CN102816240B - A fusion protein and its fusion protein expression vector - Google Patents
A fusion protein and its fusion protein expression vector Download PDFInfo
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- CN102816240B CN102816240B CN201110054917.6A CN201110054917A CN102816240B CN 102816240 B CN102816240 B CN 102816240B CN 201110054917 A CN201110054917 A CN 201110054917A CN 102816240 B CN102816240 B CN 102816240B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明提供一种融合蛋白及其融合蛋白表达载体。该融合蛋白包含人源性柯萨奇病毒-腺病毒受体胞外区与人IgG1的Fc段。该融合蛋白基因序列经密码子优化。该融合蛋白表达载体包含上述的融合蛋白优化后的基因序列和pPIC3.5K质粒基因序列。该融合蛋白能与柯萨奇病毒/腺病毒产生高亲和力的结合，阻止柯萨奇病毒/腺病毒对机体细胞特别是表达CAR的细胞如心肌细胞的感染，本融合蛋白可用于柯萨奇病毒和/或腺病毒感染性疾病的治疗。
The invention provides a fusion protein and a fusion protein expression vector thereof. The fusion protein comprises the extracellular region of human Coxsackie virus-adenovirus receptor and the Fc segment of human IgG1. The fusion protein gene sequence is codon optimized. The fusion protein expression vector comprises the above-mentioned optimized gene sequence of the fusion protein and the pPIC3.5K plasmid gene sequence. The fusion protein can bind with coxsackie virus/adenovirus with high affinity, and prevent coxsackie virus/adenovirus from infecting body cells, especially CAR-expressing cells such as cardiomyocytes. This fusion protein can be used for coxsackie virus And/or the treatment of adenovirus infectious diseases.
Description
Technical field
The present invention relates to a kind of fusion rotein and fusion protein expression vector, relate in particular to the fusion rotein of the Fc section that comprises humanized's Coxsackie-Adenoviral Receptor extracellular region and human IgG1.
Background technology
Coxsackie virus (Coxsackie enterovirus), belongs to Picornaviridae (Picornavirus), is positive chain RNA enterovirus (Enterovirus).Often cause infantile diarrhea, can cause the various diseases such as aseptic meningitis, epidemic pleurodynia, myocarditis, pericarditis, hand foot mouth disease, the Gestation period infects and can cause that non-spinal paralytic poliomyelitis venereal disease becomes, and causes fetal in utero infection and teratogenesis.Find at present, change of coxsackie b virus (Coxsackie B enterovirus, CVB) often causes infantile viral myocarditis (Viral Myocarditis, VMC), be children's and the modal pathogenic agent of teenager VMC, account for due to various virus myocarditic 50%.Adenovirus (adenovirus, Ad) be a class without coating double-stranded DNA (double-stranded DNA, dsDNA) virus, belong to mastadenovirus.Distributed more widely at occurring in nature, be the common disease substance that causes upper lower respiratory tract and eye part disease; Can cause human body tonsilla, gland sample body and other adenoid recessive persistent infection simultaneously; In addition, adenovirus is also the common disease substance of viral myocarditis.COxsackie is sick, the infectivity of adenovirus is strong, especially in densely populated place, to the patient of hypoimmunity (as the elderly, Patients Following Bone Marrowtransplantation, leukemia, AIDS patient etc.), by the spittle and contagion, in the same period can cause regional popular or outbreak of epidemic.Coxsackie virus is divided into A and B two classes, more than totally 30 serotype; Adenovirus can be divided into A～F6 subgenus, and totally 51 serotypes, there is no Coxsackie virus/adenovirus effective vaccine at present for prevention, for its specific drugs, need exploitation.
Therefore, very necessary from the safe and reliable newtype drug of preventing and treating Coxsackie virus/adenovirus of new angle development.
In recent years, some scholars have proposed this concept of " viral receptor trap therapy " (virus receptor trap therapy), so-called " viral receptor trap therapy " refers to and utilizes ectogenic soluble viral acceptor to come in conjunction with virus, thus the receptors bind on blocking virus and target cell membrane and viral endocytosis subsequently.Because exogenous acceptor molecule has identical or closely similar space structure with the acceptor molecule on cytolemma, therefore, repeatedly, heavy dose of use will not exist the problems such as immune response of body.Viral receptor trap therapy has been expanded the therapeutic strategy of virus disease, can become the new effective treatment means of acute disease toxicaemia phase.
Research shows, Coxsackie virus and adenovirus are by (the Coxsackie virus and adenovirus receptor of the Coxsackie virus/adenovirus receptor with cell surface, CAR) in conjunction with completing, infect initial adsorption process specifically, subsequently by the essence-Gan-aspartic acid on penton protein (RGD) thus the integrin alpha γ β 3 of sequence and cell surface and α γ β 5 make cell entry cell complete internalization process in conjunction with passing through pinosomes.Therefore, CAR is the key that most Coxsackie viruss and adenovirus enter host cell.
At present, the basic research of CAR is very deep.Research shows, CAR is that molecular weight is the transmembrane glycoprotein of 46KD, and the gene of encoding human source CAR (hCAR) expliciting the position is upper in karyomit(e) 21q11.2, comprises 7 exons, 365 amino acid, minute 3 structural domains.1. extracellular domain: containing two Ig structural domains (1.2), the tripolymer structure that Ad heads a ball by fiber is combined with Ig1 structural domain.2. cross-film section: containing 22 amino acid, cell-cell interaction is played to stable regulation effect.3. born of the same parents' internal area: containing 107 amino acid, wherein front two Cys residue may be esterified decorating site after translation, and in addition, born of the same parents' internal area may also exist Tyr phosphorylation site.
Based on these existing basic researchs, if pass through gene engineering method, obtain extracellular region protein (the extracellular region of CAR of CAR, exCAR), " being subject to bulk trap therapy " for Ad, can effectively stop the somatic infection of Adenovirus on Human of most types.
At present, more existing exploratory studys in " virus disease be subject to bulk trap therapy " this field, but only rest on preliminary stage of In vitro cell experiment and Research of Animal Model for Study.Trace it to its cause, most importantly the efficient expression problem of activated protein.Current correlative study one is to adopt escherichia coli prokaryotic expression system, and this system can obtain the expressing protein of high yield, but its protein expression form mostly is inclusion body, is difficult to obtain great amount of soluble activated protein; The 2nd, adopt cell eukaryotic expression system, as Chinese hamster ovary celI and 293T cell etc., this type of expression system can solve the correct folding and glycosylation problem of expressing protein, but its output is very low, and cost is high, and a large amount of preparations are difficulty very.
So " viral receptor trap therapy " applies human body therapy in the future, the key of realization is can a large amount of purifiable virus receptor Huo Qi subunits with high biological activity of manufacture.
Summary of the invention
The object of the present invention is to provide the expression vector of a kind of fusion rotein and fusion rotein.
Fusion rotein of the present invention, the Fc section that comprises humanized's Coxsackie-Adenoviral Receptor extracellular region and human IgG1.
Its sequence of gene order of described humanized's Coxsackie-Adenoviral Receptor extracellular region is as shown in SEQ IDNO:1.
Its sequence of gene order of described human IgG1's Fc section is as shown in SEQ ID NO:2.
The gene order of described fusion rotein is as shown in SEQ ID NO:3, and its aminoacid sequence is as shown in SEQ IDNO:4.
Fusion rotein of the present invention can be used for the medicine of preparation treatment Coxsackie virus and/or adenovirus.
Fusion protein expression vector provided by the invention, comprises above-mentioned antigen-4 fusion protein gene sequence and pPIC3.5K plasmid gene sequence.Wherein said antigen-4 fusion protein gene sequence is as shown in SEQ ID NO:2.
