Production of polyclonal antibodies against the human respiratory syncytial virus nucleoprotein and phosphoprotein expressed in Escherichia coli.
The nucleoprotein (N) and the phosphoprotein (P) of the human respiratory syncytial virus (HRSV), A2 strain, were cloned into pMAL-c2e vector. The proteins were expressed fused with the maltose-binding protein (MBP) and were preferentially found in the soluble fraction of the bacterial lysate. After their purification using amylose resin, almost no other protein was detected in SDS-PAGE. The fused proteins were cleaved by digestion with enterokinase and then used as antigens for BALB/c mice immunization. The obtained polyclonal antibodies were tested against HRSV infected cells in immunofluorescence assays. The results indicate that the antibodies generated against the recombinant proteins were able to recognize the virus proteins. We now intend to purify the cleaved N and P proteins and use them in structural studies. The recombinant proteins will also be tested as potential inducers of a protective immunity against the HRSV.
Simabuco
Fernando M
FM
Departamento de Microbiologia, Instituto de Ciências Biomédicas, Universidade de São Paulo, Avenida Professor Lineu Prestes, 1374, São Paulo-SP 05508-900, Brazil.
Carromeu
Cassiano
C
Farinha-Arcieri
Luis E
LE
Tamura
Rodrigo E
RE
Ventura
Armando M
AM
D000818:Animals; D000914:Antibodies, Viral / Q000096:biosynthesis* / Q000276:immunology; D000956:Antigens, Viral / Q000276:immunology; D015153:Blotting, Western; D006528:Carcinoma, Hepatocellular / Q000473:pathology; D002352:Carrier Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D004765:Enteropeptidase / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005260:Female; D019085:Fluorescent Antibody Technique, Direct; D005822:Genetic Vectors; D006801:Humans; D008113:Liver Neoplasms / Q000473:pathology; D058619:Maltose-Binding Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D009698:Nucleoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D018357:Respiratory Syncytial Virus Infections / Q000276:immunology; D018113:Respiratory Syncytial Virus, Human / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012995:Solubility; D014170:Transformation, Genetic; D014611:Vaccination
D000914:Antibodies, Viral; D000956:Antigens, Viral; D002352:Carrier Proteins; D058619:Maltose-Binding Proteins; D009698:Nucleoproteins; D010750:Phosphoproteins; D011993:Recombinant Fusion Proteins; D004765:Enteropeptidase