CN104744595A - Grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as preparation method and application thereof - Google Patents

Abstract

The invention discloses a grass carp haemorrhagic virus resisting engineered protein TAT (Trans-activating Transcriptional Activator)-VP7-TAT as well as a preparation method and application thereof. The amino acid sequence of the TAT-VP7-TAT protein is shown as SEQ ID NO:2, and a corresponding nucleotide sequence is shown as SEQ ID NO:1. By using Eschierichia coli BL21(DE3)/pET-32a-TAT-VP7-TAT to perform inducible expression for 4 to 5 hours, a TAT-VP7-TAT fusion protein can be efficiently expressed; the N end and the C end of the protein are respectively provided with protein transduction domain polypeptide TAT. The genetic engineering fusion protein provided by the invention can be used for preventing grass carp haemorrhagic virus diseases. The fusion protein has the advantages of high expression, easiness for industrial production, low cost and good safety; grass carp orally takes the fusion protein, so that better immune protection is realized.

Description

Anti-grass carp hemorrhage virus (GCHV) engineered protein TAT-VP7-TAT and preparation method and application

Technical field

The invention belongs to gene biological field of engineering technology, be specifically related to anti-grass carp hemorrhage virus (GCHV) engineered protein TAT-VP7-TAT and preparation method and application, more specifically relate to the antigen-4 fusion protein gene TAT-VP7-TAT subunit vaccine that a kind of 5 ' and 3 ' end is connected the gene order of nexin transduction domain polypeptide (TAT-PTD) respectively, also relate to a kind of N of a kind of Recombinant organism strain simultaneously and to hold and C holds with the engineering strain of grass carp hemorrhage virus (GCHV) (GCRV) the capsid protein VP7 fusion rotein of nexin transduction domain polypeptide; Also relate to the purposes of fusion rotein in the medicine of anti-grass carp hemorrhage virus (GCHV) that above-mentioned bacterial strains is expressed.

Background technology

Grass carp (Ctenopharyngodon idellus) is the main breed variety of CHINESE FRESHWATER, is one of " four large Chinese carps ".The feeding cost of grass carp is low, and cultivation scope is wide, and output accounts for 20% of CHINESE FRESHWATER cultivation ultimate production, has great importance to the development of Chinese rural economy.But its breeding process disease is more, except bacteriosis and parasitosis, hemorrhagic disease of grass carp can cause the large quantities of death of grass carp, and wherein infected mortality ratio is up to 90% the grass carp seedling stage, gives to produce cultivation and bring huge loss.

GCRV (Grass carp reovirus, GCRV) mainly causes grass carp, in the fingerling stage, hemorrhagic disease occurs also known as grass carp hemorrhage virus (GCHV) (Grass Carp Haemorrhage Virus, GCHV).This disease mainly occurs in the Grass Carp Juveniles stage, and it can cause the mortality of fingerling, has high infection rate and lethality rate, and Epidemic Scope is wide, morbidity season is long.Nineteen eighty-three isolates a strain grass carp hemorrhage virus (GCHV) from hemorrhagic disease of grass carp, and named as GCRV according to its characteristic, this virus is subordinate to Aquareovirus, is the first fishes virus that China is separated.GCRV virus particle is icosahedron, and in 5: 3: 2 symmetrical spheroidal particle, diameter is 65 ~ 72nm, and core diameter is about 50nm, without cyst membrane, has double capsid.GCRV is double-stranded RNA (dsRNA) virus, genome is made up of 11 segmented dsRNA, genomic fragment is divided into three groups by molecular weight, i.e. large fragment (L1-L3), middle fragment (M4-M6) and small segment (S7-S11).These 11 dsRNA encode 12 kinds of albumen altogether, comprise 7 kinds of structural protein and 5 kinds of Nonstructural Proteins, according to electrophoretic mobility of protein by 7 kinds of structural protein by order called after VP1-VP7 successively from big to small, wherein VP1, VP3, VP5, VP6, VP7 albumen is the component forming viral protein capside; 5 kinds of Nonstructural Protein called after NS16, NS26, NS31, NS38, NS80 respectively, they play an important role in the reproduction process of virus.

Immunoprophylaxis is the main method of effectively preventing and treating hemorrhagic disease of grass carp, although the traditional vaccine of the grass carp hemorrhage fever virus of the existing deactivation of China, application also has certain effect, this traditional vaccine, carry out amplicon virus with live body fish, its viral source is very difficult, be difficult to form industrialization; Nonetheless, as adopted injecting immune, practical application is inadvisable, soak and use, then arrived the large pool and also can not afford, and infusion method is also dubious on immunologic mechanism, therefore the research of subunit viral vaccine is most important for this disease of control as adopted seedling stage.VP5-VP7 protein complexes forms the outer protein coat of grass carp hemorrhage virus (GCHV), and wherein VP5 albumen is by M6 genes encoding, and containing 648 amino-acid residues, molecular weight is about 68.6kDa, may play the effect of such as Premeabilisation of cells in virus infection.VP7 albumen is by S10 genes encoding, and containing 276 amino-acid residues, molecular weight is about 29.8kDa, and it contains 1 zinc and refers to sequence, and can regulatory gene express, this albumen may be relevant with the interaction of virus and host cell and poisoning intrusion host.Research shows, VP5 and VP7 antibody can with antigen neutralization (Yongxing He, Hongxu Xu, Qian Yang, Dan Xu, Liqun Lu.The use of an in vitro microneutralization assay to evaluate the potential of recombinant VP5protein as an antigen for vaccinating against Grass carp reovirus [J] .Virology Journal, 20118:132) (Zhang L L, Shen J Y, Fang Q, et al.High level expression of grass carp reovirus VP7protein in prokaryotic cells [J] .Virologica Siniea, 2008, 23 (1): 51-56).VP7 albumen has good antigenicity, its antibody neutralization is 3 times of VP5, show that VP7 may be main antigenic determinant (the Shao L of GCRV, Sun X, FangQ, Antibodies against outer-capsid proteins of grass carp reovirus expressed in E.coli are capable of neutralizing viral infectivity.Virology Journal, 2011,8:347).External neutralization test also shows that VP7 polyclonal antibody has Neutralization effect, development GCRV subunit vaccine is made to become possible (He Y X, Yang Q, Xu H X, et al.Prokaryotic expression and purification of grass carp reovirus capsid protein VP7and its vaccine potential.African Journal of Microbiology Research, 2011,5 (13): 1643-1648).In aquaculture, oral vaccine is that one is is the most effectively prevented and cured diseases method.Utilize genetic engineering technique, intestinal bacteria give expression to subunit vaccine protein as Host Strains, as fodder additives after separation and purification, are the effective ways meeting aquaculture.

The various material of effective transport of protein transduction domains (protein transduction domain, PTD) to be one with peptide be carrier enters cell and nuclear system.The material carried by PTD has full length protein, DNA, chemicals, oligonucleotide, 40nm magnetic bead and 200nm liposome etc.Present most study, most widely used protein transduction domain derive from the activating transcription factor (Trans-activating transcriptional activator, Tat) of human immunodeficiency virus type 1 (HIV-1).There are 3 functional domains: the acidic region being positioned at N-end, the main function playing trans-activation in Tat albumen; DNA land between 22-37 amino acids, halfcystine is rich in this region; Basic region between 47-60 amino acids is main nexin transduction domain, participates in the cellular uptake of Tat albumen.Tat albumen has 86 amino acid, and Schwarze etc. determine that the minimum peptide section with protein transduction is 47-57, are made up of 11 amino acid, its sequence is YGRKKRRQRRR, containing 6 arginine and 2 lysine residues, iso-electric point is 12.7, is therefore the polypeptide with height positive charge.All can make to wear film activity with uncharged L-Ala any one alkaline amino acid residue substituted wherein to reduce, wear film activity during other amino-acid residues in alternative sequence then can not change, illustrate TAT wear film peptide with positive charge be that its transmembrane ability is necessary, therefore infer that these positive charges are likely and with eukaryotic cytolemma, strong electrostatic interaction can occur, thus mediated and wear membrane process.The polypeptide be attached thereto or protein can be transduceed and be entered cell by TAT in several minutes, and can be transported to cerebral tissue by blood circulation in vivo, and cross over hemato encephalic barrier and enter in neurone or spongiocyte, transduction rate is fast, and efficiency is high.Existing research display, tens kinds of compounds and protein are entered different cells by Successful transductions or cross over hemato encephalic barrier, and show corresponding biological activity (Yi Zhang, Jian-Fang Ning, Xing-qin Qu, Xiao-Lin Meng, Jin-Ping Xu.TAT-mediated oral subunit vaccine against white spot syndrome virus in crayfish, Journal of Virological Methods, 2012,181:59 – 67. patent No.: CN 102206660B).

Although for the transmembrane ability of TAT protein transduction domains study many, up to the present, the molecular mechanism that TAT protein transduction domains enters cell is not also studied clear completely.Early stage research is thought by cross-docking mechanism through cytolemma, and does not rely on energy, but nearest research also has the result different from this viewpoint.Although its mechanism entering cell is not also studied thorough completely, the application of TAT protein transduction domains is very extensive.The gene of the spanning transduction membrane regional code of TAT and foreign protein genes are carried out amalgamation and expression, are different edge proteins across cell membranes, enter cell interior and provide a new approach.Therefore, utilize head and the tail TAT and VP7 protein fusion, through the oral generation immunity of grass carp, be the effective ways of control hemorrhagic disease of grass carp, there is important scientific meaning and using value.

Summary of the invention

The object of the present invention is to provide a kind of fusion rotein TAT-VP7-TAT, its sequence is for shown in SEQ ID NO.2.

Another object of the present invention be to provide fusion rotein TAT-VP7-TAT corresponding nucleotide sequence, optimized encoding sequence is for shown in SEQ ID NO.1.

Another object of the present invention is to provide a kind of Recombinant organism strain, this bacterial strain can high expression N hold and C hold all with the grass carp hemorrhage virus (GCHV) capsid protein VP7 fusion rotein TAT-VP7-TAT of nexin transduction domain peptide T AT.Its advantage is that fusion protein expression is large, and be easy to suitability for industrialized production, cost is low, and security is good.This bacterial strain delivers to China typical culture collection center preservation on March 13rd, 2015, Classification And Nomenclature: colon bacillus Eschierichiacoli BL21 (DE3)/pET-32a-TAT-VP7-TAT, deposit number: CCTCC NO:M2015111, address: Wuhan, China Wuhan University.

Another object of the present invention is the preparation method that there are provided a kind of recombination engineered fusion protein TAT-VP7-TAT subunit vaccine, can prevent hemorrhagic disease of grass carp, and prevention hemorrhagic disease of grass carp has better protecting effect.

Last object of the present invention is that genetic engineering fusion protein TAT-VP7-TAT is preparing and preventing the application in the medicine of grass carp hemorrhage virus (GCHV).

The invention provides fusion rotein TAT-VP7-TAT provided by the invention and effectively can prevent hemorrhagic disease of grass carp.Recombination fusion protein TAT-VP7-TAT is after grass carp is oral, fusion rotein TAT-VP7-TAT can enter blood through intestinal tissue, subunit vaccine directly can be entered in blood without intramuscular injection, pass through blood circulation, make its fusion rotein arrive different tissues, initiatively stimulate body to produce antivirus action.

For achieving the above object, the present invention is by the following technical solutions:

One of technical essential of the present invention is structure and the abduction delivering of the Recombinant organism strain of expressing gene engineering recombination fusion protein TAT-VP7-TAT.

A preparation method for recombination fusion protein TAT-VP7-TAT subunit vaccine, its step is as follows:

1. obtain the GCRV outer capsid proteins TAT-VP7-TAT gene that e. coli codon is optimized.

Synthetic gene TAT-VP7-TAT, its sequence for shown in SEQ ID NO.1, and is connected on pGEM-T carrier, namely constructs plasmid pGEM-T-TAT-VP7-TAT.

By plasmid pGEM-T-TAT-VP7-TAT transformation of E. coli E.coli DH5 α, obtain bacterial strain E.coli DH5 α pGEM-T-TAT-VP7-TAT, for increment and the preservation of gene.

2. the structure of fusion gene pET32a-TAT-VP7-TAT expression vector

From bacterial strain E.coli DH5 α pGEM-T-TAT-VP7-TAT, extract plasmid pGEM-T-TAT-VP7-TAT, cut through BamH I and Hind III enzyme, obtain object fragment TAT-VP7-TAT.Cut through BamH I and Hind III enzyme by pET32a carrier, agarose gel electrophoresis, glue reclaims, and obtains carrier pET32a (+).Transformation of E. coli DH5 α competent cell after being connected by 22 DEG C, object fragment and carrier, through qualification, the positive recombinant obtained is the intestinal bacteria containing TAT-VP7-TAT gene, this plasmid called after: pET32a-TAT-VP7-TAT.

3. the preparation of colibacillus engineering

By plasmid pET32a-TAT-VP7-TAT transformation of E. coli BL21 (DE3) competent cell.The positive transformant obtained is accredited as through PCR intestinal bacteria recombination engineering bacteria BL21 (DE3) pET32a-TAT-VP7-TAT that positive bacterium colony is energy expressed fusion protein.

A kind of Recombinant organism strain Escherichia coli BL21 (DE3) pET-32a-TAT-VP7-TAT is obtained by aforesaid method, this bacterial strain delivers to China typical culture collection center preservation on March 13rd, 2015, Classification And Nomenclature: colon bacillus Eschierichia coli BL21 (DE3)/pET-32a-TAT-VP7-TAT, deposit number: CCTCC NO:M2015111, address: Wuhan, China Wuhan University.

This project bacterium has following feature: (1) basis of microscopic observation, size 1-3 micron, and whole body flagellum, can move, without gemma, be Gram-negative tyrothricin.(2) cultivate in common LB flat board, its bacterium colony colonial morphology is smooth type, and colony edge is neat, and surface is glossy, moistening, smooth, in lime look, ammonia benzyl chloramphenicol resistance, aerobic bacteria, substratum is LB substratum, fills a prescription to be: yeast extract 5g/L, Tryptones 10g/L, sodium-chlor 10g/L, agar 15g/L, pH 7.0.(3) facultative anaerobic bacterium, optimum growth temperature is 37 DEG C, IP available TG or the automatic abduction delivering of lactose, and metabolism is vigorous, and abduction delivering 4-5h can reach the maximum expression amount of albumen.