The present invention also provides a kind of Host Strains, and it contains above-mentioned fusion protein expression vector.
The present invention by humanized's Coxsackie-Adenoviral Receptor extracellular region (
extracellular
coxsackie virus and
adenovirus receptor, exCAR) gene coded sequence and human IgG1's Fc fragment gene encoding sequence has carried out codon optimized according to the inclined to one side preferendum of pichia spp genetic code, frame is formed by connecting as fusion rotein exCAR:Fc encoding gene, this fusion rotein had both had the high-bond with Coxsackie virus/adenovirus, can utilize again antibody Fc end and cytophagous Fc receptors bind, accelerate engulfing and elimination efficiency in conjunction with virus.Utilize pichia spp pPIC3.5K/GS115 expression system, complete the solution expression with high efficiency of fusion rotein exCAR:Fc, obtain a large amount of purifiable and there is the solubility exCAR:Fc albumen of high biological activity.
Pichia yeast expression system is a kind of eukaryotic expression system of maturation, has the expression that strong promoter can strictly regulate and control target protein; And processing and modification after can translating the albumen of expressing, thereby make the albumen giving expression to there is biological activity.In addition, its nutritional requirement is low, and growth is fast, and substratum is cheap, is convenient to suitability for industrialized production and high density fermentation and cultivates.The present invention has selected pichia spp pPIC3.5K/GS115 expression system to express target protein, to realize target protein, has and the solubility of the similar biologic activity of native protein, high efficient expression.
Described fusion rotein can produce with Coxsackie virus/adenovirus the combination of high-affinity, stop Coxsackie virus/adenovirus particularly to express the cell of CAR as myocardial cell's infection to body cell, this fusion rotein can be used for the treatment of Coxsackie virus and/or adenovirus infection disease.
The present invention is configured to fusion rotein by the Fc section of the extracellular region of humanized's Coxsackie virus/adenovirus receptor and IgG1, fusion rotein is had and viral high-bond, again can be effectively with phagocytic cell film on antibody Fc receptors bind, mediation phagocytic cell kills and wounds engulfing of virus, can accelerate virus elimination efficiency in vivo.
For above and other objects of the present invention, feature and advantage can be become apparent, preferred embodiment cited below particularly, and coordinate accompanying drawing, be described in detail below.
Accompanying drawing explanation
Fig. 1 is the PCR qualification result of recombinant bacterial strain pPIC3.5K-exCAR/DH5 α, wherein: M swimming lane: DNA marker; 1,2 swimming lanes: exCAR PCR negative control; 3,4,5 swimming lanes: exCAR PCR qualification result;
Fig. 2 is recombinant plasmid pPIC3.5K-exCAR:Fc PCR qualification result, wherein: M swimming lane: DNAmarker; 1,3 swimming lanes: exCAR PCR result; 2 swimming lanes: exCAR PCR negative control; 4 swimming lanes: exCAR:Fc PCR negative control; 5 swimming lanes: exCAR:Fc PCR result; 6 swimming lanes: Fc PCR result; 7 swimming lanes: Fc PCR negative control;
Fig. 3 is recombinant plasmid pPIC3.5K-exCAR:Fc/SnaB I, Not I double digestion rear electrophoresis figure; Wherein: M swimming lane: DNA marker; 1 swimming lane: positive recombinant plasmid pPIC3.5K-exCAR:Fc/SnaB I, Not I double digestion, cut out about 1350bp DNA band as seen; 2 swimming lanes: pPIC3.5K/SnaB I, Not I double digestion;
Fig. 4 is recombinant plasmid pPIC3.5K-exCAR:Fc order-checking collection of illustrative plates (result part sectional drawing), marks initiation codon and SnaB I restriction enzyme site in figure;
Fig. 5 is positive transformant pPIC3.5K-exCAR:Fc/GS115PCR evaluation figure, wherein: M swimming lane: DNA marker; 1 swimming lane: pPIC3.5K/GS115 genomic dna adopts yeast positive transformant primers designed amplification; 2 swimming lanes: pPIC3.5K-exCAR:Fc/GS115 genomic dna adopts yeast positive transformant primers designed amplification; 3,6 swimming lanes: exCAR:Fc PCR negative control; 4 swimming lanes: pPIC3.5K/GS115 genomic dna adopts exCAR:Fc upstream and downstream primer amplification result; 5 swimming lanes: pPIC3.5K-exCAR:Fc/GS115 genomic dna adopts exCAR:Fc upstream and downstream primer amplification result;
Fig. 6 is the expression of exCAR:Fc albumen, wherein: M swimming lane: albumen Marker; 1,2 swimming lane: pPIC3.5K/GS115 empty carrier bacterium expressing protein after methanol induction 96h; 3,4 swimming lanes: pPIC3.5K-exCAR:Fc/GS115 is expressing protein after methanol induction 96h;
Fig. 7 is exCAR:Fc protein purification situation, wherein: M swimming lane: albumen Marker; 1,2 swimming lane: exCAR:Fc is result after ProteinA affinity chromatography and gel chromatography; 3,4 swimming lanes: exCAR:Fc is result after ProteinA affinitive layer purification;
Fig. 8 is exCAR:Fc albumen Western-blot result figure, wherein: M swimming lane: albumen Marker (170,130,95,72,55,43,34,26,17,10KDa); 1 swimming lane: pPIC3.5K/GS115 expressing protein and primary antibodie are hatched result; 2 swimming lanes: pPIC3.5K-exCAR:Fc/GS115 expressing protein and primary antibodie are hatched result;
Fig. 9 A to Fig. 9 C is respectively before exCAR:Fc albumen blocking-up, Hela cell result figure is infected in the blocking-up adenovirus adEasy-1 strain after BSA control group, blocking-up, wherein: Hela cell 48h figure is infected in Fig. 9 A:100MOI adEasy-1 strain; Fig. 9 B: Hela cell 48h is infected in final concentration 2ug/ml BSA contrast blocking-up adEasy-1 strain; Fig. 9 C: Hela cell 48h figure is infected in final concentration 2ug/ml exCAR:Fc albumen blocking-up adEasy-1 strain;
Figure 10 is exCAR:Fc N-terminal sequencing result;
Figure 11 is exCAR:Fc albumen blocking-up CoxB3 virus infection Hela cell MTT experimental result picture.
Embodiment
One, materials and methods
(1) bacterial strain, plasmid, virus strain and cell strain
Intestinal bacteria DH
5 αby this laboratory, preserved; Pichia spp (Pichia pastoris) expression plasmid pPIC3.5K, bacterial strain GS115 and pPIC3.5K/GS115 are purchased from American I nvitrogen company; Pediatric hospital molecular diagnosis chamber doctor Huang Yi of adenovirus adEasy-1 Zhu You Medical University Of Chongqing is so kind as to give; Coxsackie virus (CoxB3 virus) m strain is purchased from Wuhan virus institute of the Chinese Academy of Sciences; Hela cell strain is preserved by this laboratory.
