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Expression and purification of the matrix protein of Nipah virus in baculovirus insect cell system
Seyedehsara Masoomi Dezfooli1, Wen Siang Tan1,2, Beng Ti Tey3,4
1Laboratory of Vaccine and Immunotherapeutic, Institute of Bioscience, Universiti Putra Malaysia, 43400 UPM Serdang, Selangor, Malaysia.
Abstract:
Nipah virus (NiV) causes fatal respiratory illness and encephalitis in humans and animals. The matrix (M) protein of NiV plays an important role in the viral assembly and budding process. Thus, an access to the NiV M protein is vital to the design of viral antigens as diagnostic reagents. In this study, recombinant DNA technology was successfully adopted in the cloning and expression of NiV M protein. A recombinant expression cassette (baculovirus expression vector) was used to encode an N-terminally His-tagged NiV M protein in insect cells. A time-course study demonstrated that the highest yield of recombinant M protein (400-500 μg) was expressed from 107 infected cells 3 days after infection. A single-step purification method based on metal ion affinity chromatography was established to purify the NiV M protein, which successfully yielded a purity level of 95.67% and a purification factor of 3.39. The Western blotting and enzyme-linked immunosorbent assay (ELISA) showed that the purified recombinant M protein (48 kDa) was antigenic and reacted strongly with the serum of a NiV infected pig.
Insights
Researchers successfully cloned and expressed Nipah virus (NiV) matrix (M) protein using recombinant DNA technology. This provides a crucial NiV M protein for developing diagnostic reagents and understanding viral assembly.
Area of Science:
- Virology
- Molecular Biology
- Protein Expression
Background:
- Nipah virus (NiV) causes severe respiratory illness and encephalitis in humans and animals.
- The NiV matrix (M) protein is essential for viral assembly and budding.
- Access to NiV M protein is vital for developing diagnostic reagents.
Purpose of the Study:
- To clone and express the Nipah virus M protein using recombinant DNA technology.
- To establish a purification method for the recombinant NiV M protein.
- To confirm the antigenicity of the purified NiV M protein.
Main Methods:
- Utilized a baculovirus expression vector for N-terminally His-tagged NiV M protein expression in insect cells.
- Conducted a time-course study to optimize protein yield.
- Employed metal ion affinity chromatography for protein purification.
- Verified protein purity and antigenicity using Western blotting and ELISA.
Main Results:
- Achieved high yield (400-500 μg) of recombinant NiV M protein 3 days post-infection.
- Purified the NiV M protein to 95.67% purity with a 3.39 purification factor.
- Confirmed the purified 48 kDa recombinant M protein is antigenic and reacts with NiV-infected pig serum.
Conclusions:
- Successfully cloned and expressed the Nipah virus M protein in insect cells.
- Developed an efficient purification strategy for the recombinant NiV M protein.
- The purified recombinant NiV M protein demonstrates antigenicity, suitable for diagnostic reagent development.
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