CN106399336B - The gene order and protein preparation method of high expression Human Glutamate Decarboxylase 65 albumen in drosophila cell - Google Patents
The gene order and protein preparation method of high expression Human Glutamate Decarboxylase 65 albumen in drosophila cell Download PDFInfo
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Abstract
The present invention provides a kind of gene order and protein preparation method of high expression Human Glutamate Decarboxylase 65 albumen, and eucaryon host is Drosophila S 2 cells.Kind Preference is used by inquiring into codon, codon optimization is carried out to the original gene sequence of Human Glutamate Decarboxylase 65, a brand-new GAD65 gene order that can improve expression of recombinant proteins efficiency is obtained, as shown in SEQ NO.1.The invention provides the drosophila cell high-expression vector of the carrying brand-new GAD65 sequences of people, the GAD65 gene orders as shown in SEQ NO.1 are included.GAD65 contains StrepII labels, by a step affinitive layer purification can obtain high-purity, high immunogenicity GAD65 albumen, substantially increase the yield of recombinant protein and reduce cost.The present invention changes traditional method for obtaining Human Glutamate Decarboxylase 65 with yeast or insect baculovirus expression system, the Drosophila S 2 cells continuous cultivating system great expression GAD65 of this high density is utilized first, and high-purity, low cost and the Human Glutamate Decarboxylase 65 albumen with good immunogenicity are quickly prepared by StrepII labels.
Description
Technical field：
The invention belongs to Human Glutamate Decarboxylase 65 albumen high efficient expression and preparing technical field, and in particular to carrying GAD65 drosophila cells
Cance high-expression gene sequence and the efficient technologies of preparing of GAD65.
Research background：
Glutamate decarboxylase (Glutamic Acid Decarboxylase, GAD65) is that a kind of important pancreas islet itself is anti-
Original, its protein product can extensively using but be not limited to islet autoantibody (glutamic acid decarboxylase antibody, GADA) detection, antigen
T lymphocyte specific detects and propagation, and autoimmune diabetes prevention and treatment.
The expression of foreign protein is often related to many factors, includes the efficiency of transcription and translation.The table in drosophila cell
Intelligent's GAD65 albumen, the difference of codon availability often reduces the expression of foreign protein between kind.The present invention passes through
Inquire into codon and use kind Preference, analyze and synthesize the optimal sequence that Human Glutamate Decarboxylase 65 albumen is expressed in insect S2 cells, most
The expression efficiency of recombinant protein is improved eventually.
By cultivating cell, the expression for obtaining recombinant protein is still one of important method of a large amount of production protein.Carefully
Bacterium, yeast, insect, mammalian cell and the correlative study of insect cell carried out at present all respectively have its limitation.
The recombinant protein expressed in bacterium lacks correct folding and posttranslational modification, the immunogenicity for often leading to recombinant protein are relatively low.
The post translational processing of Yeast expression foreign protein and mammal are different, and are not easy to carry out high density fermentation.In insect
Expressed by Baculovirus-insect System foreign protein can be used in Sf9, Sf21 and High five cells, but can quilt after cell infection
Cracking, it is impossible to which steady and continuous culture, albumen differences between batches are larger.Foreign protein cost height is expressed in mammalian cell and is expressed
Measure low.The present invention is with Drosophila S 2 cells expression Human Glutamate Decarboxylase 65 albumen, the post translational processing of albumen and unusual phase in mammal
Seemingly, and can high density continuously cultivate, substantially reduce cost and for subsequent purification technique batch between uniformity provide safeguard.
The conventional label of purification of recombinant proteins is 6His, GST label etc., but often after purification purity of protein it is not high, it is necessary to
Target protein purity is promoted to more than 90% by means such as follow-up molecular sieve, ion exchange, hydrophobic chromatographies.In the present invention
In by StrepII labels mediate affinity chromatography, can pass through a step affinity chromatography obtain restructuring GAD65 albumen (purity
More than 90% and there is good immunogenicity), substantially increase the yield of recombinant protein.
The content of the invention：
It is an object of the present invention to provide the Human Glutamate Decarboxylase 65 protein gene sequence of high expression in drosophila cell and GAD65 albumen height
The method prepared is imitated, the improved gene order can greatly improve the expression of Human Glutamate Decarboxylase 65 albumen.After High Density Cultivation
Insect cell cracking after the restructuring GAD65 albumen of purity more than 90% can be obtained by a step affinity chromatography, effectively reduce
Loss in cost and purge process.The GAD65 albumen of purifying has good immunogenicity through ELISA checkings.
The gene order of present invention Human Glutamate Decarboxylase 65 albumen of high expression in drosophila cell, as shown in SEQ NO.1.
Sequence shown in SEQ NO.2 is the sequence before the Human Glutamate Decarboxylase 65 gene order transformation of the invention as shown in SEQ NO.1
Row.