(2) main agents
KOD plus Taq archaeal dna polymerase and PCR reagent Japan TOYOBO company
Ligation high Japan TOYOBO company
SnaB I Japan Takara company
EcoR I Japan Takara company
Not I Japan Takara company
Sal I Japan Takara company
The Marker days biological company limiteds of root of DL2000 DNA
Vast Tyke, plasmid extraction kit Beijing biotech firm
Gel reclaims vast Tyke, test kit Beijing biotech firm
EB U.S. Amresco packing
Agarose U.S. BBI
Yeast powder Britain Oxoid
Tryptones Britain Oxoid
YNB Beijing ancient cooking vessel state
Agar Chengdu Tian Tai biotech firm
SDS U.S. BBI
The raw work in DTT Shanghai
The raw work in vitamin H Shanghai
Helicase Beijing ancient cooking vessel state
D-glucitol Beijing ancient cooking vessel state
Acrylamide U.S. BBI
N, N '-methylene bisacrylamide Canada BBI
Ammonium persulphate Canada BBI
The raw work in TEMED Shanghai
Tris Canada BBI
Sephadex G-250 U.S. Amersham Pharmacia
DMEM/F12 (DF) substratum HighClone
The biological company limited of foetal calf serum folium ilicis chinensis
The biological company limited of protein molecular weight standard sky root
Staphylococcal protein A,SPA chromatography column Canada BBI company
The anti-human CAR antibody of goat U.S. Santa company
Company of HRP-rabbit Kang YangIgGZhong China fir Golden Bridge
(3) key instrument equipment
ISO 9001 electronic balance Germany Sartorius companies
Milli-Q Elix water purifior U.S. Millipore company
PB-20 pH meter Germany Sartorius company
Taicang, TH2 large amplitude constant-temperature table Jiangsu experimental installation factory
DU800 spectrophotometer U.S. Beckman company
ERC-4 type clean work station skill is thought ultra-clean company limited
PTC-100
tMpCRYi U.S. MJ Research company
Gene Pulse
tMelectricity conversion instrument U.S. Bio-Rad company
Transfer printing instrument U.S. Bio-Rad company wets
DYY-11 type electrophoresis apparatus Liuyi Instruments Plant, Beijing
MDF-382E (N) Ultralow Temperature Freezer Japan Sanyo company
Chemimager
tM5500 gel imaging instrument U.S. Bio-Rad companies
The grand experimental installation of Nereid company on PK-8B electric heating constant-temperature water-bath tank
PYX-DHS-50 * 64S water isolation type electro-heating standing-temperature cultivator Shanghai leap medical apparatus and instruments factory
D-37520 type low-temperature and high-speed whizzer Germany Heraeus company
TGL-16G type desk centrifuge Town in Shanghai is enjoyed scientific instrument factory
UltrasonicsVCX-750 ultrasonic apparatus U.S. Sonics
CO2 incubator Germany Heraeus company
Fluorescent microscope Germany Leica
(4) main solution and reagent preparation
1.LB liquid nutrient medium:
Take Tryptones 10g, yeast powder 5g, NaCl10g, adds 800ml ddH
2o dissolves, and with NaOH adjust pH to 7.4, adds ddH
2o to 1000ml, autoclaving 20min, 4 ℃ save backup.It is the AMP of 100 μ g/ml that preparation adds final concentration before use containing AMP substratum.
2.LB solid medium
On the basis of LB liquid nutrient medium, adding final concentration is the agar of 15g/L, autoclaving 20min, and pour plate, solidifies the rear packaging plastic bags of using, and 4 ℃ save backup.Preparation needs to be down to after 65 ℃ until temperature containing AMP substratum, and adding final concentration is the AMP of 100 μ g/ml, shakes up rear pour plate.
3.100mmol/L CaCl
2solution
Take 14.7g CaCl
22H
2o is dissolved in 1000ml ddH
2in O, filtration sterilization, 4 ℃ save backup.
4.TAE electrophoretic buffer (50 * stock solution, pH approximately 8.5)
Take Tris alkali 242g, Na
2eDTA2H
2o37.2g, adds 57.1ml glacial acetic acid, adds ddH
2o to 1000ml, dilutes 50 times during use.
5.1% sepharose
Take 1g agarose, add in 100ml1 * TAE damping fluid, with microwave-oven-heating, dissolve, until temperature, be down to after 65 ℃, add the EB10 μ l of 10mg/ml, mix in rear impouring mould, insert comb, solidify rear use.
6．10×D
I.e. 20% glucose (Dextrose) solution.Take 200g glucose and be dissolved in 1L ddH
2in O, Entkeimung, 4 ℃ of preservations.
7．10×M
I.e. 5% methyl alcohol (Methanol) solution.Get 5ml methyl alcohol and add 95ml ddH
2in O, Entkeimung, 4 ℃ of preservations.
8．10×GY
I.e. 10% glycerine (Glycerol) solution.Get 100ml glycerine and add to 900ml ddH
2in O, 15 pounds of autoclaving 20min, 4 ℃ of preservations.
9．10×YNB
Take 34g YNB, 100g ammonium sulfate, adds ddH
2o is molten to 1L, heating for dissolving, Entkeimung, 4 ℃ of preservations.
10．500×B
I.e. 0.02% vitamin H (Biotin) solution.Take 20mg vitamin H, be dissolved in 100ml ddH
2in O, Entkeimung, 4 ℃ of preservations.
11.1M potassium phosphate buffer (pH6.0)
Measure 132ml1M K
2hPO
4, 868ml1M KH
2pO
4, pH6.0 ± 0.1 (as pH needs to adjust, with phosphoric acid or KOH, adjusting), after autoclaving, puts 4 ℃ or room temperature preservation.
12.YPD liquid nutrient medium
Get 2.0g yeast extract, 4.0g peptone is dissolved in 180ml distilled water, and 121 ℃, 30min autoclaving, adds 20ml10 * D after cooling.
13.YPD solid medium
Get 2.0g yeast extract, 4.0g peptone is dissolved in 180ml distilled water, and adding final concentration is the agar of 20g/L, and 121 ℃, 30min autoclaving, adds 20ml10 * D after cooling.
14.BMGY liquid nutrient medium
Get peptone 20g, yeast extract 10g, adds deionized water 700ml, 121 ℃, 20min autoclaving, then adds 10 aseptic * GY, 10 * YNB, each 100ml of 10 * phosphate buffered saline buffer, is chilled to and adds 500 * Biotin2ml after room temperature and be made into BMGY substratum.
15.BMMY liquid nutrient medium
Get peptone 20g, yeast extract 10g, adds deionized water 700ml, 121 ℃, 20min autoclaving, then adds 10 aseptic * M, 10 * YNB, each 100ml of 10 * phosphate buffered saline buffer, is chilled to and adds 500 * Biotin2ml after room temperature and be made into BMGY substratum.
16.MD solid medium
In 160ml water, add 3.0g agar, autoclaving, is cooled to after 60 ℃, adds successively 20ml10 * YNB, 20ml10 * D, 0.4ml500 * Biotin, immediately bed board.
17.MM solid medium
In 160ml water, add 3.0g agar, autoclaving, is cooled to after 60 ℃, adds successively 20ml10 * YNB, 20ml10 * M, 0.4ml500 * Biotin, immediately bed board.
18. yeast competence treatment solutions
100mM LiAc, 10mM DTT, 0.6M sorbyl alcohol, and10mM Tris-HCl, pH7.5
19.G418-YPD is dull and stereotyped
Get 2.0g yeast extract, 4.0g peptone is dissolved in 180ml distilled water, adding final concentration is the agar of 20g/L, 121 ℃, 30min autoclaving, the G418 (0,0.25,0.5,1.0,1.5,2.0,4.0mg/ml) that adds 20ml10 * D and different concns after cooling, fully shakes up bed board, and 4 ℃ standby.
20. film washing liquids (PBST)
Take NaCl8.0g, KCl0.2g, KH
2pO
40.2g, Na
2hPO
412H
2o2.9g is dissolved in 800ml ddH
2in O, add ddH after 500ul Tween-20
2o is settled to 1L.
21. transferring film damping fluid (pH8.3)
Take Tris3.0g, glycine 11.3g, add ddH after methyl alcohol 200ml
2o is settled to 1L.
22.CAPS electricity turns damping fluid
Take CAP2.2g and be dissolved in 800ml ddH
2in O, add 100ml methyl alcohol, NaOH adjusts PH to 11 and is settled to 1L.
23. Ponceau S staining fluids
Take Ponceau S 0.5g, add glacial acetic acid 1ml, ddH
2o is settled to 100ml.
24. confining liquids/primary antibodie, two anti-diluents
Taking skim-milk 5g is dissolved in 100ml PBST.
25. trisodium citrate substrate buffer solutions
Take citric acid 0.51g, Na
2hPO
4.12H
2o1.84g adds ddH
2o is settled to 100ml.
26. nitrite ions
Take DAB15mg, add methyl alcohol 5ml, 30%H
20
215ul and trisodium citrate substrate buffer solution 25ml.
27.0.01mol/L PBS(pH7.4) solution allocation
NaCl8.00g, KCl0.20g, KH
2pO
40.20g, NaH
2pO
4h
2o1.56g, 1000ml ddH
2o dissolves, autoclave sterilization, 4 ℃ of preservations.
embodiment 1: gene and primer synthetic
Synthesizing of 1.1exCAR and IgG1Fc gene
According to humanized CAR extracellular region gene coded sequence (exCAR) (Accession Number:NM_001338) in GenBank and IgG1Fc fragment gene encoding sequence (Accession Number:AF237583), according to the inclined to one side preferendum of pichia spp genetic code, exCAR and IgG1Fc fragment gene sequence have been carried out simultaneously codon optimized, He Cheng exCAR and IgG1Fc gene and build engineering bacteria PUC-exCAR/DH by the raw work point in Shanghai
5 α, PUC-Fc/DH
5 α, the nucleotide sequence after codon optimized is as follows:
exCAR(651bp):SEQ?ID?NO:1
ttgtccatcactactccagaagagatgattgagaaggctaagggtgagactgcctacttgccatgtaagttcactttgtctccagaagaccaaggtccattggacatcgagtggttgatttccccagctgacaatcagaaggttgatcaagtcattattttgtactctggtgacaagatttacgacgactactacccagacttgaagggtagagttcacttcacctccaatgacttgaagtctggtgatgcttctatcaatgtcaccaatttgcaattgtctgacattggtacttaccagtgtaaggtcaagaaggctccaggtgttgctaataagaagattcacttggtcgttttggttaagccatccggtgctagatgttacgttgacggttctgaggaaattggttccgacttcaagatcaagtgtgagccaaaggaaggttccttgccattgcagtacgagtggcaaaagttgtctgactcccagaagatgccaacttcttggttggctgagatgacttcctctgttatttctgtcaagaatgcttcttctgagtactctggtacttactcctgtaccgtcagaaacagagtcggttctgaccagtgtttgttgagattgaacgttgtcccaccatccaataaggct
IgG1Fc（696bp）:SEQ?ID?NO:2
gagccaaagtcttgtgacaagactcacacctgtccaccatgtccagctccagagttgttgggtggtccatccgtcttcttgttcccaccaaagccaaaggacaccttgatgatctccagaaccccagaggtcacctgcgtcgttgtcgacgtctcccacgaggacccagaggtcaagttcaactggtacgtcgacggtgttgaggtccacaatgctaagaccaagccaagagaggagcagtacaactccacctacagagtcgtctctgtcttgaccgtcttgcaccaggactggttgaatggtaaggagtacaagtgcaaggtctccaacaaggctttgccagctccaatcgagaagaccatctccaaggctaagggtcagccaagagagccacaggtctacaccttgccaccatccagagaggagatgaccaagaaccaggtctccttgacctgcttggtcaagggtttctacccatccgacatcgctgtcgagtgggagtctaatggtcagccagagaacaactacaagaccaccccaccagtcttggactccgacggttccttcttcttgtactccaagttgaccgttgacaagtctagatggcagcagggtaacgtcttctcctgctccgtcatgcacgaggctttgcacaaccactacactcagaagtccttgtccttgtccccaggtaag
The fusion rotein exCAR-Fc nucleotide sequence of Pichia anomala expression (the EcoR I restriction enzyme site for adding in frame, atg is initiator codon, tga is termination codon): SEQ ID NO:3
atggtgtccatcactactccagaagagatgattgagaaggctaagggtgagactgcctacttgccatgtaagttcactttgtctccagaagaccaaggtccattggacatcgagtggttgatttccccagctgacaatcagaaggttgatcaagtcattattttgtactctggtgacaagatttacgacgactactacccagacttgaagggtagagttcacttcacctccaatgacttgaagtctggtgatgcttctatcaatgtcaccaatttgcaattgtctgacattggtacttaccagtgtaaggtcaagaaggctccaggtgttgctaataagaagattcacttggtcgttttggttaagccatccggtgctagatgttacgttgacggttctgaggaaattggttccgacttcaagatcaagtgtgagccaaaggaaggttccttgccattgcagtacgagtggcaaaagttgtctgactcccagaagatgccaacttcttggttggctgagatgacttcctctgttatttctgtcaagaatgcttcttctgagtactctggtacttactcctgtaccgtcagaaacagagtcggttctgaccagtgtttgttgagattgaacgttgtcccaccatccaataaggct
gagccaaagtcttgtgacaagactcacacctgtccaccatgtccagctccagagttgttgggtggtccatccgtcttcttgttcccaccaaagccaaaggacaccttgatgatctccagaaccccagaggtcacctgcgtcgttgtcgacgtctcccacgaggacccagaggtcaagttcaactggtacgtcgacggtgttgaggtccacaatgctaagaccaagccaagagaggagcagtacaactccacctacagagtcgtctctgtcttgaccgtcttgcaccaggactggttgaatggtaaggagtacaagtgcaaggtctccaacaaggctttgccagctccaatcgagaagaccatctccaaggctaagggtcagccaagagagccacaggtctacaccttgccaccatccagagaggagatgaccaagaaccaggtctccttgacctgcttggtcaagggtttctacccatccgacatcgctgtcgagtgggagtctaatggtcagccagagaacaactacaagaccaccccaccagtcttggactccgacggttccttcttcttgtactccaagttgaccgttgacaagtctagatggcagcagggtaacgtcttctcctgctccgtcatgcacgaggctttgcacaaccactacactcagaagtccttgtccttgtccccaggtaagtga
The fusion rotein exCAR-Fc aminoacid sequence (451AA, M.W50.3kD, pI6.43) of Pichia anomala expression: SEQ ID NO:4
VSITTPEEMIEKAKGETAYLPCKFTLSPEDQGPLDIEWLISPADNQKVDQVIILYSGDKIYDDYYPDLKGRVHFTSNDLKSGDASINVTNLQLSDIGTYQCKVKKAPGVANKKIHLVVLVKPSGARCYVDGSEEIGSDFKIKCEPKEGSLPLQYEWQKLSDSQKMPTSWLAEMTSSVISVKNASSEYSGTYSCTVRNRVGSDQCLLRLNVVPPSNKA
EPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
1.2 primers synthesize and PCR reaction system
The amplimer of exCAR: upstream primer 5 '-tata
tacgta gtgtccatcactactccag-3 ’ – SEQID NO:5 (underscore is partly SnaB I site, is Kozak sequence in frame); Downstream primer 5 '-tata
gaattcagccttattggatggtg-3 ’ – SEQ ID NO:6 (underscore is partly EcoR I site).The amplimer of IgG1Fc: upstream primer 5 '-tata
gaattcgagccaaagtcttgtgac-3 ’ – SEQ ID NO:7 (underscore is partly EcoR I site); Downstream primer: 5 '-tatttatt
gcggccgctcacttacctggggacaagg-3 ’ – SEQ ID NO:8 (underscore is partly Not I site).Yeast positive transformant primers designed: upstream primer 5 '-gactggttccaattgacaagc-3 ’ – SEQ ID NO:9; Downstream primer 5 '-gcaaatggcattctgacatcc-3 ’ – SEQ ID NO:10.Primer is synthetic by the raw work in Shanghai.
