[Cloning, purification, and antigenic characterization of three recombinant fragments derived from SARS-CoV S1 domain].
OBJECTIVE
The present study aimed to clone and express three fragments of genomic RNA derived from SARS associated coronavirus (SARS-CoV) S1 domain and to study its immunogenicity.


METHODS
The S1 domain gene was amplified by PCR with specific primers and was inserted into the prokaryotic expression vector pQE-30. Three fragments (40-751, 746-1344 and 746-2001 bp) derived from S1 domain produced after the recombinant plasmid (pQE-30/S1) was digested by restriction endonucleases. The three fragments were cloned into pQE-30 and expressed in M15 strains of Escherichia coli. The expression products, designated S1a, S1b and S1c respectively, were purified by Ni affinity chromatography. The immunogenicity was analyzed by Western Blot and ELISA using serologically confirmed sera from SARS patients and the sera from healthy donors was used as control at the same assay.


RESULTS
Three recombinant plasmids (pQE-30/S1a, pQE-30/S1b, pQE-30/S1c) were constructed.Fusion proteins with relative molecular mass of 26,700, 22,500 and 46,000 dalton were successfully expressed with amounts of 35%, 35% and 30% of total cell protein and purified by Ni affinity chromatography, respectively. Western Blot and ELISA analysis showed that the S1c protein could be specifically recognized by the sera from SARS patients.


CONCLUSIONS
The recombinant S1c protein was a good immunogen and has the potential to be used as a vaccine against SARS-CoV infection.
Mei
Ya-bo
YB
Central Laboraory, Zhujiang Hospital, The First Military Medical University, Guangzhou 510282, China.
Liao
Zhi-yong
ZY
Wang
Ya-di
YD
Zhang
Li-ya
LY
Xu
Hua
H
Yuen
Kwok-yung
KY
Che
Xiao-yan
XY
D000914:Antibodies, Viral / Q000097:blood; D000954:Antigens, Surface / Q000235:genetics / Q000276:immunology / Q000378:metabolism; D015153:Blotting, Western; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007074:Immunoglobulin G / Q000097:blood; D007075:Immunoglobulin M / Q000097:blood; D010957:Plasmids / Q000235:genetics; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D045473:Severe acute respiratory syndrome-related coronavirus / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D045169:Severe Acute Respiratory Syndrome / Q000097:blood / Q000821:virology; D014759:Viral Envelope Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism
D000914:Antibodies, Viral; D000954:Antigens, Surface; D007074:Immunoglobulin G; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014759:Viral Envelope Proteins