The preparation method of Human Glutamate Decarboxylase 65 albumen, the sequence association StrepII labels shown in SEQ NO.1 are cloned into expression and carried
In body, then it is transfected into drosophila cell and expresses simultaneously with r plasmid, GAD65-StrepII tag fusion proteins will be obtained.
Described expression vector is pAc5.1/V5-His A.
The affinity purification label is StrepII labels；Described r plasmid is pCoBlast resistant vectors.
The drosophila cell is S2.
Specifically include following steps：
(1) according to kind preferential expression, codon is carried out to the ORF sequences of Human Glutamate Decarboxylase 65 with OptimumGene softwares
Optimization；
(2) artificial synthesized complete sequence；
(3) primer and restriction enzyme site design：
According to the sequence of synthesis and StrepII labels design primer, and KpnI and NotI restriction enzyme sites are added, primer is as follows
It is shown：
forward primer：
5'-CGGGGTACCCAAACATGTGGTCGCATCCGCAGTTCGAGAAGGCCTCGCCAGGAAGCGG-3'；
reverse primer：5’-ATAAGAATGCGGCCGCTTATTACAGGTCCTGGCCCAGG-3’；
(4) PCR amplifications obtain purpose GAD65-StrepII fragments；
(5) double digestion is carried out to purpose fragment and carrier；
(6) purpose fragment after digestion connects with carrier in the case where T4 connects enzyme effect；
(7) recombinant conversion:Recombinant expression carrier is transfected into bacterium competent cell；
(8) GAD65-StrepII carrier is expressed in a large amount of extractings after sequence verification
(9) by GAD65-StrepII carriers and pCoBlast resistant vector cotransfection drosophila cell expressing proteins
(10) albumen that step (9) obtains by obtaining high-purity GAD65 albumen after purification
Carrier in step (5) is pAc5.1/V5-His A.
Step (6) carries out restructuring connection using ligase：Junction fragment 100ng, add 50ng pAc5.1/V5-His A
Plasmid, 5 μ l 2 × reaction buffer and 1 μ l T4 ligases, supply water to 10 μ l；1h is connected at 22 DEG C, transformed competence colibacillus is thin
Born of the same parents.
The detailed process of step (9)：
Cultured healthy S2 cells are chosen, by 3 × 106Individual cell is seeded in 60mm tissue culture dishes, culture medium
For the Sf-900 II SFM Medium for S2 cells, every ware adds 3ml culture medium, trained under 28 DEG C of aseptic conditions in constant temperature
Support and cultivated in case；
Cell transfecting is carried out using calcium phosphate transfection kit K2780-01：
1. distilled water 233 μ l, calcium chloride 240mM 36 μ l, 1 μ g/ μ l are sequentially added in sterile 1.5ml centrifuge tube
GAD65-StrepII-pAc5.1/V5-His A plasmids 19 μ l, the μ l of 0.5 μ g/ μ l pCoBlast plasmids 2；
2. being transferred to after being slowly mixed together 1min in the centrifuge tube of 2 × HEPES HBS containing 200 μ l, mix；
3. mixture is placed into 30-40min in room temperature；
4. slowly adding mixture in cell culture medium, slow rotating and culturing ware is fully to mix；
5. cultivated 1 day in 28 DEG C of constant incubator；
6. cell culture medium is carried out to change liquid：
7. culture medium is sucked in 15ml centrifuge tube together with cell；
8. after 1000rpm centrifugations 3min, supernatant is removed, and add 1ml fresh cultures into 15ml centrifuge tube；
9. mixture is rejoined in new centrifuge tube；
Cleaned again once with 2ml culture mediums；
With 2ml culture medium suspension cells, and go in culture dish and cultivate；
28 DEG C of cultures add 20 μ g/ml Blasticidin screening and clonings and establish stably transfected cell line two days later.
Cell culture after step (9) transfection is in the Sf-900 II SFM Medium containing 20 μ g/ml Blasticidin
In, it is divided into 10 cell triangle blake bottles, every bottle of 200ml medium culture；After cell culture collects cracking, then pass through a step
The targeting of StrepII label affinity purifications chromatogram obtains.
The high expression optimisation strategies of GAD65 of the present invention and experimental verification
Kind preferential expression is studied
First by OptimumGene algorithms, according to the ORFs of sequence, sequence is optimized using proprietary algorithm,
The parameter that substituted for codon for mRNA structures and can optimize etc., has screened the gene of expression cloning codon optimization.
Software according to translational selection (pressure of selection be present in translation gene during, i.e., codon preference different loci (it is synonymous,
In non-synonymous and gene regions) polymorphism and difference between relation, form the G of gene, C content, mrna length, species
TRNAs abundance, conservation of amino acids, encoding gene are in the position of genomic DNA, the context relation of codon base composition
Preferential expression sequence is built Deng 7 factors, Human Glutamate Decarboxylase 65 codon is optimized.Codon adaptation indexI after optimization
(Codon Adaptation Index, CAI) rises to 0.96 from 0.63, optimization codon frequency (Frequency of
Optimal Codons, FOP) greatly improve, G/C content is optimized to 61.93% (Fig. 1) from 48.22%.
