CN104530241B - A kind of genetic engineering bacterium for expressing cell-penetrating peptide fusion protein and application - Google Patents

Abstract

The present invention provides a kind of genetic engineering bacterium for expressing cell-penetrating peptide fusion protein and application, the present invention provides a kind of genetic engineering bacterium,E. coli BL21/ pET‑32a–CSFR, CCTCC NO：M2014631.The bacterial strain can be with expressing protein transduction structural domain polypeptide（PEP‑1）With swine fever virus receptor（CD46）1 CD46 of genetic engineering fusion protein PEP.Its advantage is that expression quantity is big, and soluble protein is at low cost, and security is good.The 1 CD46 albumen of PEP of expression can pass through the mediating proteins of cell membrane as swine fever virus, improve the virus titer of cell culture swine fever virus, can solve in current swine fever virus cell vaccine that viral level is low, the shortcomings that being not achieved with seedling standard.

Description

A kind of genetic engineering bacterium for expressing cell-penetrating peptide fusion protein and application

Technical field

The invention belongs to genetic engineering field, specifically, a kind of genetic engineering bacterium for expressing cell-penetrating peptide fusion protein and Using.

Technical background

Swine fever (classical swine fever, CSF) is by swine fever virus (classical swine fever Virus, CSFV) cause acute one kind of pig, heat generation, lethal infectious diseases.The disease has high degree in contact infectiousness, Zeng Guo It is widely current on border, incidence is high, and the death rate is up to more than 90%, very harmful, and huge warp is caused to pig breeding industry at that time Ji loss, important livestock and poultry infectious disease register is included in by the World Health Organization (OIE).At present, China is hog cholera vaccine life in the world Production and most commonly used country, annual total flow is about more than 4,000,000,000 parts, and market total price is about 2,000,000,000 yuans.

The interference of prolonged vaccine immunity and other epidemic diseases, pig are gradually even generating hog cholera vaccine by generation Tolerance, responsing reaction decrease or slack-off, causes the situation of hog cholera vaccine immuning failure in recent years often to occur, mild Swine fever, inferior clinical symptom swine fever are constantly popular, annual therefore to cause nearly 10,000,000,000 yuan of economic losses to China's pig breeding industry.This shape Gesture proposes the quality of hog cholera vaccine new requirement, and there is an urgent need for improve the potency of China's hog cholera vaccine.

The potency of hog cholera vaccine and the incubation of swine fever virus have an extremely close relationship, swine fever virus rely on it is quick The surface receptor of sense cell is combined into cell, expand be released to again in culture solution in the cell [Wang Zhen, Lu Yu, Zhou Pengcheng, Wait research [J] microorganism journals of swine fever virus reproductive process in PK cells and MPK cells, 1999,39 (3):189- 194.].But in incubation, even the allogenic cell in same culture vessel, and not all cell individual is all Susceptible to the virus of inoculation, this is that is, after cell inoculation virus in same culture vessel, and in not all cell body Will have cell entry amplification, and during swine fever virus culture, only small part cell has a cell entry, most cells be Slowly increase cell quantity [Wang Zhen, Lu Yu, the bright filial piety swine fever virus China of fourth into virus with the extension of incubation time Research [J] CHINA virus of rabbitization attenuated vaccine strain multiplication characteristic in primary bull testis cell, 2000,15 (23):170- 179.].This has resulted in two results：1st, initial period is cultivated, participates in that the cell of amplicon virus is less, and virus titer is difficult to rise Height, 2, the speed of cell amplicon virus it is asynchronous, under cultivation temperature, first the viral viability that is released from cell declines, This causes culture solution to reach the time lengthening of standard poison valency.It is therefore believed that：How the disease that in same culture vessel is inoculated with is made Poison, which enters within the time as short as possible in most cell individuals, to be expanded, it would be possible to be the pass for obtaining high-titer swine fever virus Key.

PEP-1 is a kind of film transducin, is also membrane-spanning protein, which can freely come in and go out cell membrane, and people make Some drugs that cannot penetrate cell membrane are brought into the albuminoid, preferable effect is achieved in cell membrane, and CD46 albumen is then It is pig plague virus specific receptor, specificity can captures swine fever virus in culture solution.Patent of the present invention uses both albumen The mode of gene expression connects, and the swine fever virus in culture solution, then wearing by PEP-1 albumen are captured by CD46 albumen Film effect brings the swine fever virus of capture into the cell into, makes in a short time to import swine fever virus by force in cultivating system and to the greatest extent may be used Being expanded into the cell more than energy, achievees the purpose that increase substantially swine fever virus culture potency.

