Xi bao yu fen zi mian yi xue za zhi = Chinese journal of cellular and molecular immunology · 2014
[Preparation and characterization of a monoclonal antibody against the nucleocapsid protein of Schmallenberg virus].
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Zhang Yongning, Wu Shaoqiang, Wernike Kerstin, Lv Jizhou, Feng Chunyan, Lin Xiangmei
Аннотация
OBJECTIVE: The present study was conducted to prepare and characterize a monoclonal antibody (mAb) against the nucleocapsid (N) protein of Schmallenberg virus (SBV).
METHODS: The SBV N gene was cloned into pET-28a-c(+ and pMAL-c5X vectors and then transformed into E.coli BL21. Histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins were respectively induced to express by IPTG and purified by nickel-nitrilotriacetic acid (Ni-NTA) agarose and amylose resin. His-SBV-N was used to immunize BALB/c mice to prepare mAb, and MBP-SBV-N was used as the coating antigen in ELISA to screen mAb-secreting hybridomas and to determine mAb titers. The mAb against SBV N protein was purified from the ascitic fluids using protein G sepharose. Western blotting and indirect immunofluorescence assay were utilized to analyze the reactivity and specificity of the mAb.
RESULTS: One mAb specific for SBV N protein (named 1F2) was successfully screened and purified. The titer of 1F2 was 1:32 000. Besides, the isotype of 1F2 was determined to be IgG2α/κ. 1F2 reacted with both recombinant SBV N proteins and SBV isolates. It was also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein.
CONCLUSION: One mAb specific for the SBV N protein was successfully prepared, it provides a useful tool for the serological detection of SBV.
METHODS: The SBV N gene was cloned into pET-28a-c(+ and pMAL-c5X vectors and then transformed into E.coli BL21. Histidine (His)-tagged (His-SBV-N) and maltose-binding protein (MBP)-tagged (MBP-SBV-N) fusion proteins were respectively induced to express by IPTG and purified by nickel-nitrilotriacetic acid (Ni-NTA) agarose and amylose resin. His-SBV-N was used to immunize BALB/c mice to prepare mAb, and MBP-SBV-N was used as the coating antigen in ELISA to screen mAb-secreting hybridomas and to determine mAb titers. The mAb against SBV N protein was purified from the ascitic fluids using protein G sepharose. Western blotting and indirect immunofluorescence assay were utilized to analyze the reactivity and specificity of the mAb.
RESULTS: One mAb specific for SBV N protein (named 1F2) was successfully screened and purified. The titer of 1F2 was 1:32 000. Besides, the isotype of 1F2 was determined to be IgG2α/κ. 1F2 reacted with both recombinant SBV N proteins and SBV isolates. It was also cross-reactive with the N proteins of genetically related Shamonda, Douglas and Akabane viruses, but not with the Rift Valley fever virus N protein.
CONCLUSION: One mAb specific for the SBV N protein was successfully prepared, it provides a useful tool for the serological detection of SBV.