A preparation method for recombination engineered fusion protein TAT-VP7-TAT subunit vaccine is as follows:

Single colony inoculation of recombination engineering strain Escherichia coli BL21pET32a-TAT-VP7-TAT in containing in the 20ml LB liquid nutrient medium of 100 μ g/ml Amp, in 37 DEG C of shaking table 300rpm incubated overnight 10-12h.Second day, in the ratio of 1:100 be inoculated into 100ml fresh containing in 2 × YT medium liquid substratum of 100 μ g/ml Amp, 37 DEG C, 250rpm cultivates about 3h, when being 0.6-0.8 to OD600, in super clean bench, get 1ml bacterium liquid as the contrast before inducing.Adding inductor IPTG to final concentration is 0.6mM, in 37 DEG C of shaking table 250rpm abduction delivering 4-5h.By bacterium liquid 4 DEG C, the centrifugal 5min of 12000rpm, abandons supernatant, collects thalline.With the resuspended thalline of appropriate PBS damping fluid, the centrifugal 20min of 400W ultrasonication 20min, 8000rpm.Precipitation after fragmentation after 8M is urea-denatured 4 DEG C, 8000rpm, centrifugal 10min, collect supernatant, the purifying of fusion rotein TAT-VP7-TAT is undertaken by Ni-NTA specification sheets, detects protein purification result, obtain the TAT-VP7-TAT of purifying with SDS-PAGE.After TAT-VP7-TAT carries out renaturation dialysis, be the genetic engineering fusion protein with biologic activity.Its sequence, for shown in SEQ ID NO.2, is characterized in that: fusion rotein has grass carp and wears intestinal function, and the molecular weight of fusion rotein is 50.9kDa.

The application of recombination fusion protein TAT-VP7-TAT in preparation prevention or treatment grass carp hemorrhage virus disease medicine, directly by oral for this albumen grass carp of throwing something and feeding, or being mixed with into other biological oral vaccine delivery with other auxiliary materials throws something and feeds grass carp.Compared with prior art, the present invention has the following advantages:

The present invention, compared with the VP7 subunit vaccine of existing report, it is advantageous that:

1) N end involved in the present invention and C end are all with the fusion rotein TAT-VP7-TAT of nexin transduction domain peptide T AT, TAT can carry VP7 albumen and directly enter in blood without intramuscular injection, solve subunit vaccine directly to enter in blood without intramuscular injection, there is the feasibility of actually operating.Compared with single TAT-VP7 fusion rotein; TAT-VP7-TAT albumen is due to N; C two end is respectively with a TAT sequence; its with positive charge will far away higher than TAT-VP7 albumen, and TAT wear film peptide with positive charge be that its transmembrane ability is necessary, so the film activity of wearing of TAT-VP7-TAT albumen can strengthen greatly; consumption reduces; immune response is rapider, plays stronger antivirus action, has higher, more permanent protected effect for hemorrhagic disease of grass carp.

2) utilize the fusion rotein of escherichia coli expression TAT nexin transduction domain and GCRV outer capsid proteins VP7, fusion rotein is prevented and treated hemorrhagic disease of grass carp as subunit vaccine.TAT-VP7-TAT recombinant protein is by the circulation of blood in fish body, make its fusion rotein arrive different tissues, add the effect that the competition for GCRV virus neutralizes, the immunoenzyme simultaneously also enhanced in fish body is lived effect, make fish body produce stronger immune factor, resistance against diseases is stronger.

3) by nexin transduction domain peptide T AT and VP7 amalgamation and expression.TAT can carry VP7 albumen and directly enter in blood without intramuscular injection.Solve subunit vaccine directly to enter in blood without intramuscular injection, solve the herd immunity problem of vaccine, there is the feasibility of actually operating.Because TAT-VP7-TAT albumen to wear film activity higher, its consumption as antiviral will reduce, and efficiency improves, and economic benefit is better.Therefore the construction process of engineering strain disclosed by the invention, its expression amount is large, and simple to operate, cost is low, and security is good, can be applied in large-scale grass carp aquaculture.

4) the N end of the genetic engineering fusion protein TAT-VP7-TAT that provides of patent of the present invention and C hold with nexin transduction domain peptide T AT.Although TAT and protein bound are transduceed enter cell by as the method preparing multiple antiviral, as patent CN 102206660 B, and also mention TAT and VP7 protein fusion expression in patent CN 103184230 A.But, up to the present, yet there are no the report with TAT sequence the N of target protein holds and C holds while at home and abroad.Theoretically, TAT-VP7-TAT albumen is due to two TAT sequences, its with positive charge will far away higher than TAT-VP7 albumen, and TAT wear film peptide with positive charge be that its transmembrane ability is necessary, so the wearing film activity and will greatly strengthen of TAT-VP7-TAT albumen, the utilization ratio of albumen is significantly improved.

5) utilize the fusion rotein of escherichia coli expression TAT nexin transduction domain and GCRV outer capsid proteins VP7, fusion rotein is prevented and treated hemorrhagic disease of grass carp as subunit vaccine.Because the enhancing of the positive charge of TAT-VP7-TAT albumen, N end and C-terminal all can wear film, greatly improve cross-film active, its efficiency entered in grass carp body strengthens, and immune response is rapider, plays stronger antivirus action.Therefore, TAT-VP7-TAT fusion rotein can be applied to grass carp aquaculture as the pharmaceutical vaccine of anti-grass carp hemorrhage disease pathogen, can significantly improve the resistance against diseases of grass carp, have higher, more permanent protected effect.

Accompanying drawing explanation

Fig. 1 is the building process schematic diagram of a kind of recombinant expression plasmid pET-32a (+)-TAT-VP7-TAT.

Fig. 2 is that the enzyme of a kind of recombinant expression plasmid pET-32a (+)-TAT-VP7-TAT cuts qualification schematic diagram.

In Fig. 2: 1, DL2000 molecular criteria; 2, plasmid pET32a-TAT-VP7-TAT through BamH I and Hind III double digestion; 3, plasmid pET32a-TAT-VP7-TAT through Hind III single endonuclease digestion; 4, plasmid pET32a-TAT-VP7-TAT through BamH I single endonuclease digestion; 5, plasmid pET32a-TAT-VP7-TAT; 6, DNA Marker IV molecular criteria.

Fig. 3 is TAT-VP7-TAT through the purifying electrophoresis schematic diagram of intestinal bacteria abduction delivering and fusion rotein thereof.

In Fig. 3: M, albumen Marker; 2, the BL21pET32a-TAT-VP7-TAT bacterium liquid of not inducing; 3, the BL21pET32a-TAT-VP7-TAT do not induced broken liquid supernatants; 4, the BL21pET32a-TAT-VP7-TAT do not induced broken liquid precipitates; 5, the BL21pET32a-TAT-VP7-TAT bacterium liquid after induction; 6, the BL21pET32a-TAT-VP7-TAT after induction broken liquid supernatants; 7, the BL21pET32a-TAT-VP7-TAT after induction broken liquid precipitates; 8, the TAT-VP7-TAT fusion rotein of purifying; 9, the TAT-VP7 fusion rotein of purifying.

Fig. 4 is that a kind of ELISA of Grass Carp Serum detects schematic diagram.

Fig. 5 is the ELISA detection schematic diagram that intestines ability worn by a kind of recombination fusion protein TAT-VP7-TAT subunit vaccine and TAT-VP7 subunit vaccine.