SAPGTP is a kind of fusion protein connector, is also connection peptide, will both active functional protein connect, After structure forms fusion protein, each albumen can keep respective activity.Connection can correctly be folded by ensureing the polypeptide chain of the two It is together and still active, but the activated centre that can make the two is away from each other, will not form steric hindrance and influence the table of activity It is existing, activity can not be made to influence each other even and inhibited.Compared by the method for computer Blast search, it is predicted that SAPGTP connectors The expression quantity of the fusion protein can not be interfered with well by together with PEP-1 membrane-spanning proteins and CD46 protein fusions, it will not The correct of different structure territory in fusion protein is influenced to fold and corresponding biological activity.

The content of the invention

Object of the present invention is to provide a kind of recombinant protein PEP-1-CD46 that can improve swine fever virus culture potency, Its sequence is shown in SEQ ID NO.2, corresponding nucleotides sequence is classified as shown in SEQ ID NO.1.PEP-1 is worn film egg by the present invention In vain with CD46 protein fusion expressions, biologically active PEP-1-CD46 albumen is obtained, culture solution is captured using CD46 albumen In swine fever virus, then the swine fever virus of capture is brought into the cell by the membrane penetration effect of PEP-1 albumen, made in cultivating system Swine fever virus is imported by force in a short time it is as much as possible expand into the cell, reach increase substantially swine fever virus training Support the purpose of potency.

It is another object of the present invention to provide a kind of cell-penetrating peptides that can improve swine fever virus culture potency to merge egg White preparation method, this method can be applied in production of vaccine, and expression quantity is big, easy to operate, at low cost.The fusion protein can have Effect improves the cultivation effect of swine fever virus in the cell, improves the malicious valency of Virus culture.

It is yet a further object of the present invention to provide a kind of Recombinant organism strain Escherichia coli (Escherichia coli)BL21(DE3)pET-32a-PEP-1-CD46.The bacterial strain can be more with expressing protein transduction structural domain Peptide (PEP-1) and swine fever virus receptor (CD46) genetic engineering fusion protein PEP-1-CD46.Its advantage is that expression quantity is big, solvable Property albumen, at low cost, security is good.The bacterium is sent to China typical culture collection center in being preserved on December 5th, 2014 Preservation is carried out, deposit number is CCTCC NO：M2014631, address：Wuhan, China Wuhan University, Classification And Nomenclature： Escherichia coli BL21(DE3)/pET-32a–CSFR。

PEP-1-CD46 protein sequences are as follows：

KETWWETWWTEWSQPKKKRKVSAPGTPMMAFCALRKALPCRPENPFSSRCFVEILWVSLALVFLLPMPS DACDEPPKFESMRPQFLNTTYRPGDRVEYECRPGFQPMVPALPTFSVCQDDNTWSPLQEACRRKACSNLPDPLNGQV SYPNGDMLFGSKAQFTCNTGFYIIGAETVYCQVSGNVMAWSEPSPLCEKILCKPPGEIPNGKYTNSHKDVFEYNEVV TYSCLSSTGPDEFSLVGESSLFCIGKDEWSSDPPECKVVKCPYPVVPNGEIVSGFGSKFYYKAEVVFKCNAGFTLHG RDTIVCGANSTWEPEMPQCIKDSKPTDPPATPGPSHPGPPSPSDASPPKDAESLDGGIIAAIVVGVLAAIAVIAGGV YFFHHKYNKKRSK

PEP-1-CD46 nucleotide sequences：

AAGGAAACATGGTGGGAAACGTGGTGGACTGAGTGGTCCCAGCCTAAAAAGAAGAGAAAGGTTTCTGCT CCAGGTACCCCTATGATGGCTTTCTGTGCTTTGCGTAAAGCTCTGCCATGTCGTCCAGAAAACCCATTTTCTTCTCG TTGTTTCGTCGAAATATTGTGGGTTTCTTTGGCTTTGGTTTTTCTATTGCCAATGCCTTCTGATGCCTGTGATGAAC CACCCAAGTTTGAATCTATGAGACCACAGTTCTTAAACACTACTTATAGGCCAGGTGATAGAGTCGAATACGAATGT AGACCTGGTTTTCAACCAATGGTTCCAGCCTTGCCAACTTTTTCTGTTTGCCAAGACGATAATACTTGGTCTCCATT GCAAGAAGCTTGTAGGAGAAAAGCTTGCTCTAATTTGCCAGATCCATTGAACGGTCAAGTTTCTTACCCAAATGGTG ACATGTTGTTTGGTTCTAAAGCCCAATTTACGTGTAACACAGGTTTTTACATTATTGGTGCTGAAACTGTTTACTGC CAAGTCTCTGGTAATGTCATGGCATGGTCCGAACCCTCTCCCTTGTGTGAAAAGATTTTGTGTAAGCCACCCGGTGA AATTCCTAACGGTAAGTACACTAACTCTCATAAGGATGTTTTTGAGTATAACGAGGTCGTTACTTACTCTTGTTTGT CTTCCACGGGACCAGATGAGTTTTCCTTGGTTGGTGAATCTTCTCTGTTTTGTATAGGTAAGGACGAATGGTCTTCT GACCCACCCGAGTGTAAGGTTGTTAAGTGCCCATACCCAGTTGTTCCCAACGGAGAAATTGTTAGTGGTTTCGGTTC AAAGTTCTATTACAAAGCAGAAGTCGTATTTAAGTGTAACGCCGGTTTTACTTTGCATGGTAGAGATACTATTGTCT GTGGTGCTAATTCTACTTGGGAGCCAGAAATGCCACAATGTATTAAGGATTCTAAACCAACTGATCCACCAGCTACT CCTGGTCCATCCCATCCAGGCCCACCCTCCCCATCAGATGCTTCTCCACCAAAGGATGCTGAATCATTGGACGGTGG TATTATAGCCGCTATTGTTGTTGGTGTTTTAGCTGCAATAGCTGTCATTGCTGGAGGTGTGTATTTCTTCCATCACA AATACAACAAAAAGAGGTCTAAGTAA

