CN106831963B - Grouper nervous necrosis virus Coat gene, in expression in escherichia coli method and application - Google Patents
Grouper nervous necrosis virus Coat gene, in expression in escherichia coli method and application Download PDFInfo
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- 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
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
- A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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Abstract
The present invention relates to gene engineering technology field, in particular to a kind of grouper nervous necrosis virus Coat gene, in expression in escherichia coli method and application.The present invention passes through the acquisition of grouper nervous necrosis virus Coat gene, has filled up the vacancy of grouper nervous necrosis virus Coat gene studies.Simultaneously according to grouper nervous necrosis virus Coat gene order can artificial synthesized or genetically modified organism synthesize biologically active recombinant protein, facilitate us and understand grouper nervous necrosis virus gene to prepare vaccine, recombinant vaccine uses the usage amount that drug in breeding production can also be greatly lowered, it is inherently eliminated the medicament residue hidden danger of aquatic products, provides health good green aquatic product for consumer.
Description
Technical field
The present invention relates to gene engineering technology field, in particular to a kind of grouper nervous necrosis virus Coat gene,
Expression in escherichia coli method and application.
Background technique
Viral Nervous Necrosis in Fishes (viral nervous necrosis, VNN), is a kind of worldwide fish
Epidemic infectious diseases, it is very harmful to prelarva and juvenile fish, serious person in one week the death rate up to 100%.Since its is high
Infectiousness and harmfulness are classified as important fish diseases (OIE, 2001) by world organization for animal health (OIE).Currently, the disease is being removed
Countries and regions other than Africa are spread rapidly, and infected fish up to more than 40, cause huge to various countries' mariculture industry
Big harm.Lead to the pathogenic of the infectious disease is nervous necrosis virus (Nervous necrosis virus, NNV) originally, it belongs to
β-Nodavirus category in Nodavirus section (Nodaviridae), is a kind of picornavirus.
Grouper is the high-quality fish of top grade in current marine fish.With China's Artificial Rearing of Epinephelus coioides
Mature and Epinephelus coioides seed industrialization large-scale production, becomes important marine fish culture kind.But this several years lithosporics
Nervous necrosis viral disease frequently occurs for the fry kind phase, causes seed mortality, and survival rate of seedling only has 3~5%, sometimes less than
1%, or even be annihilated, causing huge economic loss also to frequently result in seed outside, supply falls short of demand, and production is formed in influence.It is domestic
Chemically drug, biovaccine, RNA interference (RNAi) and aquaculture model etc. have carried out largely the prevention and treatment of the disease to outer scholar
Research, but up to the present still do not find effectively preventing measure.
For the virosis of fish, preferred immunoprotection method, that is, vaccine most effective, safe and without environment side effect
It uses.By the protection of vaccine, infecting for various viruses has been resisted in cultured fishes, so that intensive and batch production high density water
The sustainable and stable development for producing cultivation is possibly realized.Import aquatic products vaccine product produces sub- single at present mostly from eukaryotic expression system
Position vaccine, expensive, production capacity and selling price are far from satisfying Chinese market demand, are not suitable for China's seawater and support
It grows in production and promotes, and there is presently no the vaccine products of available grouper nervous necrosis virus for the country.
Traditional vaccine (attenuated vaccine, inactivated vaccine) haves the shortcomings that safety and protecting effect are poor.
Summary of the invention
The present invention be directed to traditional vaccine deficiency, provide it is a kind of by genetic engineering means research and develop recombinant protein and recombination
The vaccine of new generation of DNA technique overcomes many disadvantages of traditional vaccine, the present invention is directed to propose a kind of grouper nerve necrosis
Viral Coat gene, in expression in escherichia coli method and application.
The technical scheme is that
A kind of grouper nervous necrosis virus Coat gene, sequence 1433bp, nucleotide sequence such as SIDNO:
1。
Another object of the present invention is to disclose a kind of grouper nervous necrosis virus Coat albumen, amino acid sequence
Column such as SID NO:2, overall length 338aa, pass through Bacillus coli expression system by above-mentioned grouper nervous necrosis virus Coat gene
System expression obtains.
Grouper nervous necrosis virus Coat full length gene 1433bp, 5 ' non-translational regions (1-26bp) including 26bp,
The open reading frame (27-1043bp) of 1017bp and the 3 ' non-translational regions (1044-1433bp) of 390bp, encode 338 amino
Acid；, the atg of double underline mark is initiation codon in sequence, and the taa of single underscore mark is terminator codon.
