[Expressing of N gene encoding nucleocapsid protein of vesicular stomatitis virus and elementary application in ELISA].
The gene encoding the nucleocapsid (N) protein of vesicular stomatitis virus (VSV-NJ) was subcloned from pMD-VN5, and inserted into pBAD/Thio TOPO vector. The recombinant plasmid was identified by restriction analysis and PCR. It was sequenced to confirm the correct sequences and the correct junctional orientations of the inserted N gene. The results of SDS-PAGE and Western immunoblotting revealed that the N protein was expressed in Escherichia coli LGM194 in a high level and the recombinant fusion protein, which contained a N-terminal HP-Thioredoxin and a C-terminal polyhistidine tag. It had a molecular mass of approximately 63.5 kD and immunologically reactive activity. The recombinant protein was characterized and tested in an enzyme-linked immunosorbent assay (ELISA) format for potential application in the serodiagnosis of vesicular stomatitis using 186 serum samples from experimentally infected goats and guinea-pigs with VSV-NJ and VSV-IN, and from field origin and reference serum samples. The sensitivity and specificity of the ELISA were compared with those of the standard microtiter serum neutralization (MTSN) tests. The ELISA and MTSN test results were highly correlated for detection of VSV antibodies. The ELISA was as sensitive as the SN assay in detecting positive serum to VSV. The correlation between SN titers and ELISA titers was statistically significant. These data suggest that the recombinant fusion N protein of VSV could be used as a recombinant test antigen for the serodiagnosis of vesicular stomatitis. The ELISA based on the reconmbinant nucleocapsid protein may offer the best combination of rapidity, sensitivity, simplicity, economy, and laboratory biosafety of any of the methods yet developed for VSV serodiagnosis. This study lay on foundation for the development of the diagnosis methods in serology for VSV.
Hua
Qun-Yi
QY
Technology Center of Yunnan Entry-Exit Inspection and Quarantine Bureau, Kunming 650228, China. ciqhua@yahoo.com.cn
Jin
Ning-Yi
NY
Xu
Zi-Zhong
ZZ
Yang
Yun-Qing
YQ
Dong
Jun
J
Yang
Jing-Yan
JY
Zhou
Xiao-Li
XL
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D019590:Nucleocapsid Proteins / Q000737:chemistry / Q000235:genetics* / Q000276:immunology / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000276:immunology / Q000302:isolation & purification; D012698:Serologic Tests; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics*; D054260:Vesicular stomatitis New Jersey virus / Q000235:genetics*
D019590:Nucleocapsid Proteins; D011994:Recombinant Proteins