Nucleocapsid protein (NP)which exists in the particle of hantavirus and surrounds the viral RNA genome is one of the major structural proteins and plays role of antigen to elicit the antibody detected predorminantly right after infection of the
virus in
the patients of hemorragic fever with renal syndrome (HFRS)or experimental animals. NP is important target antigen in serological diagnostic system of HFRS utilizing whole antigens from the native virus particle, such as IFA, ELISA and Western
blotting.
Therefore, the preparation of this protein in the level of higher quantiity and purity is desirasble for developed dianosis of the disease. The purpose of this study is the cloning of NP gene which exists in the S genome segment of Maaji (MAA)
virus and
expression of the gene to obtain qualified, genetically engineered NP to be utilized as an immunodiagnostic antigen.
First of all, for the purpose of amplifing the MAA-NP gene by PCR, the specific primers were built from the known nucleotide sequence of Hantaan viral NP gene. The viral cDNA of the NP gene was synthesized by using the primers and RNase H AMV
reverse
transcriptase. Thereafter, using this cDNA as a template, the NP gene was amplifed specifically by Taq DNA polymrerase. The pT7blue(R)T-overhang vector systems were used for cloning of the amplified NP gene. The expression system was consisted of
BL21
(DE3)pLysS and pET16b as a host and a plasmid repectively. Into Ndel site of pET16b, NP gene was ligated with cohesive end for the expression, Insertion of NP gene in the plasmid was confirmed by PCR and mini prep methods. For expression, IPTG
was
used
and the expressed protein was characterized by Western blotting, The MAA-NP was expressed as the form of inclusion body (insoluble fraction)and the protein purified by affinity and metal chealating columns reacted specifically with the sera from
patients of HFRS as to be tested by ELISA and Western blotting.
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