1. exCAR or Fc gene PCR reaction system are as follows:
Thermal circulation parameters: 94 ℃ 3 minutes; (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 60 seconds) * 32 circulations; 72 ℃ 5 minutes.
2. the PCR reaction system that exCAR-Fc gene or yeast positive transformant are identified is as follows:
Thermal circulation parameters: 94 ℃ 3 minutes; (94 ℃ 30 seconds, 55 ℃ 30 seconds, 72 ℃ 90 seconds) * 32 circulations; 72 ℃ 5 minutes.
embodiment 2: recombinant bacterial strain pPIC3.5K-exCAR/DH
5 αstructure and evaluation
2.1exCAR gene EcoR I/SnaB I enzyme is cut
By the PUC-exCAR/DH building in embodiment 1.2
5 αbacterium liquid is inoculated in LB (AMP
r) in substratum, 37 ℃, 180rpm incubated overnight, plasmid extraction kit extracts PUC-exCAR plasmid, and makes EcoR I, SnaB I substep enzyme and cut.
1. EcoR I endonuclease reaction system is as follows:
Put 37 ℃, react and after 3 hours, in system, directly add SnaB I and corresponding damping fluid to carry out second step enzyme to cut.
2. SnaB I endonuclease reaction system is as follows:
Put 37 ℃, react 3 hours, enzyme is cut product electrophoresis in 1% sepharose, reclaims exCAR enzyme and cuts rear gene fragment.
2.2pPIC3.5K plasmid EcoR I/SnaB I enzyme is cut
Plasmid extraction kit from pPIC3.5K/GS115 bacterium, extract pPIC3.5K plasmid and make EcoR I, SnaB I distribution enzyme is cut, endonuclease reaction system and step are carried out with reference to embodiment 2.1, enzyme is cut product electrophoresis in 1% sepharose, reclaims enzyme and cuts rear plasmid pPIC3.5K.
2.3 ligation
Enzyme in embodiment 2.1 and 2.2 is cut to product and by system below, connects reaction:
Connection product is put 16 ℃ of reactions and is spent the night.
The competent preparation of 2.4 bacillus coli DH 5 alpha
With reference to molecular cloning experiment guide (third edition, the 2002[of Science Press is beautiful], J. Pehanorm Brooker, D.W Russell work, Huang Peitang etc. translate), carry out: bacillus coli DH 5 alpha is inoculated in to the LB liquid nutrient medium of 2ml, 37 ℃, 160rpm jolting overnight incubation.Next day, 37 ℃, 240rpm was cultured to the early stage (OD of logarithmic growth in 1% ratio transferred species in 200ml LB liquid nutrient medium
600=0.3～0.5).Take out shaking flask, centrifugal 10 minutes of 4 ℃ of horizontal centrifuge 5000rpm, abandon supernatant, add the 100mmol/LCaCl of 20ml ice precooling in precipitation
2the resuspended bacterium of solution, ice bath is recentrifuge after 30 minutes, then uses the 100mmol/LCaCl of 20ml ice precooling
2the resuspended bacterium of solution, and add glycerine in 15% ratio, by the intestinal bacteria DH of preparation
5 αcompetent cell is distributed into 100 μ l, in-70 ℃, saves backup.
2.5 connect product transforms DH
5 αcompetence
Get 12 μ l connection products and join 100 μ l DH
5 αin competent cell, ice bath 30 minutes; Put 42 ℃ of heat-shockeds 90 seconds, after taking out, ice bath is 2 minutes; Added 900 μ l not containing in the LB liquid nutrient medium of AMP, 37 ℃, 180rpm jolting is cultivated 1 hour, gets 200 μ l converted products and coats LB (AMP
r) flat board, 37 ℃ of night incubation, the clone on picking flat board identifies.
2.6 recombinant bacterial strain pPIC3.5K-exCAR/DH
5 αpCR identify
Picking LB (AMP
r) bacterium colony that grows on flat board is in LB (AMP
r) substratum in, 37 ℃, 180rpm jolting overnight incubation, get 1 μ l bacterium liquid and carry out PCR evaluation, PCR primer and reaction system are carried out with reference to the exCAR gene condition that increases in embodiment 1.2.
Result: recombinant bacterial strain pPIC3.5K-exCAR/DH5 α identifies through PCR, has amplified the object band (exCAR is 651bp) conforming to expection size, the results are shown in Figure 1.
embodiment 3: recombinant bacterial strain pPIC3.5K-exCAR:Fc/DH
5 αstructure
3.1pPIC3.5K-exCAR plasmid EcoR/Not I double digestion
Adopt plasmid extraction kit to extract PCR in embodiment 2.6 and be accredited as positive recombined engineering bacteria plasmid pPIC3.5K-exCAR, and make EcoR I/Not I double digestion.
It is as follows that enzyme is cut system:
Put 37 ℃, react 3 hours.Enzyme is cut product electrophoresis in 1% sepharose, reclaims enzyme and cuts rear plasmid pPIC3.5K-exCAR.
3.2Fc gene Not I/EcoR I double digestion
By the PUC-Fc/DH building in embodiment 1.2
5 αbacterium liquid is inoculated in LB (AMP
r) in substratum, 37 ℃, 180rpm incubated overnight, plasmid extraction kit extracts PUC-Fc plasmid, and makes Not I/EcoR I double digestion, and reaction system is as follows:
Put 37 ℃, react 3 hours.Enzyme is cut product electrophoresis in 1% sepharose, reclaims enzyme and cuts rear IgG1Fc gene fragment.
3.3 ligation
Embodiment 3.1 is cut to product with enzyme in 3.2 to be connected by embodiment 2.3 ligation conditions.
3.4 connect product transforms DH
5 αcompetence
Method for transformation carries out with reference to embodiment 2.5, and picking LB (AMP after 37 ℃ of night incubation
r) clone on flat board identifies.
embodiment 4: the evaluation of recombinant plasmid pPIC3.5K-exCAR:Fc
The PCR of 4.1 recombinant plasmids identifies
Picking LB (AMP
r) bacterium colony that grows on flat board is in LB (AMP
r) substratum in, 37 ℃ of 180rpm jolting overnight incubation, plasmid extraction kit extracts recombinant plasmid Parallel PC R and identifies, PCR primer and reaction system are undertaken by increase in embodiment 1.2 exCAR, Fc and exCAR:Fc gene condition.
The double digestion of 4.2 recombinant plasmids is identified
To through PCR, be accredited as the capable SnaB I of positive recombinant plasmid, the evaluation of Not I double digestion, reaction system is as follows:
Put 37 ℃, react 3 hours, enzyme is cut the capable 1% agarose gel electrophoresis analysis of product.
The order-checking of 4.3 positive recombinant plasmids is identified
Positive recombinant plasmid is served to Hai Shenggong order-checking.According to sequential analysis recombinant plasmid, have or not reading frame shift and amino acid mutation.
