cattle; D003001:Cloning, Molecular; D003458:Cryptosporidium / Q000145:classification / Q000235:genetics* / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D015870:Gene Expression; D018840:HSP70 Heat-Shock Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D007106:Immune Sera / Q000032:analysis / Q000276:immunology; D007114:Immunization / Q000379:methods; D008297:Male; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D010802:Phylogeny; D015800:Protozoan Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000276:immunology; D020133:Reverse Transcriptase Polymerase Chain Reaction
D018840:HSP70 Heat-Shock Proteins; D007106:Immune Sera; D015800:Protozoan Proteins; D011994:Recombinant Proteins