WO2011056060A2 - Recombinant matrix protein of nipah virus - Google Patents
Recombinant matrix protein of nipah virus Download PDFInfo
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- WO2011056060A2 WO2011056060A2 PCT/MY2010/000211 MY2010000211W WO2011056060A2 WO 2011056060 A2 WO2011056060 A2 WO 2011056060A2 MY 2010000211 W MY2010000211 W MY 2010000211W WO 2011056060 A2 WO2011056060 A2 WO 2011056060A2
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- matrix protein
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- nipah virus
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
- C07K16/1027—Paramyxoviridae, e.g. respiratory syncytial virus
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2760/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses negative-sense
- C12N2760/00011—Details
- C12N2760/18011—Paramyxoviridae
- C12N2760/18211—Henipavirus, e.g. hendra virus
- C12N2760/18222—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
Definitions
- This invention provides a method in constructing a recombinant expression vector which harbors the matrix ( ) gene of Nipah virus (NiV) and the expression of the matrix (M) protein of Nipah virus (NiV) in Escherichia coli.
- Nipah virus is a zoonotic paramyxovirus that causes fatal encephalitic and respiratory illness in humans and livestocks.
- the virus outbreak in Peninsular Malaysia in 1998 claimed 105 human lives and resulted in massive culling of about 1 million infected swine with encephalitis and respiratory.
- Fruit bats (flying foxes) are believed to be the natural reservoir for NiV and may be introduced into pig farms through their secretions (Chua, K.B., Koh, C.L., Hooi, P.S., Wee, K.F., Khong, J.H., Chua, B.H., Chan, T.P., Lim, M.E., Lam, S.K., 2002.
- NiV outbreaks have occurred in Malaysia, Singapore, India and Bangladesh following various chains of transmission including intermediate host species (Chua, K.B., Koh, C.L., Hooi, P.S., Wee, K.F., Khong, J.H., Chua, B.H., Chan, T.P., Lim, M.E., Lam, S.K., 2002. Isolation of Nipah virus from Malaysian island flying-foxes. Microbes Infect. 4, 145-151 ), vehicle borne transmission, bat to human transmission and human-to-human transmission. Identification of the spillover into human population has now been extended to several countries in Southeast Asia including Indonesia, India and Bangladesh. It is probably much more extensive due to undiagnosed cases in many countries. The ability of NiV to infect a variety of species along with its mode of transmission coupled with its high pathogenicity demand a rapid search for possible tools for diagnosis of early infection.
- NiV has pleomorphic structures with different sizes and shapes.
- the virus contains two envelope glycoproteins: the G protein is responsible for binding to the cellular receptors, Ephrin B2 and B3 (Bonaparte, M.J., Dimitrov, A.S., Bossart, K.N., Crameri, G., Mungall, B.A., Bishop, K.A., Choudhry, V., Dimitrov, D.S., Wang, L.F., Eaton, B.T., Broder, C.C., 2005.
- Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc. Natl. Acad. Sci. U.S.A.
- the M protein is one of the abundant proteins in the virion and it is important in determining the virion architecture.
- the M gene is predicted to be 1359 nucleotides (nt) in length, with an ORF of 1059 nt, encoding the M protein (352 amino acids) with a predicted molecular mass (M r ) about 39.93 kDa. Its high hydrophobic nature coupled with high net positive charge attribute to its property of association with membranes. However, there is no information available on the production of the M protein in microbial cells.
- the present invention provides a recombinant matrix protein of Nipah virus containing matrix protein of Nipah virus which includes at least one His tag and myc epitope.
- the present invention also provides a recombinant plasmid including recombinant matrix protein of Nipah virus.
- Also provided is a method for producing recombinant matrix protein of Nipah virus which includes the steps of (a) culturing expression host containing expression vector of recombinant matrix protein in a medium which allows growth of the expression host, (b) allowing the recombinant matrix protein to be expressed in the expression host, (c) harvesting the expression host from step (b) from the medium, (d) disrupting the expression host harvested from step (c) in a medium for sonication purposes and (e) purifying the recombinant matrix protein obtained from step (d).
- Also provided is a method for purifying recombinant matrix protein of Nipah virus which includes the steps of (a) harvesting and lysing recombinant expression host containing recombinant matrix protein cells, (b) purifying the recombinant matrix protein using nickel affinity column chromatography, (c) dialyzing the purified recombinant matrix protein from step (b) with buffer solution, (d) fractionating the purified recombinant matrix protein from step (c) using centrifugation and (e) collecting and pooling the fractionated recombinant matrix protein obtained from step (d) concentration of protein.
- composition comprising a therapeutically effective amount of recombinant matrix protein of Nipah virus.
- diagnostic reagent comprising an effective amount of recombinant matrix protein matrix protein of Nipah virus.
- FIG. 1 shows the expression and purification of the NiV M protein expressed in E. coli BL21 (DE3) cells.
- SDS-PAGE and Coomassie blue staining A
- Western blot analysis [with anti- myc monoclonal antibody (B) and with swine anti-NiV serum (C)] of the protein.
- FIG. 2 shows the separation of the M protein with sucrose density gradient centrifugation.
- the M protein purified with the Ni-NTA affinity column was separated on a sucrose gradient.
- Western blot (A) and ELISA (B) results of the gradient fractions detected with anti-myc antibody (1 :5000).
- For ELISA 50 ⁇ of each fraction was used to coat the wells. Fractions correspond to the theoretical percentage of sucrose are indicated on top of the bars.
- FIG. 3 is a transmission electron micrograph showing the formation of spherical structures the purified NiV M protein. Bar represents 200 nm.
- FIG. 4 is an enzyme-linked immunosorbent assay (ELISA) showing the immunoreactivity of a panel of 18 sera (collected during Nipah virus outbreak in 1999) against NiV M protein purified with sucrose density gradient centrifugation. Samples 1 to 3 are negative sera and samples 4 to 18 are positive sera. The error bars represent standard deviations from the means. The assay was performed in triplicates. Description of the Preferred Embodiments
- the M coding region of the Nipah virus (NiV) swine isolate VRI2794/99 was prepared through reverse transcription-polymerase chain reaction (RT-PCR). Two primers were used for the amplification as given below:
- NiV-M-6 FD 5' -CCATGGCCATGGAGCCGGACATC- 3' (forward primer)
- NiV-M-5 RV 5'-GTAAGCTTCGCCCTTTAGAATTCTCCCTGT-3' (reverse primer)
- the PCR products were ligated to pTrcHis2 vector containing the coding region for the myc epitope and 6 His residues downstream of the multiple cloning site.
- the insert of the recombinant plasmid was confirmed to be in frame by DNA sequencing.