Experimental verification
Western blotting results show, the GAD65 protein expression levels after sequence alterations are apparently higher than original sequence
Arrange (Fig. 2).Prove that codon preference optimization is remarkably improved the expression quantity of recombined human GAD65 albumen.Pass through the affine layer of a step
Analysis can obtain high-purity GAD65 albumen (Fig. 3), and albumen has good immunogenicity (Fig. 4).
Brief description of the drawings
Fig. 1 .GAD65 gene orders codon after drosophila host transformation for being greatly optimized；
(B, D, F) has before codon adaptation indexI (A), optimization codon frequency (C) and G/C content (E) relatively optimize after optimization
Greatly improve.
Fig. 2 codon optimizations can greatly improve the yield of Human Glutamate Decarboxylase 65 albumen in Drosophila S 2 cells；
The plasmid transfection Drosophila S 2 cells of sequence or original series after carrying GAD65 codon optimizations, detection cell cracking
GAD65 and actin expression in thing.
The step affinity chromatographys of Fig. 3 mono- obtain high-purity restructuring GAD65 albumen；
GAD65 albumen after A, western blotting checkings before purification；
B, coomassie brilliant blue staining detect GAD65 purity of protein after purification；
C, LC/MS mass spectrum obtain sequence to albumen n end sequencing；
D, mass spectrum obtain sequence and fitted like a glove with GAD65 protein sequences in ncbi database.
The Human Glutamate Decarboxylase 65 albumen of Fig. 4 purifying has good immunogenicity；
GAD65 or commercialization the GAD65 albumen of purifying are coated with 96 orifice plates, ELISA detection negative serums and basic, normal, high drop
Spend GADA serum.The GAD65 albumen of purifying can not only distinguish GADA feminine genders and positive serum, and can effectively distinguish GADA
Basic, normal, high titer serum.
Fig. 5 .pAc5.1/V5-His A and pCoBlast plasmid (Life Technologies) collection of illustrative plates.
Embodiment：
Detailed description below is intended to further illustrate the present invention, is not intended to limit the present invention.Do not violating structure of the present invention
Any replacement made on the basis of think of belongs to protection scope of the present invention with improving.
(1) embodiment 1：Build GAD65-StrepII label protein expression vectors
1. codon optimization is carried out to the albumen coded sequence of Human Glutamate Decarboxylase 65 with OptimumGene；
2. design primer and restriction enzyme site
According to the sequence after optimization and StrepII sequence labels design primer, and add KpnI, NotI restriction enzyme site.Draw
Thing is as follows：
P1:
5'-CGGGGTACCCAAACATGTGGTCGCATCCGCAGTTCGAGAAGGCCTCGCCAGGAAGCG G-3'
P2：5’-ATAAGAATGCGGCCGCTTATTACAGGTCCTGGCCCAGG-3’
According to original series and StrepII sequence labels design primer, and add KpnI, NotI restriction enzyme site.Primer is such as
Shown in lower：P3:
5'-CGGGGTACCCAAACATGTGGTCGCATCCGCAGTTCGAGAAGGCTAGCCCAGGCTCCGGATTTTG-
3'
P4:5’-ATAAGAATGCGGCCGCTTATTATAAATCTTGTCCAAGGC-3’
3.PCR amplifications obtain purpose fragment
The DNA of synthesis is expanded with PCR and is obtained purpose fragment, and concrete operations are as follows：
Reaction condition：94 DEG C of denaturation 5min, afterwards into 40 circulations, cycling condition is 94 DEG C of denaturation 30s, and 55 DEG C are annealed
30s, 72 DEG C of extension 2min.After 40 circulations, 72 DEG C of final extension 10min.PCR product with 1% Ago-Gel
Electrophoresis detection.
4.PCR product glue reclaims
(1) after PCR reactions, pcr template, and other unwanted products are removed using gel electrophoresis；
(2) prepare 1 × TAE of 500ml (10ml 50 times of TAE are diluted in 500ml distilled water)；
(3) 1% Ago-Gel is prepared.Continue to heat when being heated to nearly seething with excitement, after mixing, until visually seeing not
Untill the particle of floating (the high fire heating 1-2min on micro-wave oven).Carefully take out the triangle equipped with Ago-Gel
Bottle, when temperature is cooled to 50 DEG C, 3 μ l ethidium bromides are added, sealing rubber die is imported into and inserts canine tooth comb；
(4) loading dye of the volume of sample 1/6 is added in the sample in proportion；
(5) loading dye and sample blending loading, on careful point sample to Ago-Gel, 90 volts of electrophoresis is about
30min；
(6) gel imaging, the band of size position consistency is then cut out and predicted, be put into 2ml Eppendorf centrifuge tubes
In (EP pipes).During cutting purpose band is positioned using ultraviolet lamp；
(7) need to weigh immediately after gel excision, and remove the weight of empty 2ml EP pipes, and remembered label；
(8) using the glue reclaim extracts kit recovery sample band of Shanghai life work biology Co., Ltd；
(9) 600 μ l binding Buffer are added in centrifuge tube, 55 DEG C of thermostatic drying chambers is put into and is dissolved.Every
1min jiggles centrifuge tube.Blob of viscose is completely dissolved after about 10min；
(10) solution after dissolving is added to liquid-transfering gun in the screen pipe that kit provides, and places 2min；
(11) high speed refrigerated centrifuge, 10000rpm centrifugations 1min；
(12) liquid is discarded, adds wash buffer 600 μ l, high speed freezing centrifuge 10000r/min centrifugations 1min；
(13) 12 steps are repeated once；
(14) liquid, 13000rpm centrifugations 2min are discarded；
(15) filter membrane is nested into centrifuge tube, 30 μ l distilled waters is added in filter membrane center with dissolving DNA, place 2min
Afterwards, 13000rpm centrifuges 1min.Remaining liquid is the DNA fragmentation containing recovery in centrifuge tube.
