CN110387385A - A kind of novel rhabdovirus expression vector - Google Patents
A kind of novel rhabdovirus expression vector Download PDFInfo
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- CN110387385A CN110387385A CN201910650551.5A CN201910650551A CN110387385A CN 110387385 A CN110387385 A CN 110387385A CN 201910650551 A CN201910650551 A CN 201910650551A CN 110387385 A CN110387385 A CN 110387385A
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Abstract
The invention discloses a kind of novel rhabdovirus expression vectors, are related to field of biotechnology.The present invention obtains the novel rhabdovirus expression vector after knocking out four on rhabdovirus expression vector continuous dispensable gene Ac84, Ac85, Ac86 and Ac87 simultaneously.Novel rhabdovirus expression vector of the invention makes exogenous protein expression level improve 80%.The multiplication characteristic of virus is unaffected simultaneously.The raising of expression yield can significantly reduce the production cost of enterprise.The rhabdovirus expression vector can be used for biological products industrial circle, especially subunit vaccine industrial circle.
Description
Technical field
The present invention relates to field of biotechnology more particularly to a kind of novel rhabdovirus expression vectors.
Background technique
Baculoviral is the double-stranded DNA virus of specific infection arthropod, autographa california multiple nuclear polyhedrosis virus
(Autographa californica nucleopolyhedrovirus, AcMNPV) is the type sepecies of baculoviral.Since
Nineteen eighty-three Smith GE etc. has expressed (Mol Cell since humanβ-interferon's gene with baculoviral for the first time in insect cell
Biol.1983；3:2156-65.), due to having many advantages, such as low cost, high yield, and have various posttranslational modification systems,
Rod string design is widely used in research and production, especially biological products and vaccine industrial circle.
However, compared to the prokaryotic expression system (Escherichia coli, bacillus subtilis) and yeast that industrially generally use
The yield of expression system, rod string design is also unsatisfactory, this leads to being produced into for baculovirus expression system
This is high.
Therefore, it is necessary to study specific technological means to improve the yield of above-mentioned expression vector system.
Summary of the invention
In view of this, main purpose is raising table the embodiment of the invention provides a kind of novel rhabdovirus expression vector
Up to yield.
In order to achieve the above objectives, invention broadly provides following technical solutions:
On the one hand, the embodiment of the invention provides a kind of novel rhabdovirus expression vectors, by baculoviral AcMNPV table
Four continuous dispensable gene Ac84, Ac85, Ac86 and Ac87 are obtained described novel rod-shaped after knocking out simultaneously on up to carrier
Virus expression carrier.
Preferably, the knockout of the Ac84 refers to that the promoter region of the Ac84 is destroyed or the code area of the Ac84
It is destroyed or the promoter region of the Ac84 is destroyed simultaneously with its code area.
Preferably, the knockout of the Ac85 refers to that the promoter region of the Ac85 is destroyed or the code area of the Ac85
It is destroyed or the promoter region of the Ac85 is destroyed simultaneously with its code area.
Preferably, the knockout of the Ac86 refers to that the promoter region of the Ac86 is destroyed or the code area of the Ac86
It is destroyed or the promoter region of the Ac86 is destroyed simultaneously with its code area.
Preferably, the knockout of the Ac87 refers to that the promoter region of the Ac87 is destroyed or the code area of the Ac87
It is destroyed or the promoter region of the Ac87 is destroyed simultaneously with its code area.
On the other hand, the present invention provides a kind of recombinant proteins, with the novel rhabdovirus expression vector building recombination
Virus, then infected insect host cell, the host cell obtain institute by cultivating, being proliferated and be expressed as recombinant protein
State recombinant protein.
Another aspect, the present invention provides the novel rhabdovirus expression vector applications industrial in vaccine.