- the recombinant expression vector was introduced into E. coli strain BL21 (DE3) for expression.
- Affinity column chromatography and sucrose gradient centrifugation were employed to purify the recombinant matrix protein.
- Induced culture was harvested and cells were lysed by sonication.
- the lysate, obtained after centrifugation was loaded onto a pre-equilibrated Ni- NTA agarose column and was incubated for 1 hour at room temperature.
- the bound recombinant M protein was eluted after washing and elute was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting.
- SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
- the partially purified recombinant protein was dialyzed and concentrated with a 30 kDa cut-off polyethersulfone membrane.
- FIG. 1 B and 1 C shows the results of Western blot and enzyme-linked immunosorbent assay (ELISA) of sucrose gradient profile of the purified M protein. Analysis of the fractions revealed that the M protein migrated into the gradient forming a bell shape peak from fractions 2-10.
- the purified recombinant M protein was absorbed to carbon-coated grids and stained with 2% uranyl acetate. Referring to FIG. 3, examination under an electron microscope revealed that M protein assembles into spherical particles.
- a total of 18 predefined sera were analyzed using the purified M protein for detecting the anti-M antibody in the swine sera obtained during the outbreak.
- About 100 ⁇ [_ of the purified M protein was added to the well. After incubation for 18 hours at 4°C, the plate was washed and blocked with 10% bovine serum albumin (BSA) incubated for 2 hours at room temperature. Subsequently, the plates were washed and incubated for 1 hour at room temperature with the appropriate dilution (1 :20) of the swine sera from infected and non-infected animals.
- BSA bovine serum albumin
- anti-swine immunoglobulin (1 :3000 dilution) conjugated to alkaline phosphatase was added and the plates were incubated further for 1 hour at room temperature.
- the enzyme substrate solution containing p-nitrophenyl phosphate (0.1 %) in diethanolamine (1 M), pH 9.5 was added. The reaction was stopped after 30 minutes incubation at room temperature, and the A 05 values were measured with a microtiter plate reader. Referring to FIG. 4, all the positive serum samples showed higher readings when compared to the negative samples, demonstrating the capability of M protein as a diagnostic reagent.
- the pharmaceutical composition which comprises of a therapeutically effective amount of the recombinant matrix protein.
- the recombinant matrix protein in the pharmaceutical composition includes the matrix protein of the Nipah virus with at least one His tag and myc epitope. Besides that, the recombinant matrix protein is useful as a diagnostic reagent which contains an effective amount of the recombinant matrix protein
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- Proteomics, Peptides & Aminoacids (AREA)
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Abstract
The present invention provides a recombinant matrix protein of Nipah virus containing matrix protein of Nipah virus which includes at least one His tag and myc epitope. Further, the present invention also provides a recombinant plasmid including recombinant matrix protein of Nipah virus. Also provided is a method for producing recombinant matrix protein of Nipah virus which includes the steps of (a) culturing expression host containing expression vector of recombinant matrix protein in a medium which allows growth of the expression host, (b) allowing the recombinant matrix protein to be expressed in the expression host, (c) harvesting the expression host from step (b) from the medium, (d) disrupting the expression host harvested from step (c) in a medium for sonication purposes and (e) purifying the recombinant matrix protein obtained from step (d). Also provided is a method for purifying recombinant matrix protein of Nipah virus which includes the steps of (a) harvesting and lysing recombinant expression host containing recombinant matrix protein cells, (b) purifying the recombinant matrix protein using nickel affinity column chromatography, (c) dialyzing the purified recombinant matrix protein from step (b) with buffer solution, (d) fractionating the purified recombinant matrix protein from step (c) using centrifugation and (e) collecting and pooling the fractionated recombinant matrix protein obtained from step (d) concentration of protein. There is also provided a pharmaceutical composition comprising a therapeutically effective amount of recombinant matrix protein of Nipah virus. Also provided is a diagnostic reagent comprising an effective amount of recombinant matrix protein matrix protein of Nipah virus.
Description
Recombinant Matrix Protein of Nipah Virus
Field of Invention
This invention provides a method in constructing a recombinant expression vector which harbors the matrix ( ) gene of Nipah virus (NiV) and the expression of the matrix (M) protein of Nipah virus (NiV) in Escherichia coli.
Background of Invention
Nipah virus (NiV) is a zoonotic paramyxovirus that causes fatal encephalitic and respiratory illness in humans and livestocks. The virus outbreak in Peninsular Malaysia in 1998 claimed 105 human lives and resulted in massive culling of about 1 million infected swine with encephalitis and respiratory. Fruit bats (flying foxes) are believed to be the natural reservoir for NiV and may be introduced into pig farms through their secretions (Chua, K.B., Koh, C.L., Hooi, P.S., Wee, K.F., Khong, J.H., Chua, B.H., Chan, T.P., Lim, M.E., Lam, S.K., 2002. Isolation of Nipah virus from Malaysian island flying-foxes. Microbes Infect. 4, 145-151 ). Other animals such as dogs, cats and horses can also be infected by the virus when they come in close contact with infected pigs (Chua, K.B., Koh, C.L., Hooi, P.S., Wee, K.F., Khong, J.H., Chua, B.H., Chan, T.P., Lim, M.E., Lam, S.K., 2002. Isolation of Nipah virus from Malaysian island flying-foxes. Microbes Infect. 4, 145-151 ). NiV outbreaks have occurred in Malaysia, Singapore, India and Bangladesh following various chains of transmission including intermediate host species (Chua, K.B., Koh, C.L., Hooi, P.S., Wee, K.F., Khong, J.H., Chua, B.H., Chan, T.P., Lim, M.E., Lam, S.K., 2002. Isolation of Nipah virus from Malaysian island flying-foxes. Microbes Infect. 4, 145-151 ), vehicle borne transmission, bat to human transmission and human-to-human transmission. Identification of the spillover into human population has now been extended to several countries in Southeast Asia including Indonesia, India and Bangladesh. It is probably much more extensive due to undiagnosed cases in many countries. The ability of NiV to infect a variety of species along with its mode of transmission
coupled with its high pathogenicity demand a rapid search for possible tools for diagnosis of early infection.