Another object of the present invention is the method that above-mentioned Coat albumen is expressed in E. coli system, including recombination
Building, recombinant vector transformed competence colibacillus cell, fungi preservation, inducing expression and the identification of carrier.
Preferably, the expression vector is pET30a, and the competent cell is BL21 (DE3).
Preferably, the specific method is as follows for the inducing expression:
Step 1: take positive colony to be inoculated into the LB culture medium containing 50 μ g/mL kanamycin sulfates in 1:100 ratio, in
37 DEG C of * 200rpm growths are stayed overnight in shaking table；
Step 2: above-mentioned bacterium solution being inoculated into the 2L containing 50 μ g/mL kanamycin sulfate resistances in 1:100 ratio in second day
In TB culture medium, it is put into 37 DEG C of * 200rpm in shaking table and grows to OD600When=0.6-0.8, shaking table temperature is down to 15-25 DEG C,
Final concentration 0.10-0.30mM IPTG induced growth 16h is added after 1-3h；
Step 3: the bacterium solution after induction is abandoned into supernatant in 4 DEG C of * 8000rpm centrifugation 15min, collects thallus, it is slow with phosphate
Fliud flushing PBS is resuspended and repeats centrifugal process to clean thallus；It is placed in -20 DEG C of preservations.
Preferably, recombinant vector conversion the specific method is as follows:
Step 1: taking out competent cell BL21 (DE3) from -80 DEG C of refrigerators, and be immediately placed in ice water, in ice
Upper thawing 2-5min；
Step 2: tube wall is flicked after melting 1-2 times so that cell is resuspended；The expression vector Plasmid DNA of about 100ng is directly added
Enter in competent cell, gentle agitation mixing is subsequently placed at 30min on ice-water bath；
Step 3: be put into water-bath after taking-up: 42 DEG C, heat shock 90s, heat shock is complete to put back to rapidly 3min in ice-water bath；It takes out
The LB liquid medium of 200 μ L non-resistants is taken to be added in the competent cell converted afterwards；
Step 4: being put into 37 DEG C of * 195rpm growth 1h in shaking table, draw 50 μ L-80 μ L suspension even spreads and contain 50 μ g/
On the LB plate of mL kanamycin sulfate, it is inverted, 37 DEG C of overnight incubations.
Preferably, the specific method is as follows for the preservation of the bacterial strain and inducing expression:
Step 1: the LB culture medium that 4mL contains 50 μ g/mL kanamycin sulfates is added in the picking monoclonal from conversion plate
In；
2:37 DEG C of * 200rpm of step is cultivated to OD600For 0.5-0.8,2-3 hours；It is taken out in growth course under aseptic condition
Sample measures OD600Value；
Step 3: taking the bacterium solution of 0.9mL in pipe to mix with 0.1mL80% sterile glycerol, be stored in -80 DEG C of refrigerators；
Step 4: final concentration 0.375mM IPTG being added into Tube propagation liquid, places 37 DEG C of induction 4h.
Preferably, the identification uses SDS-PAGE and Electronic Speculum observation.
Another object of the present invention is to disclose a kind of vaccine of anti-grouper nervous necrosis virus, contains above-mentioned lithosporic
Fish nervous necrosis virus Coat albumen or its any mixture.
Most what a purpose of the invention is a kind of open above-mentioned grouper nervous necrosis virus Coat gene anti-
Application in grouper nervous necrosis virus.
The beneficial effects of the present invention are:
The present invention passes through the acquisition of grouper nervous necrosis virus Coat gene, has filled up grouper nervous necrosis virus
The vacancy of Coat gene studies.It simultaneously can artificial synthesized or transgenosis according to grouper nervous necrosis virus Coat gene order
The biologically active recombinant protein of biosynthesis, facilitates us and understands grouper nervous necrosis virus gene to prepare vaccine,
Recombinant vaccine is inherently eliminated the medicine of aquatic products using the usage amount that drug in breeding production can also be greatly lowered
Object remains hidden danger, provides health good green aquatic product for consumer.
Temperature, revolving speed, the concentration of IPTG and time are optimized during the inducing expression of albumen of the present invention, induced
Shi Wendu is 15-25 DEG C, IPTG concentration is 0.10-0.30mM, induction time 16h, can increase the dissolubility of recombinant protein, have
Correct configuration is formed conducive to protein folding, to have biological activity.
Detailed description of the invention
Attached drawing 1 is that SDS-PAGE analyzes Coat albumen in BL21 (DE3) expression；
Lane M:SDS-PAGEProtein marker；Lane 0: control；Lane 1:15 DEG C overnight induction；Lane 2:
37 DEG C of induction 4h；
Attached drawing 2 is that VLP and NNV virus structure are compared in Electronic Speculum observation, and (A) is NNV virus structure, and (B) is VLP albumen knot
Structure；
Attached drawing 3 is that SDS-PAGE analyzes Coat albumen supernatant purification result；
Lane M:SDS-PAGEProtein marker；Lane 1: full bacterium breaks supernatant after bacterium centrifugation；Lane 2: supernatant is same
Efflux after Ni-IDA is incubated for；BufferA (being free of TritonX-100) eluent of Lane 3-4:50mM imidazoles；Lane 5-
BufferA (being free of TritonX-100) eluent of 6:100mM imidazoles；The BufferA of Lane 7-10:300mM imidazoles (is free of
TritonX-100) eluent；BufferA (being free of TritonX-100) eluent of Lane 11-12:500mM imidazoles；
Attached drawing 4 is Coat determination of protein concentration standard curve；
Attached drawing 5 is antibody test effect curve.
Specific embodiment
The technical scheme in the embodiments of the invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, this field is common
Technology
Personnel's every other embodiment obtained without making creative work, belongs to protection of the present invention
Range.Embodiment 1: grouper nervous necrosis virus Coat gene design and optimization uses the password of the safe biological recent development of moral
Sub- optimization software Max Codon TMO ptimization Program (V13), target protein Coat final optimization pass as a result,
Its sequence is 1433bp, nucleotide sequence such as SIDNO:1.
Embodiment 2: grouper nervous necrosis virus Coat protein expression and purifying
1, Coat protein expression and identification
The conversion of 1.1 expression vectors
Step 1: taking out competent cell BL21 (DE3) from -80 DEG C of refrigerators, and be immediately placed in ice water, in ice
Upper thawing 2-5min.
Step 2: tube wall is flicked after melting 1-2 times so that cell is resuspended.The expression vector Plasmid DNA of about 100ng is directly added
Enter in competent cell, gentle agitation mixing is subsequently placed at 30min on ice-water bath (can prepare 42 DEG C of water-baths at this time).
Step 3: (42 DEG C), heat shock 90s are put into water-bath after taking-up；Heat shock is complete to put back to rapidly 3min in ice-water bath；It takes
The LB liquid medium of 200 μ L non-resistants is taken to be added in the competent cell converted after out.
Step 4: being put into shaking table (37 DEG C of * 195rpm) and grow 1h, draw 50 μ L-80 μ L suspension even spreads and contain 50 μ
In the LB plate of g/mL kanamycin sulfate, it is inverted, 37 DEG C of overnight incubations.
The preservation and inducing expression of 1.2 bacterial strains
Step 1: 3 monoclonals of picking from conversion plate are added LB of the 4mL containing 50 μ g/mL kanamycin sulfates and cultivate
In base, number is labeled as " 0 " " 1 " " 2 ".
2:37 DEG C of * 200rpm of step is cultivated to OD600About 0.5-0.8 (it is generally necessary to 2-3 hours).Nothing in growth course
Sample is taken out under the conditions of bacterium measures OD600Value.
Step 3: taking the bacterium solution of each 0.9mL in " 1 " " 2 " number pipe to mix with 0.1mL80% sterile glycerol, be stored in -80 DEG C of ice
In case.
Step 4: final concentration 0.375mM IPTG (IPTG is not added in " 0 " control group) is added into Tube propagation liquid, usual " 0 "
" 1 " places 15 DEG C of overnight inductions, and " 2 " place 37 DEG C of induction 4h.
1.3SDS-PAGE identifies inducing expression result
Step 1. takes 12000rpm*10min*4 DEG C of culture solution of each 400-600 μ L centrifugation in " 0 " " 1 " " 2 " test tube respectively, goes
Except supernatant, 500 μ L ddH are added2Thallus is resuspended in O, and 12000rpm*10min*4 DEG C of centrifugation again, removes supernatant.
Phosphate buffer (PBS) mixing of 50 μ L is added so that precipitating is resuspended in step 2..