5. double digestion
Double digestion reaction is carried out to PCR primer and pAc5.1/V5-His A plasmid vectors：The μ g of glue reclaim fragment 2.0 or load
The μ g of body 2.0,10 × buffer 5 μ l, each 1 μ l of two kinds of enzymes are added, supplies aseptic double-distilled water to the μ l of reaction system 50.37 DEG C of reactions 2
Hour, and using gel electrophoresis recovery endonuclease bamhi.Fragment after recovery is by sequence verification to be defined as target fragment.
6. carry out restructuring connection using ligase
Junction fragment 100ng, add 50ng pAc5.1/V5-His A plasmids, 52 × reactions of μ l buffer and 1 μ l T4
Ligase, water is supplied to 10 μ l；1h is connected at 22 DEG C, for transformed competence colibacillus cell.
7. transformation experiment
Taking-up is stored in -80 DEG C of DH5 α Escherichia coli, is thawed on ice.There is 100 μ l competence in each centrifuge tube
Cell.For each conversion reaction, the plasmid for drawing 5 μ l is incubated into the centrifuge tube equipped with competent cell, and on ice
20-25min so that DNA is adsorbed onto cell surface.Prepare 42 DEG C of water-baths.After competent cell is ready, in 42 DEG C of water-baths
Heat shock 90s.Immediately be put into ice bath, add 1ml LB culture mediums after 2min on ice, and vibrate mixing, allow cell from
Heat shock is recovered and restarts to grow.45-60min is cultivated on 37 DEG C of shaking tables, muddiness occurs in liquid.100 μ l are coated with to containing
Have on the flat board of ampicillin.The overnight incubation in 37 DEG C of constant incubators.Single bacterium colony pipette tips after overnight incubation are careful
Choose in the EP pipes containing 800 μ l LB culture mediums.Cultivate 4 hours on 37 DEG C of shaking tables, then detected whether using round pcr
Purpose fragment.Be accredited as the positive serves the raw work biology Co., Ltd sequencing in sea.
8. plasmid largely extracts
(1) 100ml bacteriums are incubated overnight；
(2) bacterium is resuspended in 10ml P1 buffer solutions.Ensure that ribonuclease A is added in buffer solution P1；
(3) add 10ml buffer solution P2, gently overturn mixing 4-6 time, should not whirlpool shake because this will cause genome
DNA fracture.If it is necessary, continue reverse EP pipes until solution become sticky with it is transparent, not allow the cracking reaction time more than 5min；
(4) 10ml buffer solution P3 are added, and gently overturn test tube immediately 4-6 times, 20 minutes on ice, compact white precipitate
It will be formed；
(5) 4 degree >=20,000g is centrifuged 30 minutes, and supernatant is transferred into QIAGEN-tip 500；
(6) 2 30ml QC solution washings；
(7) add 15ml QF solution eluted dnas, add 10.5ml isopropanol；
(8) 4 degree >=15,000g is centrifuged 30 minutes；
(9) supernatant is outwelled, adds the ethanol of 5ml 70% washing DNA, 4 degree >=15,000g centrifugation 30 minutes；
(10) supernatant is outwelled, residual solution adds distilled water dissolving DNA after air-drying.
(2) embodiment 2：The recombination expression of Human Glutamate Decarboxylase 65 and identification in insect S2 cells
1. Drosophila S 2 cells are recovered and passage
The cell line frozen is taken out from liquid nitrogen container to thaw at room temperature；With 70% ethanol and sterilize outside cell bottle
Side, transfer cell liquid are resuspended cell and simultaneously centrifuge 2-3 minutes in 1000g, remove culture into 4ml complete mediums at room temperature
DMSO. inoculating cells in base are into 5ml fresh complete mediums 28 DEG C, no CO2In incubator cultivate cell to density 6~
20x106cells/ml。
Cell density reaches 5x106During cells/ml, cell grows into tufted, and this has no effect on the growth of cell, passage
During can blow and beat open packed cell.By 1：2 to 1：5 ratio passage cells.28 DEG C of culture cells until density reach 6~
20x106cells/ml。
2. recombinant vector transfected Drosophila cells
(Life is carried out with reference to the operation scheme of DES Drosophila expression systems and calcium phosphate transfection kit (K2780-01)
Technology companies), concrete operations are as follows:
(1) cultured healthy S2 cells are chosen, by 3 × 106Cell kind in 60mm tissue culture dishes.Culture medium
For the Sf-900II SFM for S2 cells, the addition 3ml culture medium per ware.Enter under 28 DEG C of aseptic conditions in constant incubator
Row culture.
(2) distilled water 233 μ l, calcium chloride 240mM 36 μ l, 1 μ g/ μ l are sequentially added in sterile 1.5ml centrifuge tube
GAD65-StrepII-pAc5.1/V5-His A plasmids 19 μ l, the μ l of 0.5 μ g/ μ l pCoBlast plasmids 2；
(3) it is transferred in the centrifuge tube of 2 × HEPES HBS containing 200 μ l, mixes after being slowly mixed together 1min；
(4) mixture is placed into 30-40min in room temperature；
(5) slowly add mixture in cell culture medium, slow rotating and culturing ware is fully to mix；
(6) cultivated 1 day in 28 DEG C of constant incubator；
(7) cell culture medium is carried out changing liquid；
(8) 28 DEG C of cultures add 20 μ g/ml Blasticidin screening and clonings and establish stably transfected cell line two days later.
3. a small amount of expression and identification of recombinant protein
With blasticidin S screen stable expression cell strain and cultivate 3 or 4 weeks after, be collected by centrifugation cell cracking.With
The expression (Fig. 2) of GAD65 albumen before and after the son optimization of western blotting password comparisons.
(1) preparation of western blotting separation gels
1. glass plate is cleaned up with soap and water, then cleaned again once with ethanol.And assemble；
2. 6% separation gel 10ml is prepared first.Add prepare separation gel (comprising 0.008ml TEMED and
0.1ml10%APS).Separation gel is poured among two glass plates, and fluid sealant, pour into isobutanol above separation gel so that
The top of separation gel is smooth.Prepare to pour into concentration glue after sealing 10min.
(2) western blotting concentrate the preparation of glue
1. pour out the isobutanol of separation gel upper end.And wash isobutanol with water.Inner surface is dried with filter paper；
2. adding APS/TEMED in glue is concentrated, mix, be then poured on above separation gel；
3. being inserted perpendicularly into comb, a little concentration glue is then poured into comb both sides, so that the both sides of comb are fully sealed, and is gone
Bubble removing；
4. waiting 20-30min aggegation is repeated to concentrate glue.
(3) western blotting gel electrophoresises
1. glue mould is put into electrophoresis apparatus, Tris- glycine running buffers are poured into；
2. carefully remove the bubble around electrophoresis tank；
3. carefully extracting comb, and the bubble around hole is removed with syringe；
4. heating is boiled to be denatured 5min jointly by sample and loading buffer；
5. carry out prerunning with 20mA electric current at the beginning.When stainable bands go to separation gel, electric current is gone to
30mA.Stop electrophoresis when developed band is gone near gel bottom；
6. gel is dyed with Coomassie brilliant blue.Gel is put into Coomassie brilliant blue, dyes 1h.Afterwards, add de-
Toner (40% ethanol, 10% acetic acid and 50% distilled water), decolourizes in three times, each 1h.After having decolourized, observation of taking pictures.
(4) transferring film
1. gel is taken out, transferring film instrument is put into.Transferring film liquid is poured on transferring film instrument, and (transferring film used in this experiment turns skill to be wet
Art).6 layers of filter paper are laid in bottom, acetate film is put in centre, and gel is put on upper strata, and afterwards, upper strata is then covered with 6 layers of filter paper；
2. with electric current 0.25A, 250V voltage transferring film 40min；
3. the film after taking a turn for the better carefully takes out.With carefully cleaning 3 times of TBST solution, each 5min.Subsequently enter subsequent operation.
(5) antibody incubation and colour developing
1. closed first with skim milk.5% skim milk is added, closes 30min；
2. acetate film is closed with film sealing machine；
3. 1ml 1 is added in bag is closed:GAD65 antibody or actin antibody after 1000 dilutions are incubated, at -4 DEG C
It is incubated overnight；
4. acetate film is cleaned three times with TBST, each 10min；
5. 1ml 1 is added in bag is closed:Goat anti-rabbit igg secondary antibody after 5000 dilutions, 1 hour of room temperature；
6. acetate film is cleaned three times with TBST, each 10min；
7. developed the color using HRP methods.A, after the mixing of B liquid, acetate film is put into chemical colour reaction.