NiV has pleomorphic structures with different sizes and shapes. The virus contains two envelope glycoproteins: the G protein is responsible for binding to the cellular receptors, Ephrin B2 and B3 (Bonaparte, M.J., Dimitrov, A.S., Bossart, K.N., Crameri, G., Mungall, B.A., Bishop, K.A., Choudhry, V., Dimitrov, D.S., Wang, L.F., Eaton, B.T., Broder, C.C., 2005. Ephrin-B2 ligand is a functional receptor for Hendra virus and Nipah virus. Proc. Natl. Acad. Sci. U.S.A. 102, 10652-10657) and the F protein mediates membrane fusion (Bossart, K.N., Wang, L.F., Flora, M.N., Chua, K.B., Lam, S.K., Eaton, B.T., Broder, C.C., 2002. Membrane fusion tropism and heterotypic functional activities of the Nipah virus and Hendra virus envelope glycoproteins. J. Virol. 76, 11 186-1 1 198). Lying beneath the viral envelope is the matrix (M) protein, which interacts with both the glycoproteins and the nucleocapsid (N) or ribonucleoprotein (RNP) complex.
Currently the viral proteins have been prepared by directly culturing the virus on animal cell cultures. However, NiV has been classified by the centre for Diseases Control as Biosafety level 4 pathogen to denote "the most dangerous organisms known to man". It is absolutely dangerous to culture the live virus, and it requires BSL-4 or 3 facilities. Hence, recombinant DNA technology provides an alternative means to produce the viral proteins.
The M protein is one of the abundant proteins in the virion and it is important in determining the virion architecture. The M gene is predicted to be 1359 nucleotides (nt) in length, with an ORF of 1059 nt, encoding the M protein (352 amino acids) with a predicted molecular mass (Mr) about 39.93 kDa. Its high hydrophobic nature coupled with high net positive charge attribute to its property of association with membranes. However, there is no information available on the production of the M protein in microbial cells.
It was, therefore, an object to provide a novel expression system capable of producing the NiV M protein in Escherichia coli. The M fusion protein (Mfus) bearing the myc epitope and six residues of His at their carboxy terminal end could be produced successfully in E. coli by
means of recombinant DNA technology. The M,us protein was expressed to a substantial level in the bacteria as recognized by swine anti-NiV serum indicating the usefulness of this product as a diagnostic reagent. Summary of Invention
Accordingly, the present invention provides a recombinant matrix protein of Nipah virus containing matrix protein of Nipah virus which includes at least one His tag and myc epitope.
Further, the present invention also provides a recombinant plasmid including recombinant matrix protein of Nipah virus.
Also provided is a method for producing recombinant matrix protein of Nipah virus which includes the steps of (a) culturing expression host containing expression vector of recombinant matrix protein in a medium which allows growth of the expression host, (b) allowing the recombinant matrix protein to be expressed in the expression host, (c) harvesting the expression host from step (b) from the medium, (d) disrupting the expression host harvested from step (c) in a medium for sonication purposes and (e) purifying the recombinant matrix protein obtained from step (d).
Also provided is a method for purifying recombinant matrix protein of Nipah virus which includes the steps of (a) harvesting and lysing recombinant expression host containing recombinant matrix protein cells, (b) purifying the recombinant matrix protein using nickel affinity column chromatography, (c) dialyzing the purified recombinant matrix protein from step (b) with buffer solution, (d) fractionating the purified recombinant matrix protein from step (c) using centrifugation and (e) collecting and pooling the fractionated recombinant matrix protein obtained from step (d) concentration of protein.
There is also provided a pharmaceutical composition comprising a therapeutically effective amount of recombinant matrix protein of Nipah virus.
Also provided is a diagnostic reagent comprising an effective amount of recombinant matrix protein matrix protein of Nipah virus.
Description of Figures
FIG. 1 shows the expression and purification of the NiV M protein expressed in E. coli BL21 (DE3) cells. SDS-PAGE and Coomassie blue staining (A), Western blot analysis [with anti- myc monoclonal antibody (B) and with swine anti-NiV serum (C)] of the protein. Lanes: M, Molecular weight markers in kDa; 1 , E. coli cells harboring the recombinant plasmid carrying the M gene (IPTG induced bacterial ceil lysate); 2, The M protein purified with Ni-NTA column; 3, The M protein purified with sucrose density gradient centrifugation. Arrows indicate the position of the expected protein bands.
FIG. 2 shows the separation of the M protein with sucrose density gradient centrifugation. The M protein purified with the Ni-NTA affinity column was separated on a sucrose gradient. Western blot (A) and ELISA (B) results of the gradient fractions detected with anti-myc antibody (1 :5000). For ELISA, 50 μΙ of each fraction was used to coat the wells. Fractions correspond to the theoretical percentage of sucrose are indicated on top of the bars.
FIG. 3 is a transmission electron micrograph showing the formation of spherical structures the purified NiV M protein. Bar represents 200 nm.
FIG. 4 is an enzyme-linked immunosorbent assay (ELISA) showing the immunoreactivity of a panel of 18 sera (collected during Nipah virus outbreak in 1999) against NiV M protein purified with sucrose density gradient centrifugation. Samples 1 to 3 are negative sera and samples 4 to 18 are positive sera. The error bars represent standard deviations from the means. The assay was performed in triplicates.
Description of the Preferred Embodiments
Practical and presently preferred embodiments of the present invention are illustrative as shown in the following examples. However, it will be appreciated that those skilled in the art, on consideration of this disclosure, may make modification and improvements within the scope of the present invention.
Construction of Recombinant Expression vector encoding the full length M protein
The M coding region of the Nipah virus (NiV) swine isolate VRI2794/99 was prepared through reverse transcription-polymerase chain reaction (RT-PCR). Two primers were used for the amplification as given below:
NiV-M-6 FD: 5' -CCATGGCCATGGAGCCGGACATC- 3' (forward primer)
NiV-M-5 RV: 5'-GTAAGCTTCGCCCTTTAGAATTCTCCCTGT-3' (reverse primer)
The PCR products were ligated to pTrcHis2 vector containing the coding region for the myc epitope and 6 His residues downstream of the multiple cloning site. The insert of the recombinant plasmid was confirmed to be in frame by DNA sequencing. The recombinant expression vector was introduced into E. coli strain BL21 (DE3) for expression.
Expression and Western blotting of the M protein
Transformants carrying the recombinant expression plasmids were grown in Luria
Bertani (LB) broth, and protein expression was induced by adding isopropyl-D- thiogalactopyranoside (IPTG). Cell extracts were analyzed with SDS-PAGE under denaturing conditions and the protein was transferred to a nitrocellulose membrane. The protein was detected with swine anli-NiV serum and the result is shown FIG 1.C. A protein band with apparent molecular weight of about 43 kDa was detected. The Mfus was detected by the anti- myc monoclonal antibody (FIG. 1 B).