The PEP-1-CD46 fusion proteins that final object of the present invention is to provide recombination engineering expression are improving disease Application in the malicious valency of poison culture, especially for the application in the culture of swine fever virus.

In order to achieve the above object, the present invention takes following technical measures：

The invention also discloses the methods of Prepare restructuring engineering bacteria, and this method comprises the following steps：

1) preparation of fusion PEP-1-CD46：According to the artificial synthesized PEP-1- of the gene order announced on GeneBank CD46 genes；

2) structure of expression vector：With EcoR I and BamH I double digestion PEP-1-CD46cDNA and plasmid pET-32a (purchase From Novagen companies), glutinous end connection is carried out with PET-32a plasmids under the action of T4 ligases, converts escherichia coli DH5a (being purchased from Novagen companies) competent cell, picking single bacterium colony carry out PCR identifications, plasmid, reference are extracted in a small amount with alkaline lysis 《Molecular Cloning:A Laboratory guide》It carries out, DNA output is between 100ng and 5 μ g, depending on the copy number of plasmid.To the matter of extraction Grain carries out digestion identification, and obtained positive colony is named as pET-32a-PEP-1-CD46.

3) preparation of Recombinant organism：Plasmid pET-32a-PEP-1-CD46 is converted into e. coli bl21 (DE3) competent cell (purchased from Novagen companies).PCR evaluation and screenings go out positive transformant, and picking positive colony single bacterium is dropped into Row culture, final engineering bacteria (present invention or the recombination engineered strain Esche for obtaining stability and high efficiency expression PEP-1-CD46 Richia coli BL21 (DE3) pET-32a-PEP-1-CD46), which send to China in being preserved on December 5th, 2014 Type Tissue Collection carries out preservation, and deposit number is CCTCC NO：M2014631, address：Wuhan, China Wuhan University, Classification And Nomenclature：Escherichia coli BL21(DE3)/pET-32a–CSFR.

The bacterial strain belongs to gram-Negative bacillus, no brood cell, and whole body flagellum can move.Optimum growth temperature is 37 DEG C, can With IPTG or the automatic induced expression of lactose, it is metabolized vigorous.The bacterium anabolism ability is strong, containing inorganic salts, amine salt, glucose Well-grown on ordinary culture medium, the various saccharides that can ferment production acid, aerogenesis.Its bacterium colony is rounded, and there is light on neat in edge, surface Pool moistens, is smooth, translucent.

A kind of cell-penetrating peptide fusion protein, is prepared by following steps：

The single bacterium colony of recombination engineered strain Escherichia coli BL21 (DE3) pET-32a-PEP-1-CD46 It is inoculated in the 20ml LB fluid nutrient mediums containing ampicillin (final concentration of 100 μ g/ml), in 37 DEG C of shaking tables, 300rpm Shaking is incubated overnight 10~12h.By 1：100 ratio is inoculated into the fresh lactose automatic induction culture medium ZYM5052 of 20ml (Amp+) in, 37 DEG C of constant temperature, 300rpm cultures 3h or so.Under aseptic condition, 1ml bacterium solutions are taken as the control before induction.It moves again Low temperature induction is carried out to 16 DEG C of culture shaking tables, induces 16h automatically.12000g is centrifuged, 4 DEG C, 10min collects thalline, with 20ml's Lysis Buffer are resuspended thalline and carry out ultrasonic disruption, supernatant are collected by centrifugation and with SDS-PAGE electrophoresis detection destination proteins Expression.Further expand and cultivate, after ultrasonic disruption, 12000g, 4 DEG C of centrifugations, 20min collection precipitations.

Application of the PEP-1-CD46 fusion proteins in the malicious valency for improving Virus culture, including fusion protein PEP-1- Applications of the CD46 in the production of high quality swine fever virus cell vaccine, application process are as follows：

After ST cells grow up to fine and close individual layer in Tissue Culture Flask, cell maintenance medium is outwelled, first with the pancreas egg of 5ml 0.25% Then white enzyme solutions cleaning cell surface adds the trypsin solution vitellophag of 5ml 0.25%, treat cell aggregation into Trypsin solution is outwelled by group when starting shedding off, add in 8ml cell growth mediums and terminate digestion, blow and beat mixing, expect blue dyeing with platform Viable count is carried out, adds suitable cell growth medium, adjusts cell density to 1 × 10⁵A/ml.Cell growth medium is accessed In 96 well culture plates, 200 μ l are added in per hole, each group repeats for 12 holes, 37 DEG C, 5.0%CO₂Under the conditions of quiescent culture.And it sets Blank control wells are not added with any liquid.37 DEG C, 5.0%CO₂Under the conditions of quiescent culture.Treat that cell growth is covered to 75%~80% It during lid culture plate culture plane, inhales abandon culture solution in each hole in superclean bench, 20 μ l viruses maintaining liquids (virus is accessed per hole Control group is cell maintenance medium), according to 0.1MOI dose inoculation virus liquids, and meanwhile it is above-mentioned by the final concentration addition of 40ng/ml PEP-1-CD46 fusion proteins, 37 DEG C, 5.0%CO₂Under the conditions of adsorb 1h after, viral maintaining liquid is accessed per hole to 200 μ l (viruses Control group is cell maintenance medium), 37 DEG C, 5.0%CO₂Under the conditions of quiescent culture.

Swine fever virus culture poison valency prepared by the present invention can reach 10^8.4TCID50/ml, available for swine fever attenuated cell The production of seedling.

Compared with prior art, the present invention has the following advantages：

1) fusion protein PEP-1-CD46 expression quantity according to the present invention is big, and is soluble protein, convenient for extensive Production, of low cost, operating procedure is simple；

2) present invention utilizes Bacillus coli expression PEP-1 nexin transduction domains and the fusion egg of swine fever virus receptor CD46 In vain, it is intended that capture the swine fever virus in culture solution by CD46 albumen, then by PEP-1 albumen membrane penetration effect by the pig of capture Pestivirus is brought into the cell, makes swine fever virus is imported to intracellular progress as much as possible by force in a short time in cultivating system Amplification, achievees the purpose that increase substantially swine fever virus culture potency.

3) method provided by the invention for improving swine fever virus culture potency can be solved in current swine fever virus cell vaccine Viral level is low, the shortcomings that being not achieved with seedling standard.TCID50 methods measure its culture potency and can reach 10^8.4TCID50/ml is high The weak malicious culture of swine fever of potency can be used for the classical swine fever virus vaccine for preparing high quality.

Description of the drawings

Fig. 1 is the one step growth curve that CSFV is proliferated in ST cells.

Specific embodiment

Below in conjunction with the accompanying drawings and specific embodiment the invention will be further described.All trainings being related in embodiment It supports base and molecular biology manipulations method is well known to those skilled in the art.The involved molecular biology method of this experiment is Conventional method is familiar with by those skilled in the art.What is do not elaborated in the present invention refers to《Molecular Cloning:A Laboratory guide》J. Sa The chief editors such as nurse Brooker, D.W. Russells.

Embodiment 1：PEP-1-CD46 genes it is artificial synthesized

According to CD46 (the Gene ID delivered in GeneBank:396922) sequence and the sequence of PEP-1 is manually closed Into PEP-1-CD46 genes, and add EcoR I, BamH I's restriction enzyme site respectively at its both ends, the gene order of synthesis includes S Coding region sequence shown in EQ ID NO.1.

Embodiment 2：

The structure of recombinant plasmid pET-32a-PEP-1-CD46

With restriction enzyme EcoR I, BamH I's double digestion carrier pET-32a plasmids and PEP-1-CD46cDNA.Digestion System is as follows：

Endonuclease reaction condition is 37 DEG C, 2~3 hours of reaction time.Digestion products are returned through agarose gel electrophoresis with glue Receive kit recycling open loop pET-32a and PEP-1-CD46cDNA endonuclease bamhi.

PEP-1-CD46cDNA endonuclease bamhis with open loop pET-32a are connected, obtain recombinant plasmid pET-32a-PEP-1-C D46.Linked system is as follows：

Coupled reaction liquid mixing, when 22 DEG C of coupled reactions 2 are small, store in 4 DEG C it is spare.

Calcium Chloride Method prepares competent escherichia coli cell, and step is：

1) on fresh solid LB tablets picking DH5 α single bacterium colony, be inoculated in 20ml LB fluid nutrient mediums, 37 DEG C are shaken Bed 300rpm overnight incubations.

2) the 200 μ l bacterium solutions that inoculation is activated overnight are in fresh 20ml LB fluid nutrient mediums, 37 DEG C, 300rpm cultures About 2~3h, until OD₆₀₀About 0.4~0.6.

3) under gnotobasis, bacterium solution is drawn into the 1.5ml EP pipes of precooling, 4 DEG C of centrifugations, 4000rpm, 10min.It abandons Clear culture solution collects thalline.

4) the 0.1M CaCl per effective 200 μ l precoolings₂Solution is resuspended, ice bath 30 minutes.

5) 4 DEG C of centrifugations, 4000rpm, 10min abandon supernatant culture solution, collect thalline, be placed on ice.

6) the 0.1M CaCl per effective 100 μ l precoolings₂Solution is resuspended, and competent cell is prepared and finished.

Connection product converts competent escherichia coli cell：Its step is：

1) 10 μ l of connection product is taken to add in 100 μ l competent cells under aseptic condition, gently mixing, ice bath 30min.

2) 42 DEG C of water-bath heat shock 90s move to rapidly on ice, cool down 1~2min.

3) 600 μ l LB fluid nutrient mediums are added in, the 150rpm jogs in 37 DEG C of shaking tables incubate 45min,

4) 4000rpm is centrifuged 10 minutes, is left 100 μ l supernatants, is discarded remaining bacterium solution, and thalline is resuspended with pipettor.

5) above-mentioned bacterium solution is spread evenly across containing Amp with sterile triangle spreading rod⁺(final concentration of 100 μ g/ml's) On LB tablets, forward direction is placed, and after liquid is fully absorbed, is inverted tablet, 37 DEG C of overnight incubations.

Thus E.coli DH5 α pET-32a-PEP-1-CD46 bacterium colonies can be prepared in method.

The digestion identification of positive forward direction recon pET-32a-PEP-1-CD46：

E.coli DH5 α pET-32a-PEP-1-CD46 plasmids are extracted with alkaline lysis a small amount of (15-20ug), to institute's upgrading Grain carries out single endonuclease digestion and double digestion, digestion system are as follows：

The single endonuclease digestion of recombinant plasmid pET-32a-PEP-1-CD46

The double digestion of recombinant plasmid pET-32a-PEP-1-CD46

Endonuclease reaction condition is 37 DEG C, and 2-3 hour of reaction time, digestion products are detected with agarose gel electrophoresis, The specific band that two sizes are respectively 5900bp and 1173bp is obtained, size is consistent with theoretical value, shows successfully to build weight Group plasmid pET-32a-PEP-1-CD46.

Embodiment 3：The preparation of Recombinant organism

By plasmid pET-32a-PEP-1-CD46 (1ul) conversion e. coli bl21s (DE₃) competent cell.PCR is identified Positive transformant is filtered out, gained positive colony is the recombination of the present invention for being capable of amalgamation and expression PEP-1-CD46 Engineered strain Escherichia coli BL21 (DE₃)pET-32a-PEP-1-CD46.The bacterium is in being preserved in December, 2014 It send within 5th to China typical culture collection center and carries out preservation, deposit number is CCTCC NO：M2014631, address：It is Chinese military Chinese Wuhan University, Classification And Nomenclature：Escherichia coli BL21(DE3)/pET-32a–CSFR.

Embodiment 4：The expression of genetic engineering fusion protein PEP-1-CD46

1) from solid LB (Amp⁺) picking carries the E.c of plasmid pET-32a and pET-32a-PEP-1-CD46 on tablet Oli BL21 (DE3) single bacterium colony is inoculated in the 20ml LB fluid nutrient mediums containing ampicillin (final concentration of 100 μ g/ml) In, in 37 DEG C of shaking tables, 300rpm shakings are incubated overnight 10~12h.

2) second day, the strain of activation is pressed 1：100 ratio is transferred in lactose automatic induction culture medium ZYM5052, 37 DEG C, when 300rpm shaken cultivations 3~4 are small.Under aseptic condition, 1ml bacterium solutions are taken as the control before induction.

3) 16 DEG C are then moved to, 300rpm culture shaking tables carry out low temperature induction, induce 16h automatically.

4) 4 DEG C of centrifugations, 12000rpm, 5min abandon supernatant, collect thalline.

5) the Lysis Buffer that 20ml is added in into the bacterial sediment of collection are resuspended.

6) bacteria suspension is placed in -20 DEG C of multigelations and carries out ultrasonication afterwards several times, 400W, working time 3s, during interval Between 3s.

7) when bacteria suspension becomes limpid by muddiness, show that thalline is substantially broken complete, stop ultrasonication.

8) bacterial cell disruption liquid is centrifuged into 20min in 4 DEG C, 12000rpm.

9) supernatant of centrifugation gained is collected, takes 20 μ L and isometric 2 × SDS-PAGE sample buffer mixings, sample It is placed in boiling water and boils 10min, carry out SDS-PAGE.

10) after electrophoresis, gel is unloaded, after dyeing 2-4h with Coomassie brilliant blue dye liquor, then with destainer by background colour Until taking off, to determine whether PEP-1-CD46 albumen is expressed.

SDS-PAGE shows that obtaining a size in supernatant after fusion protein PEP-1-CD46 bacterial cell disruptions is about The specific band of 46KD shows successful expression PEP-1-CD46 albumen, the sequence of the destination protein PEP-1-CD46 given expression to It is classified as shown in SEQ ID NO.2.

Embodiment 5：The purifying of genetic engineering fusion protein PEP-1-CD46

Supernatant in Ni-NTA Superflow columns affinity chromatography purifying embodiment 4 after bacterial cell disruption obtained by centrifugation, Its step are as follows：

1) 1ml Ni-NTA Agarose are drawn with pipettor and is packed into chromatography column bottom, washed with the distilled water of 10 times of column volumes Wash pillar.

2) with the Lysis Buffer balance nickel columns of 10 times of column volumes.

3) by the good protein solution of renaturation after 0.45 μm of membrane filtration, with peristaltic pump with the rate of 1ml/min, add in Into the nickel pillar balanced, make the 6-His and Ni of albumen front end²⁺Fully combine.

4) successively with suitable Imidazole containing 20mM, 40mM Imidazole, 60mM Imidazole, 80mM I The Wash Buffer of midazole elute pillar with the rate of 1ml/min, to elute lower Ni²⁺It is combined on column unstable or few Amount not with Ni²⁺With reference to non-destination protein.The OD of the efflux after elution every time is detected with nucleic acid-protein detector₂₈₀, treat OD₂₈₀ Stop washing when being continuously 0.

5) pillar is eluted with the rate of 200 μ l/min with the Elution Buffer of the Imidazole containing 250mM, used Eppen dorf pipes collect destination protein.The albumen of collection is taken to carry out 12%SDS-PAGE detection purification effects.

6) SDS-PAGE electrophoresis is carried out, detects the purification effect of collected albumen, expression quantity is big, and is soluble egg In vain, protein concentration is about 627 μ g/ml.

Embodiment 6：

Immunofluorescence test PEP-1-CD46 fusion proteins improve the research of swine fever virus culture potency

(1) prepared by cell

After ST cells grow up to fine and close individual layer in Tissue Culture Flask, cell maintenance medium is outwelled, first with the pancreas egg of 5ml 0.25% Then white enzyme solutions cleaning cell surface adds the trypsin solution vitellophag of 5ml 0.25%, treat cell aggregation into Trypsin solution is outwelled by group when starting shedding off, add in 8ml cell growth mediums and terminate digestion, blow and beat mixing, expect blue dyeing with platform Viable count is carried out, adds suitable cell growth medium, adjusts cell density to 1 × 10⁵A/ml.Cell growth medium is accessed In 96 well culture plates, 200 μ l are added in per hole, each group repeats for 12 holes, 37 DEG C, 5.0%CO₂Under the conditions of quiescent culture.

(2) viruses adsorption

When the culture plane of cell growth to 75%~80% covering cell culture well, inhale completely in superclean bench Culture solution in each hole is abandoned, adds each group cell hole of PEP-1-CD46 fusion protein groups, 20 PEP-s of the μ l containing addition are accessed per hole The cell maintenance medium of 1-CD46 fusion proteins, make addition PEP-1-CD46 fusion protein final concentrations respectively reach 20ng/ml, 40ng/ml and 60ng/ml, according to 0.1MOI dose inoculation hog cholera venom.Cell controls are set simultaneously, only access 20 μ l cells dimension Liquid is held, not virus inoculation.Virus control group accesses cells of the 20 μ l without addition PEP-1-CD46 fusion proteins per hole and maintains Liquid, according to 0.1MOI dose inoculation virus liquids, each group sets 12 hole of parallel hole.Tissue Culture Plate is placed in 37 DEG C, 5.0%CO₂Item 1h is adsorbed under part.180 μ l virus-culturing fluids (cell controls group and virus control group addition cell dimension are accessed after absorption per hole Hold liquid), it is placed in 37 DEG C, 5.0%CO₂Under the conditions of quiescent culture.