Compared with prior art, the beneficial effects of the present invention are:
The present invention in research in discovery Baculovirus Gene group there are a large amount of dispensable genes, and some nonessential bases
Because cluster adjacent to each other exists, dispensable gene existing for these clusters (containing genes with unknown function) includes Ac84 to Ac87 continuous
Four genes；After the present invention knocks out continuous four viral dispensable gene Ac84, Ac85, Ac86 and Ac87 at the same time, hair
Existing protein expression level significantly improves, the proliferation level of virus does not have significant change, illustrates the present invention by above-mentioned knockout skill
Art can provide the rhabdovirus expression vector with the excellent production traits and high yield characteristics.
Detailed description of the invention
Fig. 1 is the gene knockout strategy schematic diagram of baculovirus vector of the invention；
Fig. 2 is the green fluorescent protein GFP SDS PAGE whole cellprotein immunoblotting of baculovirus vector expression of the invention
Hybridization figure；
Fig. 3 is the green fluorescent protein GFP total fluorescence intensity histogram of baculovirus vector expression of the invention；Fig. 4 is
Growth curve of baculovirus vector of the invention.
Specific embodiment
For further illustrate the present invention to reach the technical means and efficacy that predetermined goal of the invention is taken, below with compared with
Good embodiment, to specific embodiment, technical solution, feature and its effect applied according to the present invention, detailed description is as follows.Under
Stating the special characteristic, structure or feature in multiple embodiments in bright can be combined by any suitable form.
Application principle of the invention is explained in detail with reference to the accompanying drawing.
Embodiment 1
1, the knockout of Ac84-87 gene
With with Ac84-87 DNA fragmentation upstream and downstream 50bp homology arm primer (SEQ ID NO:1 and SEQ ID NO:
2), using pTriEx1.1 plasmid as template, ampicillin resistance gene segment is expanded, the PCR product (SEQ of 1023bp is obtained
ID NO:3)。
The method of the PCR product of acquisition electrotransformation is transferred to the coli strain with RedET plasmid and Bacmid
It in HS996, is induced through arabinose and generates recombinase, the Escherichia coli amoxicillin resistant panel screening after recombination obtains
Obtain positive colony.Ac84-87 DNA fragmentation is replaced by ampicillin resistance gene segment at this time.It is mentioned from Escherichia coli
Bacmid is taken, Bacmid Δ Ac84-87 (knock out strategy and see attached drawing 1) is named as after sequencing identification.
Bacmid Δ Ac84-87 is mentioned, after the linearisation of Bsu36I digestion with restriction enzyme through plasmid is small, for subsequent experimental
It uses.
2, the protein yield of fluorescin GFP is expressed
(GFP gene fragment clone is in pTriEx1.1 with pTriEx-GFP plasmid by the Bacmid Δ Ac84-87 of linearisation
Between the site NcoI/XhoI) cotransfection Sf9 insect cell, 5 days collection P0 are for recombinant virus after transfection.P0 for virus amplification extremely
P1 generation virus, infects Sf9 cell, the 4th day collection cell after infection with 3 MOI.Polyacrylamide gel is carried out after cell cracking
Electrophoresis, transferring film, with anti-GFP antibody hybridization (attached drawing 2).The result shows that improved carrier output is apparently higher than control.
3, the activity of expressing green fluorescent protein GFP
Sf9 cell, the 4th day collection cell after infection are infected with the above-mentioned recombinant virus with GFP gene with 3 MOI.With
The green fluorescence intensity of flow cytomery cell, and computational geometry average (attached drawing 3).After comparison, it was found that green fluorescence egg
White GFP yield improves 83% relative to wild type.
4, virus multiplication characteristic
Sf9 cell is infected with 0.1 MOI using the recombinant virus with green fluorescent protein, is taken within every 24 hours later primary
Sample measures virus titer with limiting dilution method, draws a viral growth curve (attached drawing 4).Compared with wild-type virus,
The virus that Ac84-87 is knocked out has and compares similar virus multiplication characteristic.
Of the invention opens one's minds: there are a large amount of nonessential bases in research in the present invention in discovery Baculovirus Gene group
Cause, and some dispensable genes cluster adjacent to each other exists, dispensable gene (base containing unknown function existing for these clusters
Cause) it include continuous four genes of Ac84 to Ac87.
Ac84 and Ac85 is reported without any research so far, thus it is speculated that is dispensable gene.
Ac86 encodes a kind of egg with RNA ligase, 5 ' polynucleotides-kinases and 3 ' polynucleotides-phosphatase activity
White matter, it is possible to participate in RNA and repair (J Biol Chem.2004；279:18220-31.).Research thinks that it is felt in virus
Contaminate dispensable gene (the J Gen Virol.1998 of early expression；79:629-37.).
Ac87 is not reported in AcMNPV.Homologous gene Bm70 in silkworm polyhedrosis virus may encode one
Nucleocapsid protein (the Gene.1997 of 15kDa；185:69-75.), but the duplication (Virus that Bm70 has no effect on virus is knocked out
Res.2012；165:197-206.).
Generally speaking, Ac84, Ac85, Ac86 and Ac87 are the nonessential or doubtful dispensable genes of baculoviral, until
When replicating in insect cell line less, it is important to show that this four genes have the function of without public information.The present invention attempts will
Aforementioned four non-essential gene knocks out, and by examining the yield and multiplication characteristic of above-mentioned new expression vector, finds novel expression
The protein expression level of carrier significantly improves, and viral proliferation level does not have significant change, and thus explanation uses above-described embodiment 1
Method available one with it is excellent production shape and high yield characteristics rhabdovirus expression vector.
Place, those skilled in the art can not select from the prior art to the greatest extent in the embodiment of the present invention.
Disclosed above is only a specific embodiment of the invention, but scope of protection of the present invention is not limited thereto, is appointed
What those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, answer
It is included within the scope of the present invention.Therefore, protection scope of the present invention should be with above-mentioned scope of protection of the claims
It is quasi-.
Sequence table
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>a kind of novel rhabdovirus expression vector
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
tcgtaaagcg agttgaattt tgattacaaa tattttgttt atgatagcaa aacgcgcgga 60
catgag 66
<210> 2
<211> 66
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gtttacataa cattctactt taatgtaata atattcttca atttcttggg ttaccaatgc 60
ttaatc 66
<210> 3
<211> 1023
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
tcgtaaagcg agttgaattt tgattacaaa tattttgttt atgatagcaa aacgcgcgga 60
catgagacaa taaccctgat aaatgcttca ataatattga aaaaggaaga gtatgagtat 120
tcaacatttc cgtgtcgccc ttattccctt ttttgcggca ttttgccttc ctgtttttgc 180
tcacccagaa acgctggtga aagtaaaaga tgctgaagat cagttgggtg cacgagtggg 240
ttacatcgaa ctggatctca acagcggtaa gatccttgag agttttcgcc ccgaagaacg 300
ttttccaatg atgagcactt ttaaagttct gctatgtggc gcggtattat cccgtattga 360
cgccgggcaa gagcaactcg gtcgccgcat acactattct cagaatgact tggttgagta 420
ctcaccagtc acagaaaagc atcttacgga tggcatgaca gtaagagaat tatgcagtgc 480
tgccataacc atgagtgata acactgcggc caacttactt ctgacaacga tcggaggacc 540
gaaggagcta accgcttttt tgcacaacat gggggatcat gtaactcgcc ttgatcgttg 600
ggaaccggag ctgaatgaag ccataccaaa cgacgagcgt gacaccacga tgcctgtagc 660
aatggcaaca acgttgcgca aactattaac tggcgaacta cttactctag cttcccggca 720
acaattaata gactggatgg aggcggataa agttgcagga ccacttctgc gctcggccct 780
tccggctggc tggtttattg ctgataaatc tggagccggt gagcgtgggt ctcgcggtat 840
cattgcagca ctggggccag atggtaagcc ctcccgtatc gtagttatct acacgacggg 900
gagtcaggca actatggatg aacgaaatag acagatcgct gagataggtg cctcactgtt 960
tacataacat tctactttaa tgtaataata ttcttcaatt tcttgggtta ccaatgctta 1020
atc 1023
Claims (7)
1. a kind of novel rhabdovirus expression vector, which is characterized in that continuous by four on baculoviral AcMNPV expression vector
Dispensable gene Ac84, Ac85, Ac86 and Ac87 knock out simultaneously after obtain the novel rhabdovirus expression vector.