Result: recombinant plasmid pPIC3.5K-exCAR:Fc identifies and amplified the object band (exCAR:Fc is 1359bp) conforming to expection size through PCR, the results are shown in Figure 2; Adopt SnaB I/Not I double digestion recombinant plasmid pPIC3.5K-exCAR:Fc(and establish pPIC3.5K/SnaB I, Not I double digestion compares), cut out the Insert Fragment (exCAR:Fc is that 1359bp and carrier segments pPIC3.5K size are 9.0kb) conforming to expection size, enzyme is cut and be the results are shown in Figure 3.Further recombinant plasmid is carried out to nucleotide sequencing evaluation, result shows, the nucleotide sequence of goal gene exCAR:Fc and SEQ ID NO:3 meet completely.Sequencing result is shown in Fig. 4.
embodiment 5: recombinant plasmid pPIC3.5K-exCAR:Fc transforms Pichia pastoris GS115 competence
The Sal I linearizing of 5.1 recombinant plasmid pPIC3.5K-exCAR:Fc
Recombinant plasmid need be after linearizing can change or the mode of double exchange is incorporated in the genome of Pichia yeast with single cross, according to Invitrogen company operational manual, with Sal I, the plasmid of restructuring is carried out to single endonuclease digestion, and reaction system is as follows:
Reaction conditions: 37 ℃, spend the night.
Enzyme is cut capable 1% agarose gel electrophoresis of product, and glue reclaims test kit and reclaims through the linearizing pPIC3.5K-exCAR:Fc plasmid of Sal I, and-20 ℃ save backup.
The preparation of 5.2 yeast GS115 competent cells
The reference literature of preparing of GS115 competent cell carries out (S.X.Wu, G.J.Letchworth, High efficiency transformation by electroporation of pichia pastoris pretreated with lithium acetate and dithiothreitol, Bio Techiques.2004, (36): 152-154.): the single bacterium colony of picking from the YPD flat board of Pichia pastoris GS115, be inoculated in 5ml YPD substratum, 225r/min, 28 ℃ of shaking culture 8 hours.Get 500 μ l bacterium liquid and be inoculated in the culturing bottle containing 150ml YPD substratum, 225r/min, 28 ℃ of shaking culture are spent the night, and treat its OD
600reach at 2.0 o'clock, 4 ℃, 2000g collects thalline for centrifugal 5 minutes.Thalline is resuspended in room temperature in yeast competence treatment solution and places 30 minutes, and 4 ℃, 1500g, centrifugal 5 minutes, abandon supernatant thalline and wash after three times with the iceberg pears alcohol of 1M, 4 ℃, 1500g, centrifugal 5 minutes collecting precipitations, and make its cell concn be about 10 by the 1M sorbyl alcohol re-suspended cell precipitation of precooling
10individual/mL, the 80 μ l packing-80 ℃ preservation of every pipe is stand-by.
5.3 linearization plasmids transform GS115
Linearization plasmid transforms GS115 to be undertaken by Invitrogen company operational manual: get the above-mentioned GS115 competent cell of 80 μ l and mix through the linearizing pPIC3.5K-exCAR:Fc plasmid of Sal I with 5～10 μ g; move into 0.1cm electricity and transform cup; ice bath carries out electricity and transforms after 5 minutes; electricity turns parameter: 1500V; 200 Ω, 25 μ F.After electric shock, add immediately the 1M sorbyl alcohol that 1ml is ice-cold, the bacterium liquid of getting after 400 μ l transform is coated containing the MD flat board of Histidine, in 28 ℃ of incubators, cultivates 3～4 days, observes the growing state of transformant.
embodiment 6: the PCR of yeast transformant identifies
The bacterium colony growing on MD flat board in picking embodiment 5.3, extracts genomic dna and is PCR and identifies, method is: picking colony in 5ml YPD nutrient solution, 28 ℃ of constant temperature 225rpm jolting 12h; Respectively get bacterium liquid 100ul centrifugal collecting cell and add 50ul ddH
2o re-suspended cell precipitation, in liquid nitrogen and boiling water, multigelation boils 5 times, and the centrifugal 5min of 10000rpm gets supernatant and carries out PCR evaluation, and the condition that reaction system and primer are identified with reference to increase in embodiment 1.2 exCAR:Fc and yeast positive transformant is carried out.
Result: adopt upstream and downstream primer and the yeast positive transformant primers designed of exCAR:Fc simultaneously to transform bacterial strain screening, screened altogether approximately 200 recombinant bacteriums, and the two pairs of primers all can transform and bacterial strain, amplify and expect that the gene fragment that size conforms to (adopts SEQ ID NO:5 and SEQ ID NO:8 can amplify the exCAR:Fc fragment of 1359bp from pPIC3.5K-exCAR:Fc/GS115; Adopt SEQ IDNO:9 and SEQ ID NO:10 can amplify the yeast genes fragment of 220bp and the exCAR:Fc fragment of 1359bp, the i.e. gene fragment of 1579bp length), its positive transformant PCR qualification result is shown in Fig. 5.
embodiment 7: screening and the phenotypic evaluation of high copy transformant
7.1G418 the high copy of screening transformant
To be accredited as positive transformant through embodiment 6PCR, dibbling is (each transformant is established a multiple hole) in containing 96 porocyte culture plates of 200ulYPD substratum, in 28 ℃ cultivate 2 days after every hole get 10ul bacterium liquid in another containing in the Tissue Culture Plate of 190ulYPD substratum, mix and get 10ul bacterium liquid and enter the 3rd containing in the Tissue Culture Plate of 190ulYPD substratum after 28 ℃ are cultivated 2 days, cultivate each hole after 1 day for 28 ℃ and get respectively bacterium liquid 1ul dibbling in (G418 content is 0 containing on the G418-YPD flat board of different concns, 0.25, 0.5, 1.0, 1.5, 2.0, 4.0mg/ml), cultivate 3～5 days for 28 ℃, (concentration that it is generally acknowledged bacterium colony tolerance G418 is higher to observe colony growth situation, contained copy number is just higher, a copy can tolerate 0.25mg/ml G418, the like).
The phenotypic evaluation of 7.2 high copy transformants
By the height copy transformant and the Mut that filter out through G418
+, Mut
sin comparison with jolting in 28 ℃ of YPD substratum, cultivate after 12h, dibbling is on MM and MD flat board.28 ℃ cultivate 3～5 days and observe transformant and contrast between speed of growth difference: if grow on MD flat board comparatively fast, on MM flat board, poor growth or the transformant of not growing are Mut
s; The transformant that the speed of growth is suitable on MD and MM flat board is Mut
+.
Result: be accredited as approximately 200 positive recombinant bacteriums through PCR after the choosing of G418 multiple copied number sieve, finally obtained the recombination microzyme that 2 strains can tolerate 2.0mg/ml G418, called after pPIC3.5K-exCAR:Fc-1/GS115, pPIC3.5K-exCAR:Fc-2/GS115; This 2 strain transformant has the goal gene copy number of 8 series connection.
2 strain transformant dibbling MM and the MD flat board through G418-YPD flat screen, selected carry out phenotypic evaluation discovery, in 28 ℃, cultivate 3～5 days transformants and Mut
+, Mut
ssuitable to impinging upon on MD and MM flat board the speed of growth, illustrate that 2 obtained strain transformant phenotypes are Mut
+.
embodiment 8: the shaking flask abduction delivering of high copy positive transformant
The 2 strain Mut that picking obtains through embodiment 7 respectively
+high copy transformant is inoculated in 2ml YPD substratum, and 28 ℃ of shaking culture 24 hours, get this seed liquor of 1ml and be inoculated in 100ml BMGY substratum, and 28 ℃ of shaking culture 24 hours, to OD
600reach 2.0～6.0 rear standing and discard substratum, collecting cell precipitation, and with equal-volume (100ml) BMMY re-suspended cell precipitation, 28 ℃ of shaking culture abduction deliverings are about 5 days.In Induction Process, every 24h supplements a methyl alcohol to final concentration 1%.Collecting thalline every day detects for target protein expression.
Result: 2 strain Mut
+after BMGY cultivation increasing bacterium, BMMY cultivation and methanol induction are expressed, in pPIC3.5K-exCAR:Fc-2/GS115 strain abduction delivering thing, there is the protein band identical with target protein molecular weight (exCAR:Fc molecular weight of albumen is 50.3kDa) in high copy transformant pPIC3.5K-exCAR:Fc-1/GS115, pPIC3.5K-exCAR:Fc-2/GS115.Protein expression the results are shown in Figure 6.
embodiment 9:the detection of expressing protein exCAR:Fc and purifying
The SDS-PAGE of 9.1 expressing proteins analyzes
Recombination microzyme pPIC3.5K-exCAR:Fc-2/GS115 sampling through embodiment 8 abduction deliverings is carried out to SDS-PAGE analysis, electrophoresis method is with reference to the molecular cloning experiment guide (third edition, the 2002[of Science Press is beautiful] J. Pehanorm Brooker, D.W Russell work, Huang Peitang etc. translate) carry out, lamination is 5% compared with concentration, and resolving gel concentration is that 10%, 100V constant voltage is run approximately 2 hours.
The purifying of 9.2 expressing proteins
Recombination microzyme pPIC3.5K-exCAR:Fc-2/GS115 after embodiment 8 abduction deliverings, in 4 ℃, 4500rpm, is collected to thalline for centrifugal 5 minutes, and with damping fluid (20mM Na
2hPO
4, 0.15mM NaCl, pH8.0) and resuspended thalline is placed in ultrasonicly on ice, and ultrasound condition is: super 5s, stop 10s, ultrasonic 10 minutes.Bacterium supernatant is broken in the centrifugal collection of ultrasonic rear 4500rpm, and azaleine dyes and observes brokenly bacterium rate; Broken bacterium supernatant adopts Protein A affinity chromatography preliminary purification, and method by specification carries out.Thick pure protein is in 4 ℃ of 0.1M PBS(pH7.4) in dialyse 48 hours after, ultrafiltration and concentration to concentration is about 1mg/mL and adopts Sephadex G-250 to carry out gel chromatography to be further purified again.Purifying protein is-80 ℃ of preservations after ultrafiltration and concentration, filtration sterilization.
Result: recombinant bacterium pPIC3.5K-exCAR:Fc-2/GS115 is greater than 95% through its broken bacterium rate of carrying out ultrasonic bacteria breaking; Broken bacterium supernatant is after ProteinA affinity chromatography and gel chromatography, and purity of protein can reach 96%.Purification result is shown in Fig. 7.
embodiment 10: the Western-blot of expressing protein identifies
10.1 electrophoresis: the SDS-PAGE electrophoresis that carries out expressing protein by embodiment 9.1.
The transferring film of 10.2 albumen: adopt wet transfer printing transferring film, method is: by put into electricity containing gel, NC film and the filter paper of expressing protein swimming lane, turn damping fluid and soak after 10～20 minutes, by negative pole-bed course-filter paper-gel-NC film-filter paper-bed course-positive pole order, transferring film clamping plate (place each layer and all roll flat raft bubble removing with glass stick) are installed, 100V constant voltage, turns 1.5 hours in 4 ℃ of electricity.
10.3 transferring film effects check: electricity is turned to rear NC film and put into Ponceau S dye liquor and dye 5 minutes, decolour visible to protein band in water, continue to rinse to decolouring completely.
10.4 sealings: the successful NC film of dyed evaluation albumen transferring film is put into the PBST of 5% skim-milk, 37 ℃ of jolting sealing 1h, PBST jolting is washed film three times, each 10 minutes.
10.5 hatch primary antibodie: add goat-anti people CAR antibody, 1h is hatched in 37 ℃ of joltings, and PBST jolting is washed film three times, each 10 minutes.
10.6 hatch two resists: add the anti-sheep IgG of HRP-rabbit, 1h is hatched in 37 ℃ of joltings, and PBST jolting is washed film three times, each 10 minutes.
10.7 colour developings: have or not specific proteins band on the definite film of DAB colour developing, water rinses color development stopping reaction.
10.8 put 37 ℃ by film dries, and with clean filter paper, folds up preservation.
Result: identify through Western-blot, expressing protein can be special with humanized CAR antibodies, and consider the characteristic that it can be combined with staphylococcal protein A,SPA, and the molecular weight of expressing protein molecular weight and fusion rotein is in full accord, can clearly assert that expressing protein is fusion rotein exCAR:Fc.The Western-blot of expressing protein the results are shown in Figure 8.
embodiment 11: purifying protein N holds mensuration
11.1 purifying protein electrophoresis: the SDS-PAGE electrophoresis that carries out purifying protein with reference to embodiment 9.1.
The transferring film of 11.2 albumen: adopt wet transfer printing transferring film, method is: pvdf membrane is put into 100% methyl alcohol and soak after 15s, by put into CAPS electricity containing gel, pvdf membrane and the filter paper of purifying protein swimming lane, turn damping fluid and soak after 20 minutes minutes, by negative pole-bed course-filter paper-gel-pvdf membrane-filter paper-bed course-positive pole order, transferring film clamping plate (rolling flat raft bubble removing with glass stick after every placement one deck) are installed, 100V constant voltage, 4 ℃ of electricity turn 1.5 hours.
The dyeing of 11.3PVDF film and decolouring: the PVDF after electricity is turned puts into ultrapure water and cleans 2 times, in Ponceau S staining fluid, dyes after 30～50s, decolours clear to background with 50% methyl alcohol, and deionized water wash also dries.
11.4 N-terminal sequencings: encapsulate and send the order-checking of Shanghai Inst. of Life Science, CAS proteomics research analytic centre by the pvdf membrane after handling well.
Result: identify through N-terminal sequencing, 15 amino acids residues of expressing protein N end and the gal4 amino acid residue (SEQ ID NO:4) of derivation are in full accord, proof expressing protein is really that target protein exCAR:Fc and its N end are not degraded, and purifying protein N end sequencing result is shown in Fig. 9 A to Fig. 9 C.
embodiment 12: purifying is expressed the preresearch estimates of protein yield
Determination of protein concentration adopts Bradford method, and concrete steps are measured protein concn test kit specification sheets by the biological Bradford of the company limited method of sky root and carried out.It is estimated that the in the situation that of top fermentation tank not, the recombinant protein output of shake-flask culture induction is about 35mg/L.
embodiment 13: expressing protein blocking-up adenovirus infection Hela cytological effect
13.1 cell is prepared
The frozen Hela cell strain of recovering, uses perfect medium DMEM/F12(DF)+10%FBS is in 37 ℃, 5%CO
2in cell culture incubator, cultivate, when Growth of Cells to 80%, take perfect medium adjusting cell density after cell under 0.25% trysinization as 1 * 10
5/ ml, is inoculated in 24 well culture plates, 1ml/ hole; Put 37 ℃, 5%CO
2in incubator, cultivate and treat that cell grows to 70～80%.
13.2 purifying protein exCAR:Fc blocking-up adenovirus infection Hela cytological effect experiments
Hela cell is hatched and acted on to the fusion rotein exCAR:Fc of adenovirus and different concns altogether, by observation of cell fluorescence intensity, carry out the effect (started to occur fluorescence after general 24 hours, fluorescence intensity is judged with green fluorescent protein (EGFP) the positive expression rate of cell) of Preliminary detection fusion rotein blocking-up adenovirus infection.
13.2.1 experiment grouping: grow to 70～80% Hela cell and divide into groups cultivating on 24 orifice plates:
1. control group: be the Hela cell of cultivating under normal condition.