2 × SDS sample-loading buffer that 50 μ L are added in step 3. makes albuminous degeneration in 100 DEG C of heating sample 15min rapidly, so
12000rpm*5min*4 DEG C of centrifuging and taking supernatant electrophoresis afterwards.10min100V pressure stabilizing electrophoresis before electrophoresis enters to bromophenol blue indicator
After separation gel, 200V pressure stabilizing electrophoresis to bromophenol blue band is migrated to from gel bottom 1cm, takes out gel Coomassie brilliant blue dyeing liquor
Dyeing, then continues in destainer, decoloration to clear background.As described in Figure 1.
1.4 Electronic Speculum observe albumen form
The albumen VLP film-making for taking nervous necrosis virus NNV and giving expression to, and in electric the two form such as Fig. 2 under the microscope, knot
Both fruit discoveries size and form are very much like.
The amplification culture of 1.5Coat albumen
Step 1: the strain of " 1 " and " 2 " conservation being taken to be inoculated into the LB containing 50 μ g/mL kanamycin sulfates in 1:100 ratio
In culture medium, (37 DEG C of * 200rpm) growth is stayed overnight in shaking table
Step 2: above-mentioned bacterium solution being inoculated into the training of the 2L TB containing 50 μ g/mL kalamycin resistances in 1:100 ratio in second day
It supports in base, is put into shaking table (37 DEG C of * 200rpm) and grows to OD600When=0.6~0.8, shaking table temperature is down to 15-25 DEG C, about
Final concentration 0.10-0.30mM IPTG induced growth 16h is added after 1-3h
Step 3: the bacterium solution after induction is abandoned into supernatant in 4 DEG C of * 8000rpm centrifugation 15min, collects thallus, it is slow with phosphate
Fliud flushing PBS is resuspended and repeats centrifugal process to clean thallus；It is placed in -20 DEG C of preservations.(if not purified on the day of receiving bacterium,
It is placed in -20 DEG C)
2, Coat protein purification
2.1Coat albumen supernatant purifies (whole process of purification operates at low temperature)
Step 1: using BufferA:50mMTris, 150mM NaC, 1mM DTT, 1%TritonX-100,1 μ g/mL
Pepstatin A, 1 μ g/mL Leupeptin, 20mM imidazoles, bacterium mud is resuspended in pH8.0, and (usual lysate dosage is 10-15mL/g
Bacterium mud)
Step 2: ultrasound cracking carries out ultrasound with 500W power in ice bath, and every ultrasound 3s, interval 6s amount to 15min,
Broken results are observed in end under the microscope.(can suitably adjust the broken time according to broken results)
3:13000rpm*30min*4 DEG C of centrifugation of step retains supernatant, and with 0.45um membrane filtration
Step 4: BufferB:50mM Tris, 150mM NaCl, 20mM imidazoles are used, pH8.0 balances 3mL Ni-IDA column,
Balance about 5-10CV is until ultraviolet registration reaches baseline
Step 5: the Ni-IDA column after the supernatant homostasis of 3 steps filtering being mixed to be placed on rotary mixer and is incubated in 4 DEG C
Educate 60-80min
Step 6: Ni-IDA column of the chromatographic column retention containing Coat albumen used, and 10- is rinsed with Buffer B
20CV, flow control is in 1.0mL/min, until ultraviolet registration reaches baseline
Step 7: with respectively containing the Buffer B of 50mM, 100Mm, 300mM imidazoles and 500mM imidazoles elution target egg
White, flow control collects the eluant component of each imidazole gradient in 1.5mL/min
Step 8: taking each 20 μ L of component that 2 × SDS sample-loading buffer of 20 μ L is added rapidly in 100 DEG C of heating samples
10min makes albuminous degeneration, then 12000rpm*5min*4 DEG C of centrifuging and taking supernatant electrophoresis.10min100V pressure stabilizing electrophoresis before electrophoresis,
Bromophenol blue indicator enters separation gel 200V pressure stabilizing electrophoresis to bromophenol blue band and migrates to from gel bottom 1cm later, takes out gel
It is dyed, is then continued in destainer with Coomassie brilliant blue dyeing liquor, decoloration to clear background.(balance Coat albumen yield and
Purity when, can be by reducing Ni-IDA column volume suitably to obtain the target protein of high-purity) as described in Figure 3.
