Expression of Sendai virus nucleocapsid protein in a baculovirus expression system and application to diagnostic assays for Sendai virus infection.
The most common diagnostic technique for the detection of Sendai virus infection in rodents is serological evaluation by enzyme-linked immunosorbent assay (ELISA) with semipurified preparations of whole virions as antigens. This assay often suffers from a lack of specificity. The goal of the present project was to develop more specific antigens for use in diagnostic testing by producing recombinant antigens in insect cells. To identify viral proteins immunoreactive in multiple laboratory rodent species, Western blots (immunoblots) of viral polypeptides were probed with immune sera from mice, rats, and hamsters. The nucleocapsid protein (NP) reacted with immune sera from all species tested. Therefore, the NP gene was selected for cloning and expression in a baculovirus. To construct the recombinant, complementary DNA was synthesized by reverse transcription PCR from Sendai virus RNA with primers from the 5' and 3' termini of the NP-coding region. Amplified DNA was cloned into a baculovirus transfer vector (pBlueBacHis A) and was cotransfected with wild-type baculovirus into insect cells. Baculovirus recombinants containing the NP gene were identified by PCR. Evaluation of the recombinant proteins expressed in insect cells by Western blot analysis revealed specific reactivity with immune sera. In comparison with conventional ELISAs that use whole virions as the antigen, ELISAs that use recombinant NP were more specific.
Wan
C H
CH
Department of Veterinary Pathology, University of Missouri-Columbia, 65211, USA.
Riley
M I
MI
Hook
R R
RR
Franklin
C L
CL
Besch-Williford
C L
CL
Riley
L K
LK
D000818:Animals; D000830:Animals, Laboratory; D000956:Antigens, Viral / Q000235:genetics; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006224:Cricetinae; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000706:statistics & numerical data; D005814:Genes, Viral; D051379:Mice; D008969:Molecular Sequence Data; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D010222:Parainfluenza Virus 1, Human / Q000235:genetics* / Q000276:immunology; D018184:Paramyxoviridae Infections / Q000175:diagnosis* / Q000276:immunology; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D012680:Sensitivity and Specificity; D012698:Serologic Tests / Q000379:methods* / Q000706:statistics & numerical data; D018411:Spodoptera; D014758:Viral Core Proteins / Q000235:genetics* / Q000276:immunology
D000956:Antigens, Viral; D017931:DNA Primers; D004279:DNA, Viral; D019590:Nucleocapsid Proteins; D009698:Nucleoproteins; D011994:Recombinant Proteins; D014758:Viral Core Proteins