Synthesis of recombinant human parainfluenza virus 1 and 3 nucleocapsid proteins in yeast Saccharomyces cerevisiae.
Human parainfluenza virus types 1 and 3 (HPIV1 and HPIV3, respectively), members of the virus family Paramyxoviridae, are common causes of lower respiratory tract infections in infants, young children, the immunocompromised, the chronically ill, and the elderly. In order to synthesize recombinant HPIV1 and HPIV3 nucleocapsid proteins, the coding sequences were cloned into the yeast Saccharomyces cerevisiae expression vector pFGG3 under control of GAL7 promoter. A high level of recombinant virus nucleocapsid proteins expression (20-24 mg l(-1) of yeast culture) was obtained. Electron microscopy demonstrated the assembly of typical herring-bone structures of purified recombinant nucleocapsid proteins, characteristic for other paramyxoviruses. These structures contained host RNA, which was resistant to RNase treatment. The nucleocapsid proteins were stable in yeast and were easily purified by caesium chloride gradient ultracentrifugation. Therefore, this system proved to be simple, efficient and cost-effective, suitable for high-level production of parainfluenza virus nucleocapsids as nucleocapsid-like particles. When used as coating antigens in an indirect ELISA, the recombinant N proteins reacted with sera of patients infected with HPIV1 or 3. Serological assays to detect HPIV-specific antibodies could be designed on this basis.
Juozapaitis
Mindaugas
M
Institute of Biotechnology, V.A. Graiciuno 8, Vilnius LT-02241, Lithuania. jmind@ibt.lt
Zvirbliene
Aurelija
A
Kucinskaite
Indre
I
Sezaite
Indre
I
Slibinskas
Rimantas
R
Coiras
Mayte
M
de Ory Manchon
Fernando
F
López-Huertas
María Rosa
MR
Pérez-Breña
Pilar
P
Staniulis
Juozas
J
Narkeviciute
Irena
I
Sasnauskas
Kestutis
K
D000595:Amino Acid Sequence; D000914:Antibodies, Viral / Q000097:blood / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010222:Parainfluenza Virus 1, Human / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010224:Parainfluenza Virus 3, Human / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology / Q000378:metabolism*; D010253:Respirovirus Infections / Q000175:diagnosis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*
D000914:Antibodies, Viral; D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins