[Cloning and identification of the gene fragments of Paragonimus westermani].
OBJECTIVE
To screen and identify the recombinants from the cDNA library of the adult Paragonimus westermani(PwA) for immunodiagnosis and immunoprophylaxis.


METHODS
PwA cDNA library was screened with the PwA antigen immunized rabbit sera(IRS) pre-absorbed by the extract of E. coli XL1-Blue. The recombinants from positive clones were amplified by PCR, sequenced and cut off by KpmI/BamHI and, then sub-cloned into pRESETB vector. The fusion protein was expressed, analysed by SDS-PAGE and identified by Western blotting with immune rabbit serum against worm antigen of Paragonimus westermani.


RESULTS
The inserted cDNA fragment from the positive clone Pw-2 was about 800 bp, which contained an open reading frame(ORF) encoding Pw pre-procathepsin L belonging to cysteinase family. Expression product of Pw-2 was a fusion protein of 32 kDa, which can be recognized by immune rabbit serum against worm antigen of Paragominus westermani.


CONCLUSIONS
A recombinant plasmid Pw-2 encodes Pw pre-procathepsin L is constructed.
Ling
Jia-jian
JJ
Department of Pathogenic Biology and Immunology, Nanjing Medical University, Nanjing 210029.
Hou
Min
M
Liu
Jian-nan
JN
Zhang
Zi-hao
ZH
Zhang
Yao-juan
YJ
D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D056668:Cathepsin L; D002403:Cathepsins / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D017238:Genes, Helminth; D007106:Immune Sera / Q000276:immunology; D008969:Molecular Sequence Data; D010238:Paragonimus / Q000201:enzymology / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000096:biosynthesis
D018076:DNA, Complementary; D004792:Enzyme Precursors; D007106:Immune Sera; D011993:Recombinant Fusion Proteins; D002403:Cathepsins; C063905:procathepsin L; D056668:Cathepsin L