Embodiment:

Below in conjunction with drawings and the specific embodiments, the present invention is further illustrated.The all substratum related in an embodiment and molecular biology manipulations method are well known to those skilled in the art.Molecular biology method involved by this experiment is ordinary method, by those skilled in the art are familiar with.The content do not elaborated in the present invention refers to " Molecular Cloning: A Laboratory guide ", J. Pehanorm Brooker, the chief editors such as D.W. Russell.

Embodiment 1:

The preparation of fusion gene TAT-VP7-TAT, comprises the following steps:

The aminoacid sequence of the VP7 albumen of ripe GCRV is obtained from U.S.'s biotechnology center (NCBI) gene pool, convert the sequence of VP7 gene to nucleotide sequence containing intestinal bacteria preferred codons, and the gene of encoding according to nexin transduction domain peptide T AT and foreign protein genes can carry out the characteristic of amalgamation and expression, 5 ' of VP7 gene order after optimization holds with 3 ' nucleotide sequence being connected TAT respectively, called after: TAT-VP7-TAT, its sequence is the nucleotide sequence shown in SEQ IDNO:1.

Synthetic TAT-VP7-TAT gene, and be connected on pGEM-T carrier, namely synthesize plasmid pGEM-T-TAT-VP7-TAT.

Plasmid pGEM-T-TAT-VP7-TAT transformation of E. coli E.coli DH5 α (is buied by Novagen company, this laboratory conservation), obtain bacterial strain E.coli DH5 α (pGEM-T-TAT-VP7-TAT), for increment and the preservation of gene.

Calcium Chloride Method prepares competent escherichia coli cell, and competent escherichia coli cell adopts E.coli DH5 α, its step and method, instructs carry out according to Molecular Cloning: A Laboratory.

Prepared by competent escherichia coli cell, the steps include:

1. with the bacillus coli DH 5 alpha list bacterium colony of new activation on transfering loop picking solid LB flat board, be inoculated in 20ml LB liquid medium, 37 DEG C, 300rpm shakes activation and spends the night.(following steps all need aseptic technique)

2. get the intestinal bacteria of the above-mentioned activation of 200ul in fresh 20ml LB liquid medium, 37 DEG C, 300rpm shaking table cultivates that 2-3 is little is about 0.6 up to OD600 value.

3. get 1.5ml above-mentioned bacterium liquid in aseptic Eppendorf pipe, place 30min on ice.4000rpm, 4 DEG C centrifugal 10 minutes, abandons supernatant.

4. the 0.1MCaCl of 200ul ice precooling is added ₂the resuspended bacterial sediment of solution, ice bath 30 minutes, 4000rpm, 4 DEG C centrifugal 10 minutes, abandons supernatant.

5. the 0.1MCaCl of 100ul ice precooling is added ₂the resuspended precipitation of solution, is competent cell, is placed in 4 DEG C of preservations, uses and be advisable in 24h.

Get 1ul plasmid pGEM-T-TAT-VP7-TAT transformation of E. coli BL21DH5 α competent cell.PCR evaluation and screening goes out positive transformant, and gained positive colony is the E.coli DH5 α pGEM-T-TAT-VP7-TAT containing fusion gene TAT-VP7-TAT.

Embodiment 2:

The structure of expression plasmid.

In embodiment 1, plasmid pGEM-T-TAT-VP7-TAT is after BamH I and Hind III enzyme cut, electrophoresis on sepharose, the band for reclaiming is cut rapidly under ultraviolet lamp, with health be century sepharose DNA reclaim kits, single target DNA band is put into clean Eppendorf pipe, takes weight.In blob of viscose, adding the long-pending sol solutions PG of triploid, (gel is heavily 0.1g, and its volume can be considered 100uL, by that analogy).60 DEG C of water-baths 10 minutes, spin upside down Eppendorf pipe, to guarantee that blob of viscose fully dissolves every 2 minutes gentlenesses therebetween.In adsorption column, add the Buffer PS of 250 μ l, the centrifugal 2min of 12000rpm after standing 2min, outwells the liquid in collection tube.Gained solution being got 750ul joins in an adsorption column (adsorption column puts into collection tube), and room temperature (20-25 DEG C) is placed 2 minutes, and the centrifugal 2min of 12000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube.Transferred to by sol solutions in adsorption column, after the static 2min of room temperature, the centrifugal 1min of 12000rpm, outwells the liquid in collection tube.In adsorption column, add 500 μ l Buffer PG, after acting on 1min under room temperature, the centrifugal 1min of 12000rpm, outwells the liquid in collection tube.In adsorption column, add 650 μ l Buffer PW again, after left at room temperature 1min, the centrifugal 1min of 12000rpm, outwells the liquid in collection tube.Repeat to add 650 μ l Buffer PW, after left at room temperature 1min, the centrifugal 1min of 12000rpm, outwells the liquid in collection tube again.The centrifugal 2min of 12000rpm, places several minutes at 37 DEG C and thoroughly dries to ethanol again.Adsorption column is put into aseptic Eppendorf pipe, the unsettled ddH crossed through 60 DEG C of water-bath thermal pretreatment adding 30 μ l in the middle of film ₂o, ambient temperatare puts 2min, and the solution collecting acquisition in centrifugal 2min, the Eppendorf pipe of 12000rpm is digestion products.

Be connected with plasmid pET32a by the digestion products of purifying, linked system is as follows:

Connect product conversion competent escherichia coli cell, the steps include:

1. get under aseptic condition and connect product 10ul, join in 100ul bacillus coli DH 5 alpha competent cell, mix gently, ice bath 30 minutes.(following steps all need aseptic technique)

2. heat shock 90 seconds in 42 DEG C of water-baths, moves to rapidly ice bath 2-3 minute in ice.

3. add 800ul LB liquid medium, 37 DEG C, 150rpm jog, incubation 45min makes cell recovery.

4. centrifugal 10 minutes of 4000rpm, draws 800ul supernatant and discards, mixed gently by residue bacterium liquid liquid-transfering gun.

5. be uniformly coated on the solid LB flat board containing 100 μ g/ml Amp by the aseptic triangle glass rod of above-mentioned bacterium liquid, forward places 1-2 hour, until liquid is all absorbed, is inverted flat board overnight incubation in 37 DEG C of incubators.Obtain the bacterium colony of E.coli DH5 α pET32a-TAT-VP7-TAT.

The enzyme of positive recombinant pET32a-TAT-VP7-TAT cuts qualification:

With alkaline lysis method of extracting E.coli DH5 α pET32a-TAT-VP7-TAT plasmid, carry out single, double endonuclease reaction to the plasmid extracted, it is as follows that enzyme cuts system:

Endonuclease reaction condition is 37 DEG C, and the time is 3h, and product is through agarose gel electrophoresis analytical results.

Embodiment 3:

The structure of pET32a-TAT-VP7-TAT engineering bacteria.

Get 1ul plasmid pET32a-TAT-VP7-TAT transformation of E. coli BL21 (DE3) competent cell.PCR evaluation and screening goes out positive transformant, and gained positive colony is can the recombination engineering strain Escherichia coli BL21pET32a-TAT-VP7-TAT of amalgamation and expression TAT-VP7-TAT.