Purification and Electron Microscopic Analysis of M protein
Affinity column chromatography and sucrose gradient centrifugation were employed to purify the recombinant matrix protein. Induced culture was harvested and cells were lysed by sonication. The lysate, obtained after centrifugation, was loaded onto a pre-equilibrated Ni- NTA agarose column and was incubated for 1 hour at room temperature. The bound recombinant M protein was eluted after washing and elute was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The partially purified recombinant protein was dialyzed and concentrated with a 30 kDa cut-off polyethersulfone membrane. The concentrated protein was layered on a step sucrose gradient 10, 20, 30, 40 and 60% (w/v) and centrifuged. Fractions (0.5 mL) were collected and analyzed on SDS-PAGE. Positive fractions were then pooled and dialyzed against dialysis buffer. As shown in FIG. 1 B and 1 C, the M protein purified by both methods was detected by anti-myc monoclonal antibody and the swine NiV-serum. FIG. 2 shows the results of Western blot and enzyme-linked immunosorbent assay (ELISA) of sucrose gradient profile of the purified M protein. Analysis of the fractions revealed that the M protein migrated into the gradient forming a bell shape peak from fractions 2-10. The purified recombinant M protein was absorbed to carbon-coated grids and stained with 2% uranyl acetate. Referring to FIG. 3, examination under an electron microscope revealed that M protein assembles into spherical particles.
To evaluate the antigenicity of the M protein and its useful application as a diagnostic antigen, a total of 18 predefined sera (15 positives and 3 negatives) were analyzed using the purified M protein for detecting the anti-M antibody in the swine sera obtained during the outbreak. About 100 μ[_ of the purified M protein was added to the well. After incubation for 18 hours at 4°C, the plate was washed and blocked with 10% bovine serum albumin (BSA) incubated for 2 hours at room temperature. Subsequently, the plates were washed and
incubated for 1 hour at room temperature with the appropriate dilution (1 :20) of the swine sera from infected and non-infected animals. After washing, anti-swine immunoglobulin (1 :3000 dilution) conjugated to alkaline phosphatase was added and the plates were incubated further for 1 hour at room temperature. Following another washing step, the enzyme substrate solution containing p-nitrophenyl phosphate (0.1 %) in diethanolamine (1 M), pH 9.5, was added. The reaction was stopped after 30 minutes incubation at room temperature, and the A 05 values were measured with a microtiter plate reader. Referring to FIG. 4, all the positive serum samples showed higher readings when compared to the negative samples, demonstrating the capability of M protein as a diagnostic reagent.
The pharmaceutical composition, as mentioned, which comprises of a therapeutically effective amount of the recombinant matrix protein. The recombinant matrix protein in the pharmaceutical composition includes the matrix protein of the Nipah virus with at least one His tag and myc epitope. Besides that, the recombinant matrix protein is useful as a diagnostic reagent which contains an effective amount of the recombinant matrix protein
Claims
Claims
1 . A recombinant matrix protein of Nipah virus.
2. The recombinant matrix protein as claimed in claim 1 wherein the recombinant matrix protein contains matrix protein of Nipah virus.
3. The recombinant protein matrix protein as claimed in claim 2 wherein the matrix protein contains at least one His tag and myc epitope.
4. The recombinant matrix protein of Nipah virus as claimed in claim 2 wherein the matrix protein is encoded by the following nucleotide coding sequence:
1 ATGGAGCCGGACATCAAGAGTATTTCAAGTGAGTCAATGGAAGGAGTATCTGATTTCAGC 60
61 CCTAGTTCTTGGGAGCATGGTGGGTATCTTGATAAGGTTGAACCAGAAATTGATGAAAAT 120
121 GGCAGTATGATTCCAAAATACAAGATCTATACCCCAGGAGCTAACGAGAGGAAATACAAC 180
181 AACTACATGTACCTTATATGTTACGGCTTTGTTGAAGATGTTGAGAGAACCCCAGAGACA 240
GGGAAACGCAAGAAGATCAGGACAATTGCTGCCTACCCTCTGGGTGTTGGTAAGAGTGC
241 300 C
301 TCTCATCCCCAAGATCTTCTGGAGGAACTCTGTTCCCTCAAAGTTACTGTGAGAAGAACA 360
361 GCTGGATCAACTGAGAAAATTGTGTTTGGATCATCTGGCCCTCTAAATCACCTCGTTCCG 420
421 TGGAAGAAAGTACTGACTAGTGGTTCAA M i l l AATGCAGTCAAGGTTTGTCGGAACGTT 480
481 GATCAGATACAGCTTGACAAGCATCAAGCTCTGAGAATATTTTTTCTCAGTATCACAAAG 540
541 CTCAATGATTCTGGAATCTACATGATTCCACGAACCATGCTTGAGTTCAGGAGAAACAAT 600
601 GCCATTGCCTTCAATCTTCTAGTGTACTTGAAGATTGATGCTGATTTATCCAAAATGGGG 660
ATCCAGGGAAGCCTCGATAAAGATGGCTTCAAGGTTGCCTCCTTCATGCTACACTTGGG
661 720 G
721 AACTTTGTCCGTCGTGCAGGGAAGTATTACTCTGTTGATTATTGTAGGAGGAAGATTGAT 780
781 AGGATGAAATTGCAG I I I I CACTGGGTTCCATAGGCGGACTAAGTCTCCACATTAAGATC 840
841 AATGGTGTAATCAGCAAACGGCTGTTTGCTCAAATGGGATTCCAAAAAAACCTTTGTTTC 900
901 TCTTTGATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTGAGATC 960
102 1 AGCCGAGTAGCAGCTGTGTTGCAGCCTTCTATTCCAAGAGAGTTCATGATCTATGATGAT
02 108 GTCTTCATTGACAATACAGGGAGAATTCTAAAGGGCGAAGCTTACGTAGAACAAAAACTC
1 0
1 13 ATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTAA
1 4
5. The recombinant matrix protein of Nipah virus as claimed in claim 2 wherein the matrix protein has the following amino acid sequence:
MEPDIKSISS ESMEGVSDFS PSS EHGGYL DKVEPEIDEN GSMIPKYKIY TPGANERKYN 60 NYMYLICYGF VEDVERTPET GKRKKIRTIA AYPLGVGKSA SHPQDLLEEL CSLKVTVRRT 120 AGSTEKIVFG SSGPLNHLVP KKVLTSGSI FNAVKVCRNV DQIQLDKHQA LRIFFLS ITK 180 LNDSGIYMIP RTMLEFRRNN AIAFNLLVYL KIDADLSKMG IQGSLDKDGF KVASFMLHLG 240 NFVRRAGKYY SVDYCRRKID RMKLQFSLGS IGGLSLHIKI NGVI SKRLFA QMGFQKNLCF 300 SLMDINPWLN RLTWNNSCE I SRVAAVLQPS IPREFMIYDD VFIDNTGRIL KGEAYVEQKL 360 ISEEDLNSAV DHHHHHH 377
The recombinant matrix protein of Nipah virus as claimed in claim 2 wherein the matrix protein is selected from Nipah virus or Hendra virus.
