The expression of SARS-CoV M gene in P. Pastoris and the diagnostic utility of the expression product.
High-level protein expression is an important means of obtaining large amounts of viral proteins to investigate further their biological properties. To express the membrane (M) protein of SARS-CoV at high-level in vitro, the M gene fragment was amplified and cloned it into the Pichia Pastoris expression vector pPICZalphaA. SDS-PAGE and Western blotting analysis of the induced products of recombinant yeast transformant indicated that successful high-level expression of M protein was achieved, and that the expression product was similar antigenically to the natural protein. Purified recombinant M protein was used subsequently as an ELISA antigen for detection of eight serum samples screened previously by whole virus ELISA and immunofluorescence assay, and consistent results were obtained. These findings suggest that the recombinant M protein may be useful as a diagnostic reagent.
Tsinghua University and National Laboratory of Biomacromolecules, Institute of Biophysics, Chinese Academy of Science, Beijing, PR China. hanxueq@yahoo.com.cn
Xueqing
X
Han
Mark
M
Bartlam
Ying-Hua
YH
Jin
Xiangtao
X
Liu
Xiaojing
X
He
Xuepeng
X
Cai
Qingqe
Q
Xie
Zihe
Z
Rao
D000328:Adult; D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000097:blood*; D015153:Blotting, Western; D003001:Cloning, Molecular; D000086862:Coronavirus M Proteins; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D007158:Immunologic Techniques; D008297:Male; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis; D016415:Sequence Alignment; D045169:Severe Acute Respiratory Syndrome / Q000175:diagnosis* / Q000821:virology; D014763:Viral Matrix Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000276:immunology* / Q000302:isolation & purification
D000914:Antibodies, Viral; D000086862:Coronavirus M Proteins; D011994:Recombinant Proteins; D014763:Viral Matrix Proteins