[Cloning, expression and immunogenicity analysis of cathepsin L-like protease of Fasciola hepatica].
OBJECTIVE
To clone and express the cathepsin L-like protease gene of Fasciola hepatica (FhCL) and investigate the immunogenicity of the recombinant FhCL protein.


METHODS
Specific primers were designed according to the reported FhCL gene in GenBank. Using total RNA from adult worms of F. hepatica, FhCL gene was amplified by RT-PCR. The PCR product was cloned into pMD18-T vector and then subcloned into pET30a(+) vector. The recombinant plasmid was transformed into E. coli BL21 (DE3) and followed by expression of the protein induced by IPTG. The expression situation of recombinant FhCL was analyzed by SDS-PAGE. Its immunoresponse to the sera of infected goat and the antisera of SD rats against FhCL was examined by Western blotting analysis.


RESULTS
PCR and double enzyme digestion showed that the FhCL gene fragment was about 1,000 bp in length. The constructed recombinant plasmid pET30a (+)-FhCL was identified by sequencing. The recombinant protein (Mr 42,000) was expressed in the form of inclusion body. The protein was recognized respectively by the sera of infected goat and the sera from rat immunized with FhCL.


CONCLUSIONS
The recombinant plasmid pET30a(+)-FhCL has been constructed, which shows high antigenicity.
Li
Xiao-Juan
XJ
Key Laboratory of Preventive Veterinary Medicine of Heilongjiang Province, Heilongliang August-First Land Reclamation University, Daqing 163319, China.
Wen
Xiao-Bo
XB
Ran
Xu-Hua
XH
Li
Shu-Dong
SD
Wang
Chun-Ren
CR
Piao
Fan-Ze
FZ
D000818:Animals; D056668:Cathepsin L / Q000235:genetics / Q000276:immunology*; D003001:Cloning, Molecular; D005210:Fasciola hepatica / Q000201:enzymology* / Q000235:genetics* / Q000276:immunology; D015870:Gene Expression; D006041:Goats; D015801:Helminth Proteins / Q000235:genetics / Q000276:immunology*; D010957:Plasmids; D051381:Rats; D017207:Rats, Sprague-Dawley
D015801:Helminth Proteins; D056668:Cathepsin L