The recombinant matrix protein of Nipah virus as claimed in claim 6 wherein the matrix protein is selected from paramyxoviruses, orthomyxoviruses, henipaviruses, DNA viruses or RNA viruses.
8. A recombinant plasmid includes of recombinant matrix protein of Nipah virus.
9. The recombinant plasmid as claimed in claim 8 wherein the recombinant matrix protein contains matrix protein of Nipah virus.
10. The recombinant plasmid as claimed in claim 9 wherein the matrix protein contains at least one His tag and myc epitope.
1 1 . The recombinant plasmid as claimed in claim 9 wherein the matrix protein is encoded > by the following nucleotide coding sequence:
1 ATGGAGCCGGACATCAAGAGTATTTCAAGTGAGTCAATGGAAGGAGTATCTGATTTCAGC 60
61 CCTAGTTCTTGGGAGCATGGTGGGTATCTTGATAAGGTTGAACCAGAAATTGATGAAAAT 120
121 GGCAGTATGATTCCAAAATACAAGATCTATACCCCAGGAGCTAACGAGAGGAAATACAAC 180
181 AACTACATGTACCTTATATGTTACGGCTTTGTTGAAGATGTTGAGAGAACCCCAGAGACA 240
GGGAAACGCAAGAAGATCAGGACAATTGCTGCCTACCCTCTGGGTGTTGGTAAGAGTGC
241 300 C
301 TCTCATCCCCAAGATCTTCTGGAGGAACTCTGTTCCCTCAAAGTTACTGTGAGAAGAACA 360
361 GCTGGATCAACTGAGAAAATTGTGTTTGGATCATCTGGCCCTCTAAATCACCTCGTTCCG 420
421 TGG AAG AAAGTACTGACTAGTGGTTCAATTTTTAATGCAGTCAAGGTTTGTCGGAACGTT 480
481 GATCAGATACAGCTTG AC AAGCATCAAGCTCTGAGAATATTTTTTCTCAGTATCACAAAG 540
541 CTCAATGATTCTGG AATCTAC ATGATTCCACGAACCATGCTTGAGTTCAGGAGAAACAAT 600
601 GCCATTGCCTTCAATCTTCTAGTGTACTTGAAGATTGATGCTGATTTATCCAAAATGGGG 660
ATCCAGGGAAGCCTCGATAAAGATGGCTTCAAGGTTGCCTCCTTCATGCTACACTTGGG
661 720 G
721 AACTTTGTCCGTCGTGCAGGGAAGTATTACTCTGTTGATTATTGTAGGAGGAAGATTGAT 780
781 AGGATGAAATTGCAGTTTTCACTGGGTTCCATAGGCGGACTAAGTCTCCACATTAAGATC 840
841 AATGGTGTAATCAGCAAACGGCTGTTTGCTCAAATGGGATTCCAAAAAAACCTTTGTTTC 900
901 TCTTTGATGGACATC AATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTG AGATC 960
102
961 AGCCGAGTAGCAGCTGTGTTGCAGCCTTCTATTCCAAGAGAGTTCATGATCTATGATGAT
0
102 108 GTCTTCATTGACAATACAGGGAGAATTCTAAAGGGCGAAGCTTACGTAGAACAAAAACTC
1 0
108 113 ATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTAA
1 4
12. The recombinant plasmid as claimed in claim 9 wherein the matrix protein which has the following amino acid sequence:
MEPDIKSISS ESMEGVSDFS PSSWEHGGYL DKVEPEIDEN GSMIPKYKIY TPGANERKYN 60
NYMYLICYGF VEDVERTPET GKRKKIRTIA AYPLGVGKSA SHPQDLLEEL CSLKVTVRRT 120
AGSTEKIVFG SSGPLNHLVP KKVLTSGSI FNAVKVCRNV DQIQLDKHQA LRIFFLS ITK 180
LNDSGIYMIP RTMLEFRRNN AIAFNLLVYL KIDADLSKMG IQGSLDKDGF KVASFMLHLG 240
NFVRRAGKYY SVDYCRRKID RMKLQFSLGS IGGLSLHIKI NGVISKRLFA QMGFQKNLCF 300
SLMDINPWLN RLTWNNSCEI SRVAAVLQPS IPREFMIYDD VFIDNTGRIL KGEAYVEQKL 360
ISEEDLNSAV DHHHHHH 377
13. The recombinant plasmid as claimed in claim 9 wherein the matrix protein is selected from Nipah virus or Hendra virus
14. The recombinant plasmid as claimed in claim 9 wherein the matrix protein is selected from paramyxoviruses, orthomyxoviruses, henipaviruses, DNA viruses or RNA viruses. 15. The recombinant plasmid which encodes matrix protein of Nipah virus as claimed in claim 14 wherein the recombinant matrix protein is produced in Escherichia coli cells.
16. A method for producing recombinant matrix protein of Nipah virus which includes the steps of:
a. culturing expression host containing expression vector of recombinant matrix protein in a medium which allows growth of the expression host;
b. allowing the recombinant matrix protein to be expressed in the expression host; c. harvesting the expression host from step (b) from the medium;
d. disrupting the expression host harvested from step (c) in a medium for sonication purposes; and
e. purifying the recombinant matrix protein obtained from step (d).
17. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 16 wherein the recombinant matrix protein contains matrix protein of Nipah virus.
18. The method for producing recombinant matrix protein of Nipah virus as claimed claim 17 wherein the matrix protein contains at least one His tag and myc epitope.
19. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 17 wherein the matrix protein is encoded by the following nucleotide coding sequence:
1 ATGGAGCCGGACATCAAGAGTATTTCAAGTGAGTCAATGGAAGGAGTATCTGATTTCAGC 60
61 CCTAGTTCTTGGGAGCATGGTGGGTATCTTGATAAGGTTGAACCAGAAATTGATGAAAAT 120
121 GGCAGTATGATTCCAAAATACAAGATCTATACCCCAGGAGCTAACGAGAGGAAATACAAC 180
181 AACTACATGTACCTTATATGTTACGGCTTTGTTGAAGATGTTGAGAGAACCCCAGAGACA 240
241 GGGAAACGCAAGAAGATCAGGACAATTGCTGCCTACCCTCTGGGTGTTGGTAAGAGTGCC 300
301 TCTCATCCCCAAGATCTTCTGGAGGAACTCTGTTCCCTCAAAGTTACTGTGAGAAGAACA 360
361 GCTGGATCAACTGAGAAAATTGTGTTTGGATCATCTGGCCCTCTAAATCACCTCGTTCCG 420
421 TGGAAGAAAGTACTGACTAGTGGTTCAATTTTTAATGCAGTCAAGGTTTGTCGGAACGTT 480
481 GATCAGATACAGCTTGACAAGCATCAAGCTCTGAGAATATTTTTTCTCAGTATCACAAAG 540
541 CTCAATGATTCTGGAATCTACATGATTCCACGAACCATGCTTGAGTTCAGGAGAAACAAT 600
601 GCCATTGCCTTCAATCTTCTAGTGTACTTGAAGATTGATGCTGATTTATCCAAAATGGGG 660
661 ATCCAGGGAAGCCTCGATAAAGATGGCTTCAAGGTTGCCTCCTTCATGCTACACTTGGGG 720
721 AACTTTGTCCGTCGTGCAGGGAAGTATTACTCTGTTGATTATTGTAGGAGGAAGATTGAT 780
781 AGGATGAAATTGCAGTTTTCACTGGGTTCCATAGGCGGACTAAGTCTCCACATTAAGATC 840
841 AATGGTGTAATCAGCAAACGGCTGTTTGCTCAAATGGGATTCCAAAAAAACCTTTGTTTC 900
901 TCTTTGATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTGAGATC 960
961 AGCCGAGTAGCAGCTGTGTTGCAGCCTTCTATTCCAAGAGAGTTCATGATCTATGATGAT 1020
1021 GTCTTCATTGACAATACAGGGAGAATTCTAAAGGGCGAAGCTTACGTAGAACAAAAACTC 1080
1081 ATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTAA 1 134
20. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 17 wherein the matrix protein which has the following amino acid sequence: MEPDIKSISS ESMEGVSDFS PSSWEHGGYL DKVEPE IDEN GSMIPKYKIY TPGANERKYN 60 NYMYLICYGF VEDVERTPET GKRKKIRTIA AYPLGVGKSA SHPQDLLEEL CSLKVTVRRT 120 AGSTEKIVFG SSGPLNHLVP WKKVLTSGSI FNAVKVCRNV DQIQLDKHQA LRIFFLSITK 180 LNDSGIYMIP RTMLEFRRNN AIAFNLLVYL KIDADLSKMG IQGSLDKDGF KVASFMLHLG 240 NFVRRAGKYY SVDYCRRKID RMKLQFSLGS IGGLSLHIKI NGVISKRLFA QMGFQKNLCF 300 SLMDINPWLN RLT NNSCE I SRVAAVLQPS IPREFMIYDD VFIDNTGRIL KGEAYVEQKL 360 ISEEDLNSAV DHHHHHH 377
21 . The method for producing recombinant matrix protein of Nipah virus as claimed in claim 17 wherein the matrix protein is selected from Nipah virus or Hendra virus.
22. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 17 wherein the matrix protein is selected from paramyxoviruses, orthomyxoviruses, henipaviruses, DNA viruses and RNA viruses.
23. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 17 wherein the recombinant matrix protein is produced in Escherichia coli cells.
24. A method for purifying recombinant matrix protein of Nipah virus which includes the steps of:
a. harvesting and lysing recombinant expression host containing recombinant matrix protein;
b. purifying the recombinant matrix protein using nickel affinity column chromatography;
c. dialyzing the purified recombinant matrix protein from step (b) with buffer solution;
d. fractionating the purified recombinant matrix protein from step (c) using centrifugation; and
e. collecting and pooling the fractionated recombinant matrix protein obtained from step (d) concentration of protein.
25. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 24 wherein the recombinant matrix protein contains matrix protein of Nipah virus. 26. The method for producing recombinant matrix protein of Nipah virus as claimed in claim 25 wherein the matrix protein contains at least one His tag and myc epitope.
27. The method for purifying recombinant matrix protein of Nipah virus as claimed in claim 25 wherein the matrix protein is encoded by the following nucleotide coding sequence: ATGGAGCCGGACATCAAGAGTATTTCAAGTGAGTCAATGGAAGGAGTATCTGATTTCAGC 60 CCTAGTTCTTGGGAGCATGGTGGGTATCTTGATAAGGTTGAACCAGAAATTGATGAAAAT 120 GGCAGTATGATTCCAAAATACAAGATCTATACCCCAGGAGCTAACGAGAGGAAATACAAC 180 AACTACATGTACCTTATATGTTACGGCTTTGTTGAAGATGTTGAGAGAACCCCAGAGACA 240
GGGAAACGCAAGAAGATCAGGACAATTGCTGCCTACCCTCTGGGTGTTGGTAAGAGTGC
300 C TCTCATCCCCAAGATCTTCTGGAGGAACTCTGTTCCCTCAAAGTTACTGTGAGAAGAACA 360 GCTGGATCAACTGAGAAAATTGTGTTTGGATCATCTGGCCCTCTAAATCACCTCGTTCCG 420 TGGAAGAAAGTACTGACTAGTGGTTCAATTTTTAATGCAGTCAAGGTTTGTCGGAACGTT 480 GATCAGATACAGCTTG AC AAGCATCAAGCTCTGAGAATATTTTTTCTCAGTATCACAAAG 540 CTCAATGATTCTGGAATCTACATGATTCCACGAACCATGCTTGAGTTCAGGAGAAACAAT 600 GCCATTGCCTTCAATCTTCTAGTGTACTTGAAGATTGATGCTGATTTATCCAAAATGGGG 660
ATCCAGGGAAGCCTCGATAAAGATGGCTTCAAGGTTGCCTCCTTCATGCTACACTTGGG
720 G AACTTTGTCCGTCGTGCAGGGAAGTATTACTCTGTTGATTATTGTAGGAGGAAGATTGAT 780
1 AGGATGAAATTGCAGTTTTCACTGGGTTCCATAGGCGGACTAAGTCTCCACATTAAGATC 840 1 AATGGTGTAATCAGCAAACGGCTGTTTGCTCAAATGGGATTCCAAAAAAACCTTTGTTTC 900 1 TCTTTGATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTG AGATC 960
1021 AGCCGAGTAGCAGCTGTGTTGCAGCCTTCTATTCCAAGAGAGTTCATGATCTATGATGAT
0 108 GTCTTCATTGACAATACAGGGAGAATTCTAAAGGGCGAAGCTTACGTAGAACAAAAACTC
0 1 13 ATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTAA
4
28. The method for purifying recombinant matrix protein of Nipah virus as claimed in claim 25 wherein the matrix protein which has the following amino acid sequence:
EPDIKSISS ESMEGVSDFS PSSWEHGGYL DKVEPEIDEN GSMIPKYKIY TPGANERKYN 60 NYMYLICYGF VEDVERTPET GKRKKIRTIA AYPLGVGKSA SHPQDLLEEL CSLKVTVRRT 120 AGSTEKIVFG SSGPLNHLVP WKKVLTSGSI FNAVKVCRNV DQIQLDKHQA LRIFFLSITK 180 LNDSGIYMIP RTMLEFRRNN AIAFNLLVYL KIDADLSKMG IQGSLDKDGF KVASFMLHLG 240 NFVRRAGKYY SVDYCRRKID RMKLQFSLGS IGGLSLHIKI NGVI SKRLFA QMGFQKNLCF 300 SLMDINP LN RLTWNNSCEI SRVAAVLQPS IPREFMIYDD VFIDNTGRIL KGEAYVEQKL 360 I SEEDLNSAV DHHHHHH 377
29. The method for purifying recombinant matrix protein of Nipah virus as claimed in claim 25 wherein the matrix protein is selected from Nipah virus or Hendra virus.