4. the great expression of recombinant protein, purifying and identification
After the optimum condition of protein purification has been groped, by the stable S2 cell culture for expressing GAD65 in 10 triangles
Tissue Culture Flask, in every bottle of 200ml culture medium.Pass through StrepII label affinity purification colors after cell culture is collected into cracking
Spectrum targeting obtains GAD65.Affinity chromatography uses GE Healthcare companies StrepTrap HP splitters (GE
Healthcare, 28-9075-47).Its principle is that StrepTactin can be specifically bound with StrepII labels, makes albumen
Fixed on solid phase carrier, target protein is eluted with 2.5mM desthiobiotin after elution impurity.Albumen is used after purification
Western blotting, coomassie brilliant blue staining and LC/MS Mass Spectrometric Identifications (Fig. 3).
5. the measure of protein concentration
Determination of protein concentration is carried out (Pierce, 23227) using sour (BCA) method of dihomocinchonine according to kit specification.
Sample reads light absorption value at 562nm, prepares the standard curve of absorbance and protein first, and determines that fitting is bent
Line formula.
According to the extension rate of sample, original protein concentration is determined.
(3) embodiment 3：Purify the immunogenicity of GAD65 albumen
GAD65 incomplete antigen epitope is space conformation type epitope, and GAD65 albumen after purification must be able to correct fold simultaneously
Forming correct space conformation could be identified by GADA autoantibodies.To determine that the GAD65 albumen of purifying has good immunogene
Property, we choose commercially available best GAD65 albumen in the world and compareed.GAD65 and commercialization GAD65 the albumen coating 96 of purifying
Orifice plate is stayed overnight, and is closed 1 hour with 1%BSA after washing, is then added negative serum, the basic, normal, high titer serum nurture of GADA, is washed
Biotinylated GAD65 (RSR) is added after washing, nurture adds the Streptavidin of POD couplings, added after washing after 1 hour
Substrate TMB shows, 2M H2SO4Terminating reaction.The GAD65 albumen of purifying has good immunogenicity, can not only distinguish
GADA feminine genders and positive serum, and can effectively distinguish the serum (Fig. 4) of the basic, normal, high titres of GADA.
SEQ NO.1 (sequence, optimization codon are indicated with underscore after GAD65 optimizations)：
ATGGCCTCGCCAGGAAGCGGATTCTGGTCCTTCGGATCGGAGGATGGAAGCGGCGACTCCGAGAACCCA GGAACCGCCCGCGCCTGGTGCCAGGTGGCCCAGAAGTTCACCGGCGGCATCGGCAATAAGCTGTGCGCCCTGCTGTA
CGGCGATGCCGAGAAGCCCGCCGAGTCGGGAGGAAGCCAGCCACCACGCGCCGCCGCCCGCAAGGCCGCCTGCGCCT
GCGATCAGAAGCCATGCAGCTGCTCCAAGGTGGACGTGAACTACGCCTTCCTGCACGCCACCGATCTGCTGCCAGCC TGCGACGGAGAGCGCCCAACCCTGGCCTTCCTGCAGGATGTGATGAATATCCTGCTGCAGTACGTGGTGAAGAGCTT
CGATCGCTCCACCAAGGTCATCGACTTCCACTACCCCAACGAGCTGCTGCAGGAGTACAATTGGGAGCTGGCCGACC AGCCACAGAACCTGGAGGAGATCCTGATGCACTGCCAGACCACCCTGAAGTACGCCATCAAGACCGGCCACCCGCGC
TACTTCAATCAGCTGAGCACCGGACTGGATATGGTGGGACTGGCCGCCGACTGGCTGACCTCCACCGCCAACACCAA TATGTTCACCTACGAGATCGCCCCCGTGTTCGTGCTGCTGGAGTACGTGACCCTGAAGAAGATGCGCGAGATCATCG GATGGCCAGGAGGATCGGGCGACGGAATCTTCAGCCCAGGAGGAGCCATCTCCAACATGTACGCCATGATGATCGCC