The dialysis packing of 3Coat albumen
The target protein of above-mentioned collection is carried out dialysis 500mL Buffer C:1 × PBS with 3.5kDa bag filter by 3.1, and 10%
It in Glycerol, pH 7.4, is rotated with 4 DEG C of magnetic stirring apparatus, is changed the liquid once every 2h, total is changed liquid 4 times.
It is filtered after 3.2 dialysis with 0.22um film, and dispenses jelly in -80 DEG C.
4.Coat albumen quality inspection
4.1Coat protein stability tests (frozen process experiment)
Freeze after taking a packing in -80 DEG C of Coat albumen, is placed in mixture of ice and water and slowly melts to it, after thawing
No obvious suspended matter and 13000rpm*30min*4 DEG C centrifugation also without precipitating, illustrate that Coat albumen frozen process experiment is normal.
4.2Coat determination of protein concentration
Protein concentration is measured to useBradford determination of protein concentration kit.Its standard curve is as shown in Figure 4.Measure it
OD=0.275, final converted score are 0.380mg/mL.
Embodiment 3:
Vaccine VLP direct injection fish body is made in the acquisition grouper nervous necrosis virus Coat albumen of embodiment 2, selection is big
The small fish body for 4-6cm.
Injection system is intramuscular injection；VLP injection volume is 0.2-8.5 μ g/g fish；
VLP7D takes blood examination to survey antibody level for fish body 7 days after representing VLP injection, and VLP14D takes blood examination to survey after representing 14 days anti-
Body is horizontal, and NNV7D represents fish body injection nervous necrosis virus (NNV) and blood examination is taken to survey antibody level after 7 days, and NNV14D represents fish body
Injection nervous necrosis virus (NNV) takes blood examination to survey antibody level (NNV is equivalent to positive control) after 14 days, control 7d represents injection
Blood is taken within physiological saline 7 days, control 14d, which is represented, takes blood examination to survey antibody water body for injecting normal saline 14 days.
Antibody test effect is as shown in Figure 5: injection VLP reaches tens of thousands of with NNV virus liquid antibody titer, control group antibody effect
Valence is 90 or so, illustrates that VLP vaccine prepared by the present invention can generate almost the same potency with virus stock solution used.
Claims (3)
1. a kind of method that grouper nervous necrosis virus Coat albumen is expressed in E. coli system, which is characterized in that packet
Include building, recombinant vector transformed competence colibacillus cell, fungi preservation, inducing expression and the identification of recombinant vector；
The expression vector is pET30a, and the competent cell is BL21 (DE3)；
The grouper nervous necrosis virus Coat albumen is that the nucleotide sequence as described in SID NO:1 expresses to obtain；The lithosporic
The amino acid sequence such as SID NO:2 of fish nervous necrosis virus Coat albumen；
The specific method is as follows for the inducing expression:
Step 1: taking positive colony to be inoculated into the LB culture medium of the kanamycin sulfate containing 50ug/mL in 1:100 ratio, in shaking table
In 37 DEG C, 200rpm growth overnight；
Step 2: training above-mentioned bacterium solution in the 2L TB that 1:100 ratio is inoculated into the resistance of kanamycin sulfate containing 50ug/mL within second day
Support base in, be put into shaking table 37 DEG C, 200rpm grow to OD600When=0.6-0.8, shaking table temperature is down to 15-25 DEG C, l-3h
Final concentration 0.10-0.30mM IPTG induced growth 16h is added afterwards；
Step 3: the bacterium solution after induction being abandoned into supernatant in 4 DEG C, 8000rpm centrifugation 15min, thallus is collected, uses phosphate buffer
PBS is resuspended and repeats centrifugal process to clean thallus；It is placed in -20 DEG C of preservations.
2. the method that Coat albumen according to claim 1 is expressed in E. coli system, which is characterized in that the mirror
Surely SDS-PAGE and Electronic Speculum observation are used.
3. a kind of vaccine of anti-grouper nervous necrosis virus, which is characterized in that prepared containing method as claimed in claim 1 or 2
Grouper nervous necrosis virus Coat albumen.
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Inventor after: Ge Hui Inventor after: Lin Kebing Inventor after: Huang Zhongchi Inventor after: Zhu Zhihuang Inventor before: Lin Kebing Inventor before: Ge Hui Inventor before: Huang Zhongchi Inventor before: Zhu Zhihuang |