[Study on the recombinant expression of Hantaan virus protein N and the establishment and application of rNP-IgM direct capture ELISA].
OBJECTIVE
To clone the gene encoding nucleocapsid protein (NP) of hantavirus strain Z10 (HV-Z10), to construct its prokaryotic expression system as well as to establish a rNP-IgM direct capture ELISA based on HRP-labeled recombinant NP (rNP), in order to detect serum samples of patients suffering from hemorrhagic fever with renal syndrome (HFRS) and to evaluate the effects of detection.


METHODS
Gene encoding NP of strain HV-Z10 was amplified by PCR and then its prokaryotic expression system pET28a-Z10N-E. coli BL21DE3 was constructed, using routine genetic engineering method. SDS-PAGE was applied to measure the expression of rNP and ion-exchange plus Ni-NTA-affinity chromatography was performed to purify the recombinant product. Western blot assay was used to determine the specific immuno-reactivity of rNP while HRP-labeled rNP-IgM direct capture ELISA was established to detect the serum samples from 95 cases of confirmed HFRS patients. The detection effect was compared with that by routine HV-IgM indirect capture ELISA method.


RESULTS
pET28a-Z10N-E. coli BL21DE3 was able to express rNP with high efficiency. The purified rNP only showed a single protein fragment in the gel after SDS-PAGE. HV-IgG could efficiently recognize rNP and hybridize with the recombinant protein. 94.73% (90/95) of HFRS patients' serum samples were positively confirmed by rNP-IgM direct capture ELISA, while a positive rate of 92.63% (88/95) in the same samples was confirmed by HV-IgM indirect capture ELISA. The distributions of A450 values of the serum samples detected by the two IgM capture ELISAs as well as the changes of the A450 mean values from several serum samples with different dilutions were similar.


CONCLUSIONS
We successfully constructed a high efficient prokaryotic expression system of NP encoding gene of hantavirus strain HV-Z10. The rNP-IgM direct capture ELISA that established in this study could be used as a new serological test for HFRS diagnosis because of its simplicity, safety, with high sensitivity and specificity.
Zhu
Han-Ping
HP
Department of Medical Microbiology and Parasitology, College of Medicine, Zhejiang University, Hangzhou 310058, China.
Yao
Ping-Ping
PP
Xu
Fang
F
Weng
Jing-Qing
JQ
Xie
Rong-Hui
RH
Lu
Qun-Ying
QY
Zhu
Zhi-Yong
ZY
Yan
Jie
J
D015153:Blotting, Western; D036022:Capsid Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007075:Immunoglobulin M / Q000276:immunology*; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*; D014758:Viral Core Proteins / Q000235:genetics / Q000276:immunology* / Q000378:metabolism*
D036022:Capsid Proteins; D007075:Immunoglobulin M; D011994:Recombinant Proteins; D014758:Viral Core Proteins; C111782:nucleocapsid protein, Hantaan virus