2. adenovirus infection group: the every hole of Hela cell adds 6 μ l containing the adEasy-1 adenovirus of 100MOI, 37 ℃, 5%CO
2hatch 1h, abandon supernatant, add the DF nutrient solution 1ml/ hole containing 5%FBS; 37 ℃, 5%CO
2cultivate.
3. BSA control group: the every hole of Hela cell adds respectively the DF nutrient solution containing BSA, makes its final concentration be respectively 0.01ug/ml, 0.1ug/ml, 0.5ug/ml, 1ug/ml, 2ug/ml, 4ug/ml, 8ug/ml, 16ug/ml, then each hole adds 6 μ l containing the adEasy-1 adenovirus of 100MOI, 37 ℃, 5%CO
2hatch 1 hour, abandon supernatant, add the DF nutrient solution 1ml/ hole containing 5%FBS, 37 ℃, 5%CO
2cultivate, it is parallel that every concentration gradient is done 2 holes.
4. fusion rotein exCAR:Fc blocking-up group: the every hole of Hela cell adds the DF nutrient solution containing exCAR:Fc albumen, make its final concentration gradient be respectively 0.01ug/ml, 0.1ug/ml, 0.5ug/ml, 1ug/ml, 2ug/ml, 4ug/ml, 8ug/ml, 16ug/ml, then each hole adds 6 μ l containing the adEasy-1 adenovirus of 100MOI, 37 ℃, 5%CO
2hatch 1h, abandon supernatant, add the DF nutrient solution 1ml/ hole containing 5%FBS, 37 ℃, 5%CO
2cultivate, it is parallel that every concentration gradient is done 2 holes.
13.2.2 the selection of phase point during best fluorescence observation: take 12h, 24h after virus infection, 48h, 72h as time phase point observation of cell fluorescence intensity, and while selecting wherein best observation phase point decision fusion albumen to viral barrier effect.
Result: adopt the experiment of purifying protein exCAR:Fc blocking-up adEasy-1 adenovirus infection Hela cell, discovery is when selected best Fluirescence observation during phase point 48h, selected protein concentration is at 2～16ug/ml, compare cell fluorescence intensity (being EGFP expression level) obviously reduces with BSA control group, show that this fusion rotein exCAR:Fc can obviously block the infection of adenovirus to Hela cell, has very high biological activity.Refer to Figure 10.
embodiment 14: expressing protein blocking-up Coxsackie virus infection Hela cytological effect
The preparation step of 14.1 cells is with embodiment 13.1.
14.2 experiment groupings: the Hela cell of cultivating on 24 orifice plates is divided into groups:
1. normal cell control group: by through cultivate grow to hole at the bottom of 70～80% Hela cell abandon substratum, every hole adds 200 μ l DF substratum, 37 ℃, 5%CO
2after hatching 2h, add 200 μ l containing the DF substratum of 2%FBS, 37 ℃, 5%CO
2hatch.
2. CoxB3 infection group and viral infection group: by through cultivate grow to hole at the bottom of 70～80% Hela cell abandon substratum, every hole adds 200 μ l, and containing 0.2 μ l CoxB3 virus, (TCID50 is 10
-5/ 0.1ml) DF substratum, 37 ℃, 5%CO
2hatching after 2 hours adds 200 μ l containing the DF substratum of 2%FBS, 37 ℃, 5%CO
2hatch.
3. BSA control group: by through cultivate grow to hole at the bottom of 70～80% Hela cell abandon substratum, every hole adds respectively containing different B SA concentration (0.01ug/ml, 0.1ug/ml, 0.5ug/ml, 1ug/ml, 2ug/ml, 4ug/ml, 8ug/ml, 16ug/ml) and (TCID50 is 10 containing 0.2 μ l CoxB3 virus
-5/ 0.1ml) DF substratum 200 μ l, 37 ℃, 5%CO
2after hatching 2h, add 200 μ l containing the DF substratum of 2%FBS, 37 ℃, 5%CO
2hatch, it is parallel that every concentration gradient is done 2 holes.
4. fusion rotein exCAR:Fc blocking-up group: by through cultivate grow to hole at the bottom of 70～80% Hela cell abandon substratum, every hole adds respectively containing different e xCAR:Fc protein concentration (0.01ug/ml, 0.1ug/ml, 0.5ug/ml, 1ug/ml, 2ug/ml, 4ug/ml, 8ug/ml, 16ug/ml) and (TCID50 is 10 containing 0.2 μ l CoxB3 virus
-5/ 0.1ml) DF substratum 200 μ l, 37 ℃, 5%CO
2hatching after 2 hours adds 200 μ l containing the DF substratum of 2%FBS, 37 ℃, 5%CO
2hatch, it is parallel that every concentration gradient is done 2 holes.
14.3MTT method is surveyed cytoactive
Until CoxB3 infection group and viral infection group cell, there is approximately 80% cytopathy and normal group cell state when good, observe BSA control group and the blocking-up of exCAR:Fc albumen and organize each porocyte pathology situation, and add 37 ℃ of lucifuges of 100 μ lMTT (5mg/ml) to hatch 4h, abandoning the every hole of most supernatant adds 500 μ l DMSO vibrations to mix 10min crystallisate is fully melted, every hole is inhaled 100 μ l and is entered 96 orifice plates, and microplate reader is measured A
570absorbance value.
14.4 calculate albumen to viral inhibiting rate (IR)
Albumen is to viral inhibiting rate formula:
Result: adopt the infection of purifying protein exCAR:Fc blocking-up CoxB3 virus to Hela cell; each concentration BSA control group is to the equal unprotect effect of virus infection; and protein protection group can reach 62.3% left and right to viral inhibiting rate when protein concentration reaches 2ug/ml; show that this fusion rotein exCAR:Fc can obviously block the infection of CoxB3 virus to Hela cell, has very high biological activity.Protein concentration and corresponding inhibiting rate refer to table 1, Figure 11.
Table 1 albumen exCAR:Fc is to CoxB3 virus infection Hela inhibiting rate
This albumen can produce with Coxsackie virus/adenovirus the combination of high-affinity, stop Coxsackie virus/adenovirus particularly to express the cell of CAR as myocardial cell's infection to body cell, this fusion rotein can be used for the treatment of Coxsackie virus and/or adenovirus infection disease.
The present invention is configured to fusion rotein by the Fc section of the extracellular region of humanized's Coxsackie virus/adenovirus receptor and IgG1, fusion rotein is had and viral high-bond, again can be effectively with phagocytic cell film on Fc receptors bind promote engulfing and removing of virus.
Although the present invention discloses as above with preferred embodiment; so it is not in order to limit the present invention; any person of ordinary skill in the field; without departing from the spirit and scope of the invention; when doing a little change and improvement, so the present invention's protection domain is when being as the criterion depending on the claim person of defining.
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| CN104725515B (en) * | 2015-03-20 | 2017-11-14 | 扬州大学 | One species elastin polypeptide and Coxsackie Adenovirus Receptor fusion protein and its preparation method and application |
| EP3660044A1 (en) * | 2015-10-09 | 2020-06-03 | Miltenyi Biotec Technology, Inc. | Chimeric antigen receptors and methods of use |
| CN110357945B (en) * | 2019-06-21 | 2021-07-13 | 中国药科大学 | A tumor-targeted coxsackie virus/adenovirus mimetic peptide and its application |
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| CN1657541A (en) * | 2004-02-18 | 2005-08-24 | 雅臣药业集团（远东）有限公司 | Preparation method of specific composite IgY for resisting Coxsackie virus myocarditis and its combined preparation |
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