(3) it is fixed

After viruses adsorption 20min, 30min, 40min, 60min, 90min, 180min, 210min, 12h, 15h, 18h, 21h, for 24 hours, 36h, 48h, 60h, 72h, 84h, 96h and 108h respectively take 1 piece of cell plates, with the soft board-washings 2 of the PBS of pH7.2 It after secondary, inhale abandon liquid completely, 30min is fixed with 80% acetone of -20 DEG C of cooling 10min.

(4) fluorescent antibody staining

After outwelling acetone, with pH7.2PBS board-washings 2 times, each 10min.After board-washing, room temperature naturally dry connects per hole Enter the 30 μ l of pig pestivirus fluorescence antibody working solution diluted, be put into wet box, be protected from light under the conditions of 37 DEG C and be incubated 40min.

(5) fluorescence microscopy

After incubation, liquid, pH7.2PBS board-washings 2 times are outwelled, drying is placed under fluorescence microscope and amplifies 200 times of sights It examines.It tests and sets up when cell controls group unstressed configuration particle occurs.Utilize fluorescence after fluorescence colour detection addition PEP-1-CD46 It is outer in the cell the variation of speed occur, illustrate to add PEP-1-CD46 to viruses adsorption, wear the shadow of film speed and albumen synthesis It rings, measures the titre of culture solution generation of neutrons virus, make the one step growth curve that swine fever virus is proliferated in ST cells, analysis adds PEP-1-CD46 is added to improve the effect of virus titer.

1 Immunofluorescence test of table

Note：- represent not observe fluorescent grain ,+represent that a small amount of fluorescent grain can be observed, fluorescent brightness is smaller, ++ table Show and more fluorescent grain can be observed, fluorescent brightness is larger.

PEP-1-CD46 accelerates viruses adsorption and wears the speed of film as can be seen from Table 1, promotes the synthesis of virus protein, And viruses adsorption also increased with the virion quantity for wearing film.Wherein add 40ng/ml PEP-1-CD46 experimental groups virus Absorption 30min just has virion to be combined with receptor-specific on cell membrane, and absorption 90min just has a large amount of virion completions to wear Membrane process is into the cell, and 18h just has new virus protein synthesis, addition 20ng/ml, 60ng/ml PEP-1- after virus inoculation The CD46 experimental group viruses adsorption times are slower, and virus control group then just has a viral specific adsorption in 60min, during 90min Only a small amount of virion completion wears membrane process into the cell, and 21h just has new virus protein synthesis.

PEP-1-CD46 can significantly improve titre viral in culture solution as seen from Figure 1, infect 60h virus titers Reach top.Wherein, addition 40ng/ml PEP-1-CD46 experimental groups can make the titre of virus be up to 10^8.4TCID50/ Ml, lower slightly addition 20ng/ml, 60ng/ml PEP-1-CD46 experimental group virus titers (are respectively 10^7.4TCID50/ml and 10^7.6TCID50/ml), but still higher than the titre of virus control group (titre of virus control group is 10^7.1TCID50/ml).This is carried Show that we add the fusion protein of suitable concentration and can effectively facilitate multiplication rates of the CSFV in ST cells, so as to improve disease Toxic effect valency.

SEQUENCE LISTING

<110>Wuhan Inst of Veterinary Science

<120>A kind of genetic engineering bacterium for expressing cell-penetrating peptide fusion protein and application

<130>A kind of genetic engineering bacterium for expressing cell-penetrating peptide fusion protein and application

