Characterization of recombinant foot-and-mouth disease virus pentamer-like structures expressed by baculovirus and their use as diagnostic antigens in a blocking ELISA.
Non-infectious recombinant pentamer-like structures of the foot-and-mouth disease virus (FMDV) were expressed by baculovirus, and the antigenicity and immunogenicity of the proteins were analyzed in a blocking ELISA for the detection of FMDV antibodies. The recombinant pentamer-like structures were produced in insect (Sf9) cells that were inoculated with recombinant baculoviruses that expressed, simultaneously, the genes for the P1 and 3C proteins of FMDV from individual promoters. The FMDV pentamer-like structures were processed by viral 3C protease, as shown in Western blots, and were antigenic, as revealed by their reactivities in an indirect ELISA. Analysis by CsCl gradient centrifugation showed that the pentamer-like structures were similar to authentic pentameric subunits from FMDV in terms of sedimentation velocity. Furthermore, the pentamer-like structures induced high levels of FMDV-specific antibodies in mice following immunization. Observations made under the electron microscope revealed that the pentamer-like structures expressed by insect cells self-assembled to form pentameric subunits of 7-8 nm in diameter, which resemble the authentic FMDV (23+/-2 nm in diameter). The results indicate that these pentamer-like structures are as antigenic and immunogenic as authentic FMDV, although the former are smaller in size. Based on these results, a blocking ELISA was developed using the recombinant pentamer-like structure. The ELISA showed specificity of 99.5% and sensitivity of 98.5% when tested with FMDV antibody-negative and -positive sera, respectively. This blocking ELISA is highly specific and offers many advantages over the current ELISAs that use inactivated FMDV antigen. This is the first report of the production and diagnostic application of recombinant pentameric subunits of FMDV.
Oem
Jae-Ku
JK
National Veterinary Research and Quarantine Service, 480 Anyang-6-dong, Anyang, Kyong-gi 430-824, Republic of Korea. jku0622@nvrqs.go.kr
Park
Jong-Hyeon
JH
Lee
Kwang-Nyeong
KN
Kim
Yong-Joo
YJ
Kye
Soo-Jeong
SJ
Park
Jee-Yong
JY
Song
Hee-Jong
HJ
D000818:Animals; D000956:Antigens, Viral / Q000096:biosynthesis / Q000235:genetics / Q000276:immunology*; D016367:Baculoviridae / Q000235:genetics* / Q000276:immunology; D002417:Cattle; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D005260:Female; D029544:Foot-and-Mouth Disease Virus / Q000235:genetics* / Q000276:immunology*; D051379:Mice; D008807:Mice, Inbred BALB C; D018411:Spodoptera / Q000821:virology; D013552:Swine
D000956:Antigens, Viral