30. The method for purifying recombinant matrix protein of Nipah virus as claimed in claim 25 wherein the matrix protein is selected from paramyxoviruses, orthomyxoviruses, henipaviruses, DNA viruses and RNA viruses.
31 . The method in purifying recombinant matrix protein of Nipah virus as claimed in claim 25 wherein the recombinant matrix protein is produced in Escherichia coli cells.
32. The method for purifying recombinant matrix protein of Nipah virus as claimed in claim 24 wherein the centrifugation used is sucrose density gradient centrifugation. 33. A pharmaceutical composition comprising a therapeutically effective amount of recombinant matrix protein of Nipah virus.
34. The pharmaceutical composition as claimed in claim 34 wherein the recombinant matrix protein contains matrix protein of Nipah virus.
35. The pharmaceutical composition as claimed in claim 34 wherein the matrix protein contains at least one His tag and myc epitope.
36. The pharmaceutical composition as claimed in claim 34 wherein the matrix protein is encoded by the following nucleotide coding sequence:
ATGGAGCCGGACATCAAGAGTATTTCAAGTGAGTCAATGGAAGGAGTATCTGATTTCAGC 60 CCTAGTTCTTGGGAGCATGGTGGGTATCTTGATAAGGTTGAACCAGAAATTGATGAAAAT 120 GGCAGTATGATTCCAAAATACAAGATCTATACCCCAGGAGCTAACGAGAGGAAATACAAC 180 AACTACATGTACCTTATATGTTACGGCTTTGTTGAAGATGTTGAGAGAACCCCAGAGACA 240
GGGAAACGCAAGAAGATCAGGACAATTGCTGCCTACCCTCTGGGTGTTGGTAAGAGTGC
300 C TCTCATCCCCAAG ATCTTCTGGAGGAACTCTGTTCCCTCAAAGTTACTGTGAGAAGAACA 360 GCTGGATCAACTGAGAAAATTGTGTTTGGATCATCTGGCCCTCTAAATCACCTCGTTCCG 420 TGGAAGAAAGTACTGACTAGTGGTTCAATTTTTAATGCAGTCAAGGTTTGTCGGAACGTT 480 GATCAGATACAGCTTG ACAAGCATCAAGCTCTGAGAATATTTTTTCTCAGTATCACAAAG 540 CTCAATGATTCTGGAATCTACATGATTCCACGAACCATGCTTGAGTTCAGGAGAAACAAT 600 GCCATTGCCTTCAATCTTCTAGTGTACTTGAAGATTGATGCTGATTTATCCAAAATGGGG 660
ATCCAGGGAAGCCTCGATAAAGATGGCTTCAAGGTTGCCTCCTTCATGCTACACTTGGG
720 G
1 AACTTTGTCCGTCGTGCAGGGAAGTATTACTCTGTTGATTATTGTAGGAGGAAGATTGAT 7801 AGGATGAAATTGCAGTTTTCACTGGGTTCCATAGGCGGACTAAGTCTCCACATTAAGATC 8401 AATGGTGTAATCAGCAAACGGCTGTTTGCTCAAATGGGATTCCAAAAAAACCTTTGTTTC 900 1 TCTTTGATGGACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTG AGATC 960
1021 AGCCGAGTAGCAGCTGTGTTGCAGCCTTCTATTCCAAGAGAGTTCATGATCTATGATGAT
02 108 GTCTTCATTGACAATACAGGGAGAATTCTAAAGGGCGAAGCTTACGTAGAACAAAAACTC
1 08 1 13 ATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTAA
1 4
37. The pharmaceutical composition as claimed in claim 34 wherein the matrix protein which has the following amino acid sequence:
MEPDIKSISS ESMEGVSDFS PSSWEHGGYL DKVEPEIDEN GSMIPKYKIY TPGANERKYN 60 NYMYLICYGF VEDVERTPET GKRKKIRTIA AYPLGVGKSA SHPQDLLEEL CSLKVTVRRT 120 AGSTEKIVFG SSGPLNHLVP WKKVLTSGSI FNAVKVCRNV DQIQLDKHQA LRIFFLS I TK 180 LNDSGIYMIP RTMLEFRRNN AIAFNLLVYL KIDADLSKMG IQGSLDKDGF KVASFMLHLG 240 NFVRRAGKYY SVDYCRRKID RMKLQFSLGS IGGLSLHIKI NGVI SKRLFA QMGFQKNLCF 300 SLMDINPWLN RLTWNNSCE I SRVAAVLQPS IPREFMIYDD VFIDNTGRIL KGEAYVEQKL 360 I SEEDLNSAV DHHHHHH 377
38. The pharmaceutical composition as claimed in claim 34 wherein the matrix protein is selected from Nipah virus or Hendra virus.
39. The pharmaceutical composition as claimed in claim 34 wherein the matrix protein is selected from paramyxoviruses, orthomyxoviruses, henipaviruses, DNA viruses and RNA viruses.
40. The pharmaceutical composition as claimed in claim 34 wherein the recombinant matrix protein is produced in Escherichia coli cells.
41 . A diagnostic reagent comprising an effective amount of recombinant matrix protein of Nipah virus.
42. The diagnostic reagent as claimed in claim 41 wherein the recombinant matrix protein contains matrix protein of Nipah virus.
43. The diagnostic reagent as claimed in claim 42 wherein the matrix protein contains at least one His tag and myc epitope.