CGCTTCAAGATGTTCCCGGAGGTGAAGGAGAAGGGAATGGCCGCCCTGCCACGCCTGATCGCCTTCACCTCCGAGCA CTCGCACTTCAGCCTGAAGAAGGGAGCCGCCGCCCTGGGAATCGGAACCGATAGCGTGATCCTGATCAAGTGCGACG
AGCGCGGCAAGATGATCCCGAGCGATCTGGAGCGCCGCATCCTGGAGGCCAAGCAGAAGGGCTTCGTGCCATTCCTG
GTGTCGGCCACCGCCGGAACCACCGTGTACGGAGCCTTCGATCCACTGCTGGCCGTGGCCGACATCTGCAAGAAGTA CAAGATCTGGATGCACGTGGACGCCGCCTGGGGAGGAGGACTGCTGATGAGCCGCAAGCACAAGTGGAAGCTGTCGG GAGTGGAGCGCGCCAACAGCGTGACCTGGAATCCCCACAAGATGATGGGCGTGCCGCTGCAGTGCTCCGCCCTGCTGGTGCGCGAGGAGGGACTGATGCAGAACTGCAATCAGATGCACGCCTCCTACCTGTTCCAGCAGGATAAGCACTACGA
CCTGTCGTACGATACCGGCGACAAGGCCCTGCAGTGCGGACGCCACGTGGATGTGTTCAAGCTGTGGCTGATGTGGC GCGCCAAGGGAACCACCGGATTCGAGGCCCACGTGGACAAGTGCCTGGAGCTGGCCGAGTACCTGTACAACATCATC AAGAATCGCGAGGGCTACGAGATGGTGTTCGATGGCAAGCCACAGCACACCAACGTGTGCTTCTGGTACATCCCACC CTCCCTGCGCACCCTGGAGGACAATGAGGAGCGCATGTCCCGCCTGTCCAAGGTGGCCCCCGTGATCAAGGCCCGCA
TGATGGAGTACGGCACCACCATGGTGTCCTACCAGCCACTGGGCGATAAGGTGAACTTCTTCCGCATGGTCATCTCG AATCCCGCCGCCACCCACCAGGATATCGACTTCCTGATCGAGGAGATCGAGCGCCTGGGCCAGGACCTGTAA
SEQ NO.2 (GAD65 original series)：
ATGGCATCTCCGGGCTCTGGCTTTTGGTCTTTCGGGTCGGAAGATGGCTCTGGGGATTCCGAGAATCCCGGCACAGC
GCGAGCCTGGTGCCAAGTGGCTCAGAAGTTCACGGGCGGCATCGGAAACAAACTGTGCGCCCTGCTCTACGGAGACG
CCGAGAAGCCGGCGGAGAGCGGCGGGAGCCAACCCCCGCGGGCCGCCGCCCGGAAGGCCGCCTGCGCCTGCGACCAG
AAGCCCTGCAGCTGCTCCAAAGTGGATGTCAACTACGCGTTTCTCCATGCAACAGACCTGCTGCCGGCGTGTGATGG
AGAAAGGCCCACTTTGGCGTTTCTGCAAGATGTTATGAACATTTTACTTCAGTATGTGGTGAAAAGTTTCGATAGAT
CAACCAAAGTGATTGATTTCCATTATCCTAATGAGCTTCTCCAAGAATATAATTGGGAATTGGCAGACCAACCACAA
AATTTGGAGGAAATTTTGATGCATTGCCAAACAACTCTAAAATATGCAATTAAAACAGGGCATCCTAGATACTTCAA
TCAACTTTCTACTGGTTTGGATATGGTTGGATTAGCAGCAGACTGGCTGACATCAACAGCAAATACTAACATGTTCA
CCTATGAAATTGCTCCAGTATTTGTGCTTTTGGAATATGTCACACTAAAGAAAATGAGAGAAATCATTGGCTGGCCA
GGGGGCTCTGGCGATGGGATATTTTCTCCCGGTGGCGCCATATCTAACATGTATGCCATGATGATCGCACGCTTTAA
GATGTTCCCAGAAGTCAAGGAGAAAGGAATGGCTGCTCTTCCCAGGCTCATTGCCTTCACGTCTGAACATAGTCATT
TTTCTCTCAAGAAGGGAGCTGCAGCCTTAGGGATTGGAACAGACAGCGTGATTCTGATTAAATGTGATGAGAGAGGG
AAAATGATTCCATCTGATCTTGAAAGAAGGATTCTTGAAGCCAAACAGAAAGGGTTTGTTCCTTTCCTCGTGAGTGC
CACAGCTGGAACCACCGTGTACGGAGCATTTGACCCCCTCTTAGCTGTCGCTGACATTTGCAAAAAGTATAAGATCT
GGATGCATGTGGATGCAGCTTGGGGTGGGGGATTACTGATGTCCCGAAAACACAAGTGGAAACTGAGTGGCGTGGAG
AGGGCCAACTCTGTGACGTGGAATCCACACAAGATGATGGGAGTCCCTTTGCAGTGCTCTGCTCTCCTGGTTAGAGA
AGAGGGATTGATGCAGAATTGCAACCAAATGCATGCCTCCTACCTCTTTCAGCAAGATAAACATTATGACCTGTCCT
ATGACACTGGAGACAAGGCCTTACAGTGCGGACGCCACGTTGATGTTTTTAAACTATGGCTGATGTGGAGGGCAAAG
GGGACTACCGGGTTTGAAGCGCATGTTGATAAATGTTTGGAGTTGGCAGAGTATTTATACAACATCATAAAAAACCG
AGAAGGATATGAGATGGTGTTTGATGGGAAGCCTCAGCACACAAATGTCTGCTTCTGGTACATTCCTCCAAGCTTGC
GTACTCTGGAAGACAATGAAGAGAGAATGAGTCGCCTCTCGAAGGTGGCTCCAGTGATTAAAGCCAGAATGATGGAG
TATGGAACCACAATGGTCAGCTACCAACCCTTGGGAGACAAGGTCAATTTCTTCCGCATGGTCATCTCAAACCCAGC
GGCAACTCACCAAGACATTGACT TCCTGATTGAAGAAATAGAACGCCTTGGACAAGATTTATAA。
Claims (10)
1. encode the gene of the Human Glutamate Decarboxylase 65 albumen of high expression in drosophila cell, it is characterised in that described gene order is such as
Shown in SEQ ID NO.1.