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 1173

<212> DNA

<213>Artificial sequence

<400> 1

aaggaaacat ggtgggaaac gtggtggact gagtggtccc agcctaaaaa gaagagaaag 60

gtttctgctc caggtacccc tatgatggct ttctgtgctt tgcgtaaagc tctgccatgt 120

cgtccagaaa acccattttc ttctcgttgt ttcgtcgaaa tattgtgggt ttctttggct 180

ttggtttttc tattgccaat gccttctgat gcctgtgatg aaccacccaa gtttgaatct 240

atgagaccac agttcttaaa cactacttat aggccaggtg atagagtcga atacgaatgt 300

agacctggtt ttcaaccaat ggttccagcc ttgccaactt tttctgtttg ccaagacgat 360

aatacttggt ctccattgca agaagcttgt aggagaaaag cttgctctaa tttgccagat 420

ccattgaacg gtcaagtttc ttacccaaat ggtgacatgt tgtttggttc taaagcccaa 480

tttacgtgta acacaggttt ttacattatt ggtgctgaaa ctgtttactg ccaagtctct 540

ggtaatgtca tggcatggtc cgaaccctct cccttgtgtg aaaagatttt gtgtaagcca 600

cccggtgaaa ttcctaacgg taagtacact aactctcata aggatgtttt tgagtataac 660

gaggtcgtta cttactcttg tttgtcttcc acgggaccag atgagttttc cttggttggt 720

gaatcttctc tgttttgtat aggtaaggac gaatggtctt ctgacccacc cgagtgtaag 780

gttgttaagt gcccataccc agttgttccc aacggagaaa ttgttagtgg tttcggttca 840

aagttctatt acaaagcaga agtcgtattt aagtgtaacg ccggttttac tttgcatggt 900

agagatacta ttgtctgtgg tgctaattct acttgggagc cagaaatgcc acaatgtatt 960

aaggattcta aaccaactga tccaccagct actcctggtc catcccatcc aggcccaccc 1020

tccccatcag atgcttctcc accaaaggat gctgaatcat tggacggtgg tattatagcc 1080

gctattgttg ttggtgtttt agctgcaata gctgtcattg ctggaggtgt gtatttcttc 1140

catcacaaat acaacaaaaa gaggtctaag taa 1173

<210> 2

<211> 390

<212> PRT

<213>Artificial sequence

<400> 2

Lys Glu Thr Trp Trp Glu Thr Trp Trp Thr Glu Trp Ser Gln Pro Lys

1 5 10 15

Lys Lys Arg Lys Val Ser Ala Pro Gly Thr Pro Met Met Ala Phe Cys

20 25 30

Ala Leu Arg Lys Ala Leu Pro Cys Arg Pro Glu Asn Pro Phe Ser Ser

35 40 45

Arg Cys Phe Val Glu Ile Leu Trp Val Ser Leu Ala Leu Val Phe Leu

50 55 60

Leu Pro Met Pro Ser Asp Ala Cys Asp Glu Pro Pro Lys Phe Glu Ser

65 70 75 80

Met Arg Pro Gln Phe Leu Asn Thr Thr Tyr Arg Pro Gly Asp Arg Val

85 90 95

Glu Tyr Glu Cys Arg Pro Gly Phe Gln Pro Met Val Pro Ala Leu Pro

100 105 110

Thr Phe Ser Val Cys Gln Asp Asp Asn Thr Trp Ser Pro Leu Gln Glu

115 120 125

Ala Cys Arg Arg Lys Ala Cys Ser Asn Leu Pro Asp Pro Leu Asn Gly

130 135 140

Gln Val Ser Tyr Pro Asn Gly Asp Met Leu Phe Gly Ser Lys Ala Gln

145 150 155 160

Phe Thr Cys Asn Thr Gly Phe Tyr Ile Ile Gly Ala Glu Thr Val Tyr

165 170 175

Cys Gln Val Ser Gly Asn Val Met Ala Trp Ser Glu Pro Ser Pro Leu

180 185 190

Cys Glu Lys Ile Leu Cys Lys Pro Pro Gly Glu Ile Pro Asn Gly Lys

195 200 205

Tyr Thr Asn Ser His Lys Asp Val Phe Glu Tyr Asn Glu Val Val Thr

210 215 220

Tyr Ser Cys Leu Ser Ser Thr Gly Pro Asp Glu Phe Ser Leu Val Gly

225 230 235 240

Glu Ser Ser Leu Phe Cys Ile Gly Lys Asp Glu Trp Ser Ser Asp Pro

245 250 255

Pro Glu Cys Lys Val Val Lys Cys Pro Tyr Pro Val Val Pro Asn Gly

260 265 270

Glu Ile Val Ser Gly Phe Gly Ser Lys Phe Tyr Tyr Lys Ala Glu Val

275 280 285

Val Phe Lys Cys Asn Ala Gly Phe Thr Leu His Gly Arg Asp Thr Ile

290 295 300

Val Cys Gly Ala Asn Ser Thr Trp Glu Pro Glu Met Pro Gln Cys Ile

305 310 315 320

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

325 330 335

Pro Gly Pro Pro Ser Pro Ser Asp Ala Ser Pro Pro Lys Asp Ala Glu

340 345 350

Ser Leu Asp Gly Gly Ile Ile Ala Ala Ile Val Val Gly Val Leu Ala

355 360 365

Ala Ile Ala Val Ile Ala Gly Gly Val Tyr Phe Phe His His Lys Tyr

370 375 380

Asn Lys Lys Arg Ser Lys

385 390

Claims (1)

1. A kind of 1. method for improving swine fever virus infection titre, which is characterized in that thin with cell growth medium culture swine fever virus ST Born of the same parents by the use of the fusion protein that genetic engineering bacterium is expressed as Virus culture accelerating agent, add in cell maintenance medium, are inoculated with hog cholera Poison improves the virus titer of cell culture swine fever virus；

   The fusion protein is CCTCC NO by deposit number：The bacterial strain of M2014631 expresses to obtain.