44. The diagnostic reagent as claimed in claim 42 wherein the matrix protein is encoded by the following nucleotide coding sequence:
ATGGAGCCGGACATCAAGAGTATTTCAAGTGAGTCAATGGAAGGAGTATCTGATTTCAGC 60 CCTAGTTCTTGGGAGCATGGTGGGTATCTTGATAAGGTTGAACCAGAAATTGATGAAAAT 120 GGCAGTATGATTCCAAAATACAAGATCTATACCCCAGGAGCTAACGAGAGGAAATACAAC 180 AACTACATGTACCTTATATGTTACGGCTTTGTTGAAGATGTTGAGAGAACCCCAGAGACA 240
GGGAAACGCAAGAAGATCAGGACAATTGCTGCCTACCCTCTGGGTGTTGGTAAGAGTGC
300 C TCTCATCCCCAAGATCTTCTGGAGGAACTCTGTTCCCTCAAAGTTACTGTGAGAAGAACA 360 GCTGGATCAACTGAGAAAATTGTGTTTGGATCATCTGGCCCTCTAAATCACCTCGTTCCG 420 TGGAAGAAAGTACTGACTAGTGGTTCAATTTTTAATGCAGTCAAGGTTTGTCGGAACGTT 480 GATCAGATACAGCTTG AC AAGCATCAAGCTCTGAGAATATTTTTTCTCAGTATCACAAAG 540 CTCAATGATTCTGGAATCTACATGATTCCACGAACCATGCTTGAGTTCAGGAGAAACAAT 600 GCCATTGCCTTCAATCTTCTAGTGTACTTGAAGATTGATGCTGATTTATCCAAAATGGGG 660
ATCCAGGGAAGCCTCGATAAAGATGGCTTCAAGGTTGCCTCCTTCATGCTACACTTGGG
720 G AACTTTGTCCGTCGTGCAGGGAAGTATTACTCTGTTGATTATTGTAGGAGGAAGATTGAT 780 AGGATGAAATTGCAGTTTTCACTGGGTTCCATAGGCGGACTAAGTCTCCACATTAAGATC 840 AATGGTGTAATCAGCAAACGGCTGTTTGCTCAAATGGGATTCCAAAAAAACCTTTGTTTC 900
1 TCTTTGATGG ACATCAATCCTTGGCTCAACAGATTGACCTGGAACAACAGTTGTG AGATC 960
1021 AGCCGAGTAGCAGCTGTGTTGCAGCCTTCTATTCCAAGAGAGTTCATGATCTATGATGAT
02 108 GTCTTCATTGACAATACAGGGAGAATTCTAAAGGGCGAAGCTTACGTAGAACAAAAACTC
1 08 1 13 ATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTAA
1 4
45. The diagnostic reagent as claimed in claim 42 wherein the matrix protein which has the following amino acid sequence:
MEPDIKSISS ESMEGVSDFS PSSWEHGGYL DKVEPEIDEN GSMIPKYKIY TPGANERKYN 60 NYMYLICYGF VEDVERTPET GKRKKIRTIA AYPLGVGKSA SHPQDLLEEL CSLKVTVRRT 120 AGSTEKIVFG SSGPLNHLVP WKKVLTSGSI FNAVKVCRNV DQIQLDKHQA LRIFFLSITK 180 LNDSGIYMIP RTMLEFRRNN AIAFNLLVYL KIDADLSKMG IQGSLDKDGF KVASFMLHLG 240 NFVRRAGKYY SVDYCRRKID RMKLQFSLGS IGGLSLHIKI NGVI SKRLFA QMGFQKNLCF 300 SLMDINP LN RLTWNNSCEI SRVAAVLQPS IPREFMIYDD VFIDNTGRIL KGEAYVEQKL 360 ISEEDLNSAV DHHHHHH 377 46. The diagnostic reagent as claimed in claim 42 wherein the matrix protein is selected from Nipah virus or Hendra virus.
47. The diagnostic reagent as claimed in claim 42 wherein the matrix protein is selected from paramyxoviruses, orthomyxoviruses, henipaviruses, DNA viruses and RNA viruses.
48. The diagnostic reagent as claimed in claim 42 wherein the recombinant matrix protein is produced in Escherichia coli cells.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| MYPI20094720 | 2009-11-06 | ||
| MYPI20094720 | 2009-11-06 |
Publications (3)
| Publication Number | Publication Date |
|---|---|
| WO2011056060A2 true WO2011056060A2 (en) | 2011-05-12 |
| WO2011056060A3 WO2011056060A3 (en) | 2011-12-15 |
| WO2011056060A8 WO2011056060A8 (en) | 2012-06-14 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| PCT/MY2010/000211 WO2011056060A2 (en) | 2009-11-06 | 2010-10-19 | Recombinant matrix protein of nipah virus |
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| WO (1) | WO2011056060A2 (en) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011014051A1 (en) * | 2009-07-30 | 2011-02-03 | Universiti Putra Malaysia | A method for controlling proteolytic degradation of recombinant proteins |
| WO2011061849A1 (en) * | 2009-11-20 | 2011-05-26 | アリジェン製薬株式会社 | Recombinant measles virus useful as bivalent vaccine against measles and nipah virus infection |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2011014051A1 (en) * | 2009-07-30 | 2011-02-03 | Universiti Putra Malaysia | A method for controlling proteolytic degradation of recombinant proteins |
| WO2011061849A1 (en) * | 2009-11-20 | 2011-05-26 | アリジェン製薬株式会社 | Recombinant measles virus useful as bivalent vaccine against measles and nipah virus infection |
Non-Patent Citations (4)
| Title |
|---|
| 'An efficient cloning and site-specific transposition system to generate recombinant baculovirus for high-level protein expression' INVITROGEN CATALOG NOS. A11101, A11100 USER MANUAL, BAC-TO-BAC ® TOPO ® EXPRESSION SYSTEM 15 December 2008, * |
| CARA THERESIA PAGER ET AL.: 'A mature and fusogenic form of the Nipah virus fusion protein requires proteolytic processing by cathepsin L' VIROLOGY vol. 346, 07 February 2006, pages 251 - 257 * |
| HANA M. WEINGARTL ET AL.: 'Recombinant Nipah Virus Vaccines Protect Pigs against Challenge' J OF VIROLOGY vol. 80, no. 16, 08 August 2006, pages 7929 - 7938 * |
| MAJID ESHAGHI ET AL.: 'Purification and Characterization of Nipah Virus Nucleocapsid Protein Produced in Insect Cells' JOURNAL OF CLINICAL MICROBIOLOGY vol. 43, no. 7, 31 July 2005, pages 3172 - 3177 * |
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| Publication number | Publication date |
|---|---|
| WO2011056060A3 (en) | 2011-12-15 |
| WO2011056060A8 (en) | 2012-06-14 |
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