This bacterial strain delivers to China typical culture collection center preservation on March 13rd, 2015, Classification And Nomenclature: colon bacillus Eschierichia coli BL21 (DE3)/pET-32a-TAT-VP7-TAT, deposit number: CCTCC NO:M2015111, address: Wuhan, China Wuhan University.

Embodiment 4:

The expression of genetic engineering fusion protein TAT-VP7-TAT.

1. single colony inoculation of recombination engineering strain Escherichia coli BL21pET32a-TAT-VP7-TAT is in containing in the 20ml LB liquid nutrient medium of 100 μ g/ml Amp, in 37 DEG C of shaking table 300rpm incubated overnight 10-12h.

2. second day, in the ratio of 1:100 be inoculated into 100ml fresh containing in 2 × YT liquid nutrient medium of 100 μ g/ml Amp, 37 DEG C, 250rpm cultivates about 3h, when being 0.6-0.8 to OD600, in super clean bench, get 1mL bacterium liquid as the contrast before inducing.Adding inductor IPTG to final concentration is 0.6mM, in 37 DEG C of shaking table 250rpm abduction delivering 4-5h.

3. by bacterium liquid 4 DEG C, the centrifugal 5min of 12000rpm, abandons supernatant, collects thalline.With the resuspended thalline of appropriate PBS damping fluid, 3s is broken in 400W ultrasonication, stops 5s, 20min, then 4 DEG C, the centrifugal 2min of 12000rpm, gets 50 μ L supernatants and isopyknic 2 × SDS-PAGE sample buffer mixes, obtained broken Supernatant samples.Supernatant discarded, by the resuspended precipitation of appropriate amounts of sterilized water, gets 50 μ L and isopyknic 2 × SDS-PAGE sample buffer mixes, obtained broken deposit sample.

4. be placed in boiling water boil 5-10min by often organizing sample, carry out SDS-PAGE electrophoresis, detect TAT-VP7-TAT albumen and whether express, and be solubility expression or form inclusion body.After electrophoresis terminates, unload gel, in coomassie brilliant blue staining liquid (2.0g coomassie brilliant blue R_250,450ml methyl alcohol, 450ml ddH2O, 100ml Glacial acetic acid), dyeing 3 hours, then uses destainer (40ml methyl alcohol, 10ml acetic acid, 50mlddH ₂o) decolour, changed liquid once every 30 minutes, till background takes off totally.

5. observe gel and find in precipitation, have the target protein band be consistent with expection size 50.9kDa, and major part exists with inclusion bodies, expression amount is higher.Accompanying drawing 3 is shown in the SDS-PAGE qualification of fusion rotein TAT-VP7-TAT.

Embodiment 5:

The purifying of fusion rotein TAT-VP7-TAT.

(1) extraction of fusion rotein TAT-VP7-TAT and sex change.

1. by the bacterium liquid of 100ml that ferments in embodiment 4 at the centrifugal 30min of 4000rpm, collect thalline, with the resuspended thalline of 10mlLysis Buffer ,-20 DEG C of freeze thawing once, then add 20ml Lysis Buffer, 400W sonicated cells (broken 3s, stop 5s, 40min), 4 DEG C, the centrifugal 10min of 12000rpm, abandons supernatant.

2. precipitation 2M urea washes twice, 4 DEG C, after the centrifugal 10min of 12000rpm abandons supernatant, add appropriate inclusion body lysate, 4 DEG C of cracking are spent the night, and solution becomes clarification, and by the membrane filtration of supernatant liquor through 0.45 μm, the solution obtained is the good protein solution of sex change.

(2) Ni-NTA Superflow post affinity chromatography purification of recombinant proteins.

1. drawing 1ml Ni-NTA Agarose with pipettor loads bottom chromatography column, with the deionized water wash pillar of 10 times of column volumes, then balances pillar with the Lysis Buffer of 10 times of column volumes.

2. joined with the speed of 0.2ml/min by protein solution peristaltic pump good for above-mentioned sex change in the nickel post balanced, 4 DEG C of three circulation loadings, make His6 and the Ni of fusion rotein front end ²⁺abundant combination.

3. wash post with 50ml Lysis Buffer with the speed of 1ml/min, with in wash-out lower prop not with Ni ²⁺in conjunction with albumen.

4. successively with containing 20mM Imidazole, 40mM Imidazole, 60mM Imidazole Wash Buffer with the speed wash-out pillar of 0.5ml/min, wash-out Ni ²⁺the non-target protein that post combines.The OD of the effluent liquid after each wash-out is detected with nucleic acid-protein detector ₂₈₀, time steady to baseline, stop washing.

5. with containing the Elution Buffer of 250mM Imidazole with the speed wash-out pillar of 0.2ml/min, target protein is collected, until OD with Eppendorf pipe ₂₈₀stop collecting when being continuously 0.Carry out SDS-PAGE electrophoresis, the purification effect of the albumen collected by detection.

(3) renaturation of fusion rotein TAT-VP7-TAT.

1. the dialysis tubing newly bought (molecular weight that dams is 8-14kDa) is placed in containing 10mM NaHCO ₃, 1mM EDTA solution in boil 10min, removing dialysis tubing surface some impurity.

2. load in the dialysis tubing handled well by the above-mentioned target protein collected, dialysis tubing two ends clip clips, and is placed in protein renaturation liquid 4 DEG C of dialysis renaturation 24h.

3. in order to except the urea of Deproteinization middle and high concentration, dialysis tubing is put into PBS damping fluid, 4 DEG C of gradients are dialysed about 24h.Period changes dialysis buffer liquid once or twice, urea can be removed completely.

4. the protein soln PEG20000 after dialysis is concentrated into and is about 1/3 of original volume, be stored in after packing-20 DEG C for subsequent use.

5. measuring the TAT-VP7-TAT concentration after purifying with BandScan software analysis is 480 μ g/ml, its sequence for shown in SEQID NO.2, for following examples.

Embodiment 6:

The preparation and purification of polyclonal antibody and titration thereof.

(1) preparation of polyclonal antibody.

Subcutaneous injection immunize New Zealand White Rabbit after being mixed with Freund's complete adjuvant equal-volume by 400-600 μ g TAT-VP7-TAT purifying protein, each immunization interval cycle is 2 weeks, altogether immunity 3 times.Within 11st day, get blood, whole blood 37 DEG C of standing 1h after third time immunity, then 4 DEG C of hold over night, 4 DEG C, 2000g, centrifugal 20min, get supernatant frozen for subsequent use in-20 DEG C.

(2) purifying of polyclonal antibody.

1. draw 1ml Protein A Sepharose with pipettor and load Protein A Sepharose TMCL-4B column bottom, with a large amount of deionized water rinsing filler, then balance pillar with the Binding Buffer of 10 times of column volumes.

2. get 1ml antiserum(antisera) Binding Buffer and be diluted to 10ml, 4 DEG C, the centrifugal 10min of 8000g, collect supernatant also with 0.45 μm of membrane filtration.

3. the sample handled well is joined with the speed of 0.2ml/min in the pillar balanced, be cycled to repeat loading 2 times.

4. rinse pillar with Binding Buffer with the speed of 0.5ml/min again, effluent liquid protein nucleic acid detector detects OD ₂₈₀, treat OD ₂₈₀value stops washing when being continuously 0.

5. finally use Elution Buffer with the speed wash-out polyclonal antibody of 0.25ml/min, Fraction collection elutriant.For keeping the activity of antibody, in every 1ml elutriant, adding 200 μ l Neutralizing Buffer while collecting elutriant and mix immediately.

