[Construction and expression of prokaryotic expression plasmids of Pneumocystis carinii p55 antigen gene fragment].
OBJECTIVE
To construct prokaryotic recombinant expression plasmid carrying Pneumocystis carinii Mr 55 000 antigen (p55) gene fragment and express the recombinant protein.


METHODS
P. carinii pneumonia (PcP) rat models were established by subcutaneous injection of dexamethasone for 14 weeks. Total RNA was extracted from lung of P. carinii rat and p55 antigen gene fragment was cloned by RT-PCR, which was identified by sequencing. The 690 bp fragment was cloned to pGEX-4T-1, the recombinant plasmid was screened and identified by restriction analysis and PCR. The recombinant plasmid was finally induced with IPTG to express a new fusion protein, and the products were analyzed by SDS-PAGE and Western blot.


RESULTS
A fragment of 690 bp was obtained by RT-PCR. The recombinant pGEX-4T-1/690 was constructed. SDS-PAGE revealed that the molecular weight of the recombinant protein was approximately Mr 62,000, the maximum amount of the fusion protein produced was 11.6% of the total protein. The recombinant protein can be recognized by GST antibody and by the sera from P. carinii infected rats using Western blotting.


CONCLUSIONS
Prokaryotic expression plasmid pGEX-4T-1/690 has been constructed and the recombinant fusion protein shows antigenicity.
Wang
Jian-xin
JX
School of Medicine, Nantong University, Nantong 226001, China.
Duan
Yi-nong
YN
Chen
Jin-ling
JL
Dong
Yong-sheng
YS
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D003907:Dexamethasone; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D015870:Gene Expression; D007106:Immune Sera / Q000276:immunology; D008297:Male; D010446:Peptide Fragments / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D010957:Plasmids; D045363:Pneumocystis carinii / Q000235:genetics* / Q000276:immunology / Q000378:metabolism; D011020:Pneumonia, Pneumocystis / Q000097:blood / Q000139:chemically induced / Q000382:microbiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000276:immunology / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction
D005656:Fungal Proteins; D007106:Immune Sera; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003907:Dexamethasone