2. a kind of novel rhabdovirus expression vector as described in claim 1, which is characterized in that the knockout of the Ac84 refers to
The promoter region of the Ac84 is destroyed or the code area of the Ac84 is destroyed or the promoter region of the Ac84 is encoded with it
Area is destroyed simultaneously.
3. a kind of novel rhabdovirus expression vector as described in claim 1, which is characterized in that the knockout of the Ac85 refers to
The promoter region of the Ac85 is destroyed or the code area of the Ac85 is destroyed or the promoter region of the Ac85 is encoded with it
Area is destroyed simultaneously.
4. a kind of novel rhabdovirus expression vector as described in claim 1, which is characterized in that the knockout of the Ac86 refers to
The promoter region of the Ac86 is destroyed or the code area of the Ac86 is destroyed or the promoter region of the Ac86 is encoded with it
Area is destroyed simultaneously.
5. a kind of novel rhabdovirus expression vector as described in claim 1, which is characterized in that the knockout of the Ac87 refers to
The promoter region of the Ac87 is destroyed or the code area of the Ac87 is destroyed or the promoter region of the Ac87 is encoded with it
Area is destroyed simultaneously.
6. a kind of recombinant protein, which is characterized in that use the described in any item novel rhabdovirus expression vector structures of claim 1-5
Recombinant virus is built, then infected insect host cell, the host cell is by cultivating, being proliferated and be expressed as recombinant protein, i.e.,
Obtain the recombinant protein.
7. the application industrial in vaccine of the described in any item novel rhabdovirus expression vectors of claim 1-5.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113106125A (en) * | 2021-04-16 | 2021-07-13 | 陕西杆粒生物科技有限公司 | Baculoviral vector with Ac129-131 deletion |
| CN113416751A (en) * | 2021-07-01 | 2021-09-21 | 陕西杆粒生物科技有限公司 | Ac96 inactivated baculovirus vector |
| CN114292877A (en) * | 2021-01-11 | 2022-04-08 | 陕西杆粒生物科技有限公司 | Baculoviral expression vector for Ac63-64 gene knockout |
| CN114292878A (en) * | 2021-04-13 | 2022-04-08 | 陕西杆粒生物科技有限公司 | Ac68-72 knockout baculovirus expression vector |
| CN114317608A (en) * | 2020-12-28 | 2022-04-12 | 陕西杆粒生物科技有限公司 | Gene knockout type baculovirus expression vector |
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Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114317608A (en) * | 2020-12-28 | 2022-04-12 | 陕西杆粒生物科技有限公司 | Gene knockout type baculovirus expression vector |
| CN114317608B (en) * | 2020-12-28 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Gene knockout type baculovirus expression vector |
| CN114292877A (en) * | 2021-01-11 | 2022-04-08 | 陕西杆粒生物科技有限公司 | Baculoviral expression vector for Ac63-64 gene knockout |
| CN114292877B (en) * | 2021-01-11 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Baculovirus expression vector for Ac63-64 gene knockout |
| CN114292878A (en) * | 2021-04-13 | 2022-04-08 | 陕西杆粒生物科技有限公司 | Ac68-72 knockout baculovirus expression vector |
| CN114292878B (en) * | 2021-04-13 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Baculovirus expression vector with Ac68-72 knocked out |
| CN113106125A (en) * | 2021-04-16 | 2021-07-13 | 陕西杆粒生物科技有限公司 | Baculoviral vector with Ac129-131 deletion |
| CN113106125B (en) * | 2021-04-16 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Ac129-131 deleted baculovirus vector |
| CN113416751A (en) * | 2021-07-01 | 2021-09-21 | 陕西杆粒生物科技有限公司 | Ac96 inactivated baculovirus vector |
| CN113416751B (en) * | 2021-07-01 | 2023-08-22 | 陕西杆粒生物科技有限公司 | Ac96 inactivated baculovirus vector |
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