6. the antibody collected is loaded dialysed overnight in dialysis tubing, then uses PEG20000 concentrated antibody solution to 1/3rd of original volume, be stored in after packing-20 DEG C for subsequent use.With the purification effect of SDS-PAGE electrophoresis detection antibody.

(3) indirect elisa method is adopted to detect antibody titer.

1. the antibody gradient dilution 100-25600 obtained by purifying doubly.

2. diluting TAT-VP7-TAT albumen to the final concentration of purifying with PBST is 10 μ g/ml, and every hole 100 μ l adds in enzyme plate, and 4 DEG C of bags are spent the night.

3. discard the sample in enzyme plate aperture, in every hole, add about 300 μ l PBST damping fluids, soak 5min, and the washing of intermittent control shaking enzyme plate, and repeated washing three times.

4. 100 μ l 0.5%BSA are added, 37 DEG C of closed 2h in every hole.

5. repeating step 3., and every hole adds the rabbit anti-serum of 100 μ L100-25600 times gradient dilutions, and negative control is the preimmune serum of dilution 100 times, and blank is PBS, and each concentration 3 is parallel, hatches 2h for 37 DEG C.

6. repeating step 3., and every hole adds the goat anti-rabbit igg that horseradish peroxidase (HRP) that 100 μ l dilute 10000 times marks, and hatches 2h for 37 DEG C.

7. repeating step 3., and every hole adds 100 μ l OPD nitrite ions, 37 DEG C of lucifuge reaction 10-20min.

8. every hole adds 50 μ l stop buffers, color development stopping.

9. detect the light absorption value in every hole under 490nm wavelength by the full-automatic microplate reader of Bio-Rad450, average, occur that the greatest dilution of positive reaction is tiring of antibody.

After measured, the rabbit anti-serum of this experiment preparation is tired as 1:12800.

Embodiment 7:

What ELISA detected TAT-VP7-TAT albumen wears intestinal function.

(1) experiment material:

Experiment is with grass carp purchased from cultivation base, Liang Zi island, Ezhou, and body weight 50g ± 5, raise in this laboratory after buying back, raising temperature about 28 DEG C, and every day, feeding changed water once simultaneously.The grass carp of new purchase at least feeds 15 days in laboratory, treats that the in stable condition rear of grass carp is for experiment.

(2) preparation of serum:

By purifying and the TAT-VP7-TAT fusion rotein having measured concentration (480 μ g/ml) is thrown something and fed grass carp, every grass carp is thrown something and fed 200 μ L protein solutions, throws something and feeds continuously 3 days.Simultaneously using the grass carp of PBS damping fluid of only throwing something and feeding as negative control.Grass carp is raised at 30 DEG C of constant temperature, and after TAT-VP7-TAT fusion rotein of throwing something and feeding the last time, respectively at 2h, 6h, 12h, 24h tail venous blood sampling, room temperature places 1h, 4 DEG C of hold over night.By blood in centrifuges after the centrifugal 5min of 3000g, Aspirate supernatant is used for ELISA and detects.

(3) what double-antibody sandwich elisa detected TAT-VP7-TAT albumen wears intestinal function.

1. mouse-anti His6 antibody PBST is diluted 1000 times, join in enzyme plate hole with liquid-transfering gun, every hole 100 μ l.With the aperture of the enzyme plate of ParafilmTM application of sample, 4 DEG C of overnight incubation, make anti-His6 antibody fully be coated in bottom enzyme plate.

2. outwell the His6 antibody in enzyme plate hole, every hole adds 100 μ l 0.5%BSA, is placed on 37 DEG C, closes 3-4h with sealed membrane sealing.

3. outwell the liquid in enzyme plate aperture, in every hole, add about 300 μ l PBST damping fluids, soak 5min, and the washing of intermittent control shaking enzyme plate, repeated washing three times.

4. by the serum of above-mentioned preparation, every hole 100 μ l joins in enzyme plate, and simultaneously only to add the enzyme plate aperture of PBS damping fluid as blank, each sample adds three holes.With the aperture of the enzyme plate of ParafilmTM application of sample, 4 DEG C of overnight incubation, make the antigen in serum fully be combined with anti-His6 antibody.

5. repeating step 3., adds the VP7 antibody of 1:500 dilution, with the aperture of the enzyme plate of ParafilmTM application of sample, hatch 2h for 37 DEG C in every hole.

6. repeating step 3., and what every hole added that horseradish peroxidase (HRP) that 100 μ l dilute by 1:10000 marks two resists, and with the aperture of the enzyme plate of ParafilmTM application of sample, hatches 2h for 37 DEG C.

7. repeating step 3., and every hole adds 100 μ l nitrite ions, is placed on lucifuge colour developing 20min in 37 DEG C of constant incubators with sealed membrane sealing.

8. every hole adds 50 μ l reaction terminating liquids, and color development stopping is reacted.Detect the light absorption value of every hole at 490nm place by microplate reader, result is as Fig. 4.

ELISA detects data declaration, compares with blank group, and the oral TAT-VP7-TAT subunit vaccine of grass carp, after 2 hours, can detect VP7 antigen in Blood of Ctenopharyngodon, shows that TAT can carry VP7 albumen and directly enter in blood without intramuscular injection.After feeding albumen 3-12h, the content of TAT-VP7-TAT in serum raises to some extent, but starts to decline at 12h-24h, illustrates that TAT-VP7-TAT starts degradation in vivo after oral 12h.

Embodiment 8:

Fusion rotein TAT-VP7-TAT and TAT-VP7 wears the comparison of intestines ability:

(1) preparation of TAT-VP7 fusion rotein

Artificial directly synthesis TAT-VP7 fusion rotein, its sequence is for shown in SEQ ID NO.3.

(2) preparation of intestines model is worn

Experiment is with grass carp purchased from cultivation base, Liang Zi island, Ezhou, and body weight 50g ± 5, raise in our company's experiment pool after buying back, raising temperature about 28 DEG C, and every day, feeding changed water once simultaneously.The grass carp of new purchase at least feeds 15 days in experiment pool, puts to death after grass carp is in stable condition, gets 5cm grass carp intestinal tube for experiment.Rinse grass carp intestinal tube with PBS, intestinal tube one end is tightened, draws 100 μ l solution to be measured to the grass carp intestinal tube tied with liquid-transfering gun, tie the other end of intestinal tube, put in the test tube that 10ml PBS damping fluid is housed, and make PBS damping fluid complete submergence grass carp intestinal tube.Sample divides 3 groups, TAT-VP7-TAT group, TAT-VP7 group, and all use PBS damping fluid Function protein concentration to 200 μ g/ml, PBS group is as negative control.Often organize solution all to operate by above step, every root intestinal tube is all immersed in independently in PBS test tube.

(3) preparation of intestines sample is worn

The PBS damping fluid test tube of submergence intestinal tube leaves standstill at 30 DEG C, draws PBS solution in test tube, get 100 μ l at every turn respectively at 1h, 2h, 4h, 8h, 12h.

Intestinal function is worn by what wear that intestines model E LISA detects TAT-VP7-TAT, TAT-VP7 albumen.Method is implemented according to the ELISA method described in example 7, and the data preparation of last acquired results is Fig. 5.

