Serodiagnosis using recombinant nipah virus nucleocapsid protein expressed in Escherichia coli

Abstract

Nipah virus nucleocapsid (NiV-N) protein was expressed in Escherichia coli and purified by histidine tag-based affinity chromatography. An indirect immunoglobulin G (IgG) enzyme-linked immunosorbent assay (ELISA) for human and swine sera and an IgM capture ELISA for human sera were established using the recombinant NiV-N protein as an antigen. One hundred thirty-three suspected patient sera and 16 swine sera were used to evaluate the newly established ELISA systems in comparison with the CDC inactivated-virus-based ELISA systems. For the human sera, the NiV-N protein-based indirect IgG ELISA had a sensitivity of 98.6% and a specificity of 98.4%, and the NiV-N protein-based IgM capture ELISA had a sensitivity of 91.7% and a specificity of 91.8%, with reference to the CDC ELISA systems. The NiV-N-based IgM ELISA was found to be more sensitive than the inactivated-virus-based ELISA in that it captured eight additional cases. For the swine sera, the two test systems were in 100% concordance. Our data indicate that the Nipah virus nucleocapsid protein is a highly immunogenic protein in human and swine infections and a good target for serodiagnosis. Our NiV-N protein-based ELISA systems are useful, safe, and affordable tools for diagnosis of Nipah virus infection and are especially fit to be used in large-scale epidemiological investigations and to be applied in developing countries.

FIG. 1.

Expression and purification of recombinant NiV-N protein. A recombinant plasmid containing the full-length Nipah virus N gene was transformed into E. coli XL1-Blue and induced with IPTG. E. coli cells were collected and dissolved in 10 mM PBS (pH 7.5)-500 mM NaCl. After sonication, the E. coli cell lysate was centrifuged and the recombinant protein was purified from the supernatant by use of a Talon immobilized metal affinity column. The E. coli cell lysate and purified recombinant protein were analyzed in a 10% SDS-PAGE gel and revealed with Coomassie brilliant blue staining. Lane M, protein marker (SDS-7B); lane 1, supernatant of sonicated E. coli cell lysate after centrifugation; lane 2, pellet of sonicated E. coli cell lysate; lane 3, purified recombinant protein.

FIG. 2.

Western blot analysis of purified NiV-N protein. A prestained protein marker and purified recombinant NiV-N protein were separated by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. Each membrane was incubated with diluted mouse antihistidine serum or human patient serum followed by horseradish peroxidase-conjugated anti-mouse IgG or anti-human IgG (1:1,000 dilution) and the results visualized by DAB staining. (A) Reactivity of recombinant protein to mouse antihistidine serum. (B) Reactivity of recombinant protein to Nipah virus patient serum. Lanes M, protein marker (SDS-7B); lanes 1, purified NiV-N protein.