2. the preparation method of Human Glutamate Decarboxylase 65 albumen, it is characterised in that by gene described in claim 1 as shown in SEQ ID NO.1
Sequence association StrepII labels be cloned into expression vector, then be transfected into drosophila cell and express simultaneously with r plasmid, will
Obtain GAD65-StrepII tag fusion proteins.
3. according to the method for claim 2, it is characterised in that described expression vector is pAc5.1/V5-HisA.
4. according to the method for claim 2, it is characterised in that described r plasmid is pCoBlast resistant vectors.
5. according to the method for claim 2, it is characterised in that the drosophila cell is S2.
6. according to the method described in Claims 2 or 3 or 4 or 5, it is characterised in that specifically include following steps：
(1) according to kind preferential expression, codon optimization is carried out to the ORF sequences of Human Glutamate Decarboxylase 65 with software；
(2) artificial synthesized complete sequence；
(3) primer and restriction enzyme site design：
According to the sequence of synthesis and StrepII labels design primer, and KpnI and NotI restriction enzyme sites are added, the following institute of primer
Show：
forward primer：
5'-CGGGGTACCCAAACATGTGGTCGCATCCGCAGTTCGAGAAGGCCTCGCCAGGAAGCGG-3'；
reverse primer：
5’-ATAAGAATGCGGCCGCTTATTACAGGTCCTGGCCCAGG-3’；
(4) PCR amplifications obtain purpose GAD65-StrepII fragments；
(5) double digestion is carried out to purpose fragment and carrier；
(6) purpose fragment after digestion connects with carrier in the case where T4 connects enzyme effect；
(7) recombinant conversion:Recombinant expression carrier is transfected into bacterium competent cell；
(8) GAD65-StrepII carrier is expressed in a large amount of extractings after sequence verification；
(9) by GAD65-StrepII carriers and pCoBlast resistant vector cotransfection drosophila cell expressing proteins；
(10) albumen that step (9) obtains by obtaining high-purity GAD65 albumen after purification.
7. according to the method for claim 6, it is characterised in that the carrier in step (5) is pAc5.1/V5-HisA.
8. according to the method for claim 6, it is characterised in that step (6) carries out restructuring connection using ligase：Connection sheet
Section 100ng, the pAc5.1/V5-His A plasmids added after 50ng digestions, 5 μ l 2 × reaction buffer and 1 μ l T4 ligases,
Water is supplied to 10 μ l；1h, transformed competence colibacillus cell are connected at 22 DEG C.
9. according to the method for claim 6, it is characterised in that the detailed process of step (9)：
Cultured healthy S2 cells are chosen, by 3 × 106Individual cell is seeded in 60mm tissue culture dishes, culture medium be for
The Sf-900II SFM Medium of S2 cells, 3ml culture medium is added per ware, under 28 DEG C of aseptic conditions in constant incubator
Cultivated；
Cell transfecting is carried out using calcium phosphate transfection kit K2780-01:
1. distilled water 233 μ l, calcium chloride 240mM 36 μ l, 1 μ g/ μ l GAD65- are sequentially added in sterile 1.5ml centrifuge tube
StrepII-pAc5.1/V5-His A plasmids 19 μ l, the μ l of 0.5 μ g/ μ l pCoBlast plasmids 2；
2. being transferred to after being slowly mixed together 1min in the centrifuge tube of 2 × HEPES HBS containing 200 μ l, mix；
3. mixture is placed into 30-40min in room temperature；
4. slowly adding mixture in cell culture medium, slow rotating and culturing ware is fully to mix；
5. cultivated 1 day in 28 DEG C of constant incubator；
6. cell culture medium is carried out to change liquid；
7. culture medium is sucked in 15ml centrifuge tube together with cell；
8. after 1000rpm centrifugations 3min, supernatant is removed, and add 1ml fresh cultures into 15ml centrifuge tube；
9. mixture is rejoined in new centrifuge tube；
10. cleaned again once with 2ml culture mediums；
With 2ml culture medium suspension cells, and go in culture dish and cultivate；
28 DEG C of cultures add 20 μ g/ml Blasticidin screening and clonings and establish stably transfected cell line two days later.
10. according to the method for claim 6, it is characterised in that the cell culture after step (9) transfection is containing 20 μ g/ml
In Blasticidin Sf-900II SFM Medium, it is divided into 10 cell triangle blake bottles, every bottle of 200ml culture mediums training
Support；After cell culture collects cracking, then targetted and obtained by StrepII label affinity purifications chromatogram.
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