Result shows, and can wear existence VP7 antigen being detected in intestines sample after 2 hours at TAT-VP7-TAT, and this illustrates compared with TAT-VP7, and TAT-VP7-TAT can more early faster through goldbeater's skin; And in same time, the 0.6-1.1 larger than TAT-VP7 of the optical density value through the TAT-VP7-TAT albumen of goldbeater's skin.Illustrate within the unit time, the amount through the TAT-VP7-TAT albumen of goldbeater's skin is larger than TAT-VP7.

Embodiment 9:

The application of fusion rotein TAT-VP7-TAT in the anti-GCRV infection medicine of preparation

(1) test sample and source:

Preparation method is as follows for TAT-VP7-TAT fusion rotein subunit vaccine: the protein solution by the concentration of TAT-VP7-TAT fusion rotein being 480 μ g/ml, according to 1mL protein solution and 4g feed (honest feed, 088 model) mixing, namely TAT-VP7-TAT fusion rotein subunit vaccine is prepared, for subsequent use.

The feed used of feminine gender and positive control is for be mixed to get with 1mL PBS solution and 4g feed.

GCRV viral extract is carried out 10 successively ^-1, 10 ^-2, 10 ^-3, 10 ^-4, 10 ^-5, 10 ^-6, 10 ^-7dilution, infects grass carp with 200 μ l/ dosage abdomen injection only, and in 15 days, mortality ratio reaches the most highly diluted multiple of more than 90% as experiment injection concentration, and the present embodiment injection concentration used is 10 ^-6the virus stock solution used of dilution.

(2) test design:

1. choose health and live Grass carps' fries, body weight 50g ± 5, be divided into 3 groups by its average body length and body weight, often organize 60 tails.Raise in the water tank of the same specification in same indoor, room temperature controls to lead to oxygen at 20-30 DEG C, 24h, and feeding time is 40-50 days.Test is provided with negative control, positive control, and TAT-VP7-TAT fusion rotein subunit vaccine is totally 3 groups of process, and often three repetitions are established in group process, and each process cultivating water box sticks respective labels.The forage volume of every day is 2% of fish body weight, and the amount of fusion rotein subunit vaccine is every g fish 2 μ g fusion rotein.

The feed that first group of (negative control) feeding PBS mixes, injects 200 μ l 0.65% stroke-physiological saline solution after 20 days.

The fish meal that second group of (positive control) feeding PBS mixes, after 20 days, (virus concentration is 10 of former extracting solution to direct injection GCRV virus ^-6doubly dilution, injection volume is 200 μ l/).

The feed of the TAT-VP7-TAT fusion rotein subunit vaccine that the 3rd group of (experimental group) feeding every day step (1) is obtained, after 20 days, (virus concentration is 10 of former extracting solution to direct injection GCRV virus ^-6doubly dilution, injection volume is 200 μ l/).

2. investigate and add up: every day, observed and recorded respectively organized the dead fish in water tank sooner or later, the accumulative death toll of statistics.After experiment in 20 days terminates, add up remaining live fish in each process box for breeding, calculate last survival rate, the results are shown in Table 1.

3. test-results and analysis:

The water tank of oral genetic engineering fusion protein TAT-VP7-TAT subunit vaccine can be found out in table 1, through injection GCRV virus infection after 20 days, the survival rate of Grass carps' fries is 80.00-86.67%, the positive control of non-oral administration genetic engineering fusion protein TAT-VP7-TAT subunit vaccine, the survival rate through injection GCRV virus infection Grass carps' fries after 20 days is only 20.00-28.33%.This can effectively prevent infecting of GCRV after showing grass carp oral genetic engineering fusion protein TAT-VP7-TAT subunit vaccine.

The each processing item of table 1 adds up dead mantissa and survival rate statistics table thereof

Sequence table

SEQUENCE LISTING

<110> Hubei Taiyanghong Biological Engineering Co., Ltd.

<120> anti-grass carp hemorrhage virus (GCHV) engineered protein TAT-VP7-TAT and preparation method and application

<130> anti-grass carp hemorrhage virus (GCHV) engineered protein TAT-VP7-TAT and preparation method and application

<160> 3

<170> PatentIn version 3.1

 

<210> 1

<211> 1392

<212> DNA

<213> recombinant DNA

 

<220>

<221> VP7 gene

<222> (529)..(1356)

<223>

 

<220>

<221> TAT

<222> (496)..(528)

<222> (1357)..(1389)

<223>

 

<400> 1

atgagcgata aaattattca cctgactgac gacagttttg acacggatgt actcaaagcg 60

gacggggcga tcctcgtcga tttctgggca gagtggtgcg gtccgtgcaa aatgatcgcc 120

ccgattctgg atgaaatcgc tgacgaatat cagggcaaac tgaccgttgc aaaactgaac 180

atcgatcaaa accctggcac tgcgccgaaa tatggcatcc gtggtatccc gactctgctg 240

ctgttcaaaa acggtgaagt ggcggcaacc aaagtgggtg cactgtctaa aggtcagttg 300

aaagagttcc tcgacgctaa cctggccggt tctggttctg gccatatgca ccatcatcat 360

catcattctt ctggtctggt gccacgcggt tctggtatga aagaaaccgc tgctgctaaa 420

ttcgaacgcc agcacatgga cagcccagat ctgggtaccg acgacgacga caaggccatg 480

gctgatatcg gatcctatgg ccgtaaaaaa cgtcgtcagc gtcgtcgtat gccacttcac 540

atgattccgc aagtcgccca cgctatggtg cgtgcagccg ctgcaggtcg tcttacctta 600

tacacaaaaa ccaaaactga gactactaac tttgatcacg ccgagtacgt cacctgcggt 660

cgttacacca tctgcgcctt ctgtctcacc actttggctc ctcatgctaa cgttaagacc 720

atccaggact cccacgcttg ttcccgtcag ccaaatgaag ccattcgctc cttagtcgaa 780

gtgagtgaca aggcgcagat cgcactcgtt ggtagccgca ctgtggacta tcacgaattg 840

gatgtcaaag ccggtttcgt cgccccaacc gccgatgaga cagtcgtccc gtccaaggac 900

atcgtcgaac tcccgtttcg cacctgtgac ttggatgatt cctctgccac cgcctgcgtc 960

cgtaatcact gccaggccgg tcacgacggc gtcacccacc tcccgatcct ctccggtgat 1020

ttcaaattac cgaacgagca ccctactaaa ccgttggacg atacccatcc gcacgacaag 1080

gtgctgactc gctgcccgaa gactggtctc ctgcttgtcc acgacactca cgcacacgct 1140

accgccgtag ttgccaccgc cgctacccgt gccatcctca tgcatgatct ccttacatcg 1200

gcgaacgtgg atgatggtca tcaagcgcgt tccgcttgtt acggtccaac cttcagcaac 1260

ctgacctttg cttgccactc cacctgcgct tcagatatgg ctcatttcga ctgcggccag 1320

atcgttggtc tcgacttgca tgtggagcca tccgattatg gccgtaaaaa acgtcgtcag 1380

cgtcgtcgtt aa 1392

 

 

<210> 2

<211> 463

<212> PRT

<213> polypeptide

 

<220>

<221> TAT-VP7-TAT PEPTIDE

<222> (1)..(463)

<223>

 

<220>

<221> VP7

<222> (177)..(452)

<223>

 

<220>

<221> TAT

<222> (166)..(176)

<222> (453)..(463)

<223>

 

<400> 2

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

165 170 175

Met Pro Leu His Met Ile Pro Gln Val Ala His Ala Met Val Arg Ala

180 185 190

Ala Ala Ala Gly Arg Leu Thr Leu Tyr Thr Lys Thr Lys Thr Glu Thr

195 200 205

Thr Asn Phe Asp His Ala Glu Tyr Val Thr Cys Gly Arg Tyr Thr Ile

210 215 220

Cys Ala Phe Cys Leu Thr Thr Leu Ala Pro His Ala Asn Val Lys Thr

225 230 235 240

Ile Gln Asp Ser His Ala Cys Ser Arg Gln Pro Asn Glu Ala Ile Arg

245 250 255

Ser Leu Val Glu Val Ser Asp Lys Ala Gln Ile Ala Leu Val Gly Ser

260 265 270

Arg Thr Val Asp Tyr His Glu Leu Asp Val Lys Ala Gly Phe Val Ala

275 280 285

Pro Thr Ala Asp Glu Thr Val Val Pro Ser Lys Asp Ile Val Glu Leu

290 295 300

Pro Phe Arg Thr Cys Asp Leu Asp Asp Ser Ser Ala Thr Ala Cys Val

305 310 315 320

Arg Asn His Cys Gln Ala Gly His Asp Gly Val Thr His Leu Pro Ile

325 330 335

Leu Ser Gly Asp Phe Lys Leu Pro Asn Glu His Pro Thr Lys Pro Leu

340 345 350

Asp Asp Thr His Pro His Asp Lys Val Leu Thr Arg Cys Pro Lys Thr

355 360 365

Gly Leu Leu Leu Val His Asp Thr His Ala His Ala Thr Ala Val Val

370 375 380

Ala Thr Ala Ala Thr Arg Ala Ile Leu Met His Asp Leu Leu Thr Ser

385 390 395 400

Ala Asn Val Asp Asp Gly His Gln Ala Arg Ser Ala Cys Tyr Gly Pro

405 410 415

Thr Phe Ser Asn Leu Thr Phe Ala Cys His Ser Thr Cys Ala Ser Asp

420 425 430

Met Ala His Phe Asp Cys Gly Gln Ile Val Gly Leu Asp Leu His Val

435 440 445

Glu Pro Ser Asp Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

450 455 460 463

<210> 3

<211> 452

<212> PRT

<213> polypeptide

 

<220>

<221> TAT-VP7 PEPTIDE

<222> (1)..(452)

<223>

 

<220>

<221> VP7

<222> (177)..(452)

<223>

 

<220>

<221> TAT

<222> ((166)..(176)

<223>

 

<400> 3

Met Ser Asp Lys Ile Ile His Leu Thr Asp Asp Ser Phe Asp Thr Asp

5 10 15

Val Leu Lys Ala Asp Gly Ala Ile Leu Val Asp Phe Trp Ala Glu Trp

20 25 30

Cys Gly Pro Cys Lys Met Ile Ala Pro Ile Leu Asp Glu Ile Ala Asp

35 40 45

Glu Tyr Gln Gly Lys Leu Thr Val Ala Lys Leu Asn Ile Asp Gln Asn

50 55 60

Pro Gly Thr Ala Pro Lys Tyr Gly Ile Arg Gly Ile Pro Thr Leu Leu

65 70 75 80

Leu Phe Lys Asn Gly Glu Val Ala Ala Thr Lys Val Gly Ala Leu Ser

85 90 95

Lys Gly Gln Leu Lys Glu Phe Leu Asp Ala Asn Leu Ala Gly Ser Gly

100 105 110

Ser Gly His Met His His His His His His Ser Ser Gly Leu Val Pro

115 120 125

Arg Gly Ser Gly Met Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln

130 135 140

His Met Asp Ser Pro Asp Leu Gly Thr Asp Asp Asp Asp Lys Ala Met

145 150 155 160

Ala Asp Ile Gly Ser Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg

165 170 175

Met Pro Leu His Met Ile Pro Gln Val Ala His Ala Met Val Arg Ala

180 185 190

Ala Ala Ala Gly Arg Leu Thr Leu Tyr Thr Lys Thr Lys Thr Glu Thr

195 200 205

Thr Asn Phe Asp His Ala Glu Tyr Val Thr Cys Gly Arg Tyr Thr Ile

210 215 220

Cys Ala Phe Cys Leu Thr Thr Leu Ala Pro His Ala Asn Val Lys Thr

225 230 235 240

Ile Gln Asp Ser His Ala Cys Ser Arg Gln Pro Asn Glu Ala Ile Arg

245 250 255

Ser Leu Val Glu Val Ser Asp Lys Ala Gln Ile Ala Leu Val Gly Ser

260 265 270

Arg Thr Val Asp Tyr His Glu Leu Asp Val Lys Ala Gly Phe Val Ala

275 280 285

Pro Thr Ala Asp Glu Thr Val Val Pro Ser Lys Asp Ile Val Glu Leu

290 295 300

Pro Phe Arg Thr Cys Asp Leu Asp Asp Ser Ser Ala Thr Ala Cys Val

305 310 315 320

Arg Asn His Cys Gln Ala Gly His Asp Gly Val Thr His Leu Pro Ile

325 330 335

Leu Ser Gly Asp Phe Lys Leu Pro Asn Glu His Pro Thr Lys Pro Leu

340 345 350

Asp Asp Thr His Pro His Asp Lys Val Leu Thr Arg Cys Pro Lys Thr

355 360 365

Gly Leu Leu Leu Val His Asp Thr His Ala His Ala Thr Ala Val Val

370 375 380

Ala Thr Ala Ala Thr Arg Ala Ile Leu Met His Asp Leu Leu Thr Ser

385 390 395 400

Ala Asn Val Asp Asp Gly His Gln Ala Arg Ser Ala Cys Tyr Gly Pro

405 410 415

Thr Phe Ser Asn Leu Thr Phe Ala Cys His Ser Thr Cys Ala Ser Asp

420 425 430

Met Ala His Phe Asp Cys Gly Gln Ile Val Gly Leu Asp Leu His Val

435 440 445

Glu Pro Ser Asp

450 452

 

Claims (6)

1. an albumen for artificial preparation, its sequence is for shown in SEQ ID NO.2.

2. the nucleotide sequence that albumen described in claim 1 is corresponding.

3. sequence according to claim 2, is characterized in that: described nucleotides sequence is classified as shown in SEQ ID NO.1.

4. express a genetic engineering bacterium for albumen described in claim 1, it is characterized in that: colon bacillus eschierichia colibL21 (DE3)/pET-32a-TAT-VP7-TAT, deposit number: CCTCC NO:M2015111.

5. the preparation method of albumen described in claim 1, comprise genetic engineering bacterium IPTG or the automatic abduction delivering 4-5h of lactose, described genetic engineering bacterium is: colon bacillus eschierichia colibL21 (DE3)/pET-32a-TAT-VP7-TAT, deposit number: CCTCC NO:M2015111.

6. albumen according to claim 1 or nucleotide sequence according to claim 2 or the application of genetic engineering bacterium according to claim 4 in preparation treatment or prevention grass carp hemorrhage virus (GCHV) medicine.