CN101275147B - Expression vector and fused protein of human differential initiator 3 and preparation thereof - Google Patents
Expression vector and fused protein of human differential initiator 3 and preparation thereof Download PDFInfo
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Abstract
The present invention belongs to genetic engineering and medical biotechnology field, providing a human differentiation inhibitor 3 expression vector, fusion protein and their preparing method. After PCR amplification, the expression vector makes hId3 gene clone between sites EcoR I and Sal I of fusion expression vector pET32a, recombinant expression vector hId3/pET32a is produced. The invention makes hId3 encoding gene directly clone in prokaryotic expression vector, hId3 fusion protein expression vector is produced, realizing a high level expression of hId3 in escherichia coli, the expression accounts for 27% of bacterial protein, a whole set engineering bacteria induced expression and purification process are provided at the same time, rabbit anti-hId3 polyclonal antibody is produced by using hId3 fusion protein as an immunogen, laying a good foundation for researching biological function of hId3 protein and its application in clinical diagnose.
Description
Technical field
The invention belongs to genetically engineered and field of medical biotechnology, relate to a kind of human differential initiator 3 (hId3) expression vector, hId3 fusion rotein, anti-hId3 polyclonal antibody and preparation method thereof.
Background technology
Differentiation inhibiting factor (inhibitors of differentiation/DNA binding, Id), belong to one of helix-loop-helix (HLH) transcription factor family member, alkaline helix-loop-helix (bHLH) transcription factor activity is played down regulation, thereby suppress cytodifferentiation
[1-4]Mammalian cell contains four kinds of Id factors (Id1～Id4).Id3 is found in l cell system first as serum inductive immediate early gene
[5]Its participates in various kinds of cell biological procedures, comprises propagation, the embryo's of differentiation, the vascular smooth muscle of T cell and B cells whose development, skeletal muscle the blood vessel generation etc. of nerve formation, osteogenesis and tumor inducing
[5-12]The unconventionality expression of Id3 and tumour, amyotrophy, atherosclerosis and inflammation, sjogren syndrome are relevant
[12]
The same with other members of Id family, being expressed in of Id3 is the dynamic regulation state in the embryo development procedure, and in the cell development process, Id gene great expression is in undifferentiated, proliferative cell; Along with cytodifferentiation and maturation, Id genetic expression reduces gradually.Uncertain and the multiple disease that Id3 expresses is relevant with pathological state, comprises cancer, aging, arteriosclerosis, amyotrophy and inflammation etc.In the newborn process behind tissue injury, the expression pattern of Id3 also can change.And Id3 has distinct express spectra in the tumour of different tissues kind.Id3 is high expression level in colorectal cancer, astrocytoma; And in some malignant tumour as being decline expression trend in thyroid carcinoma, the ovarian cancer on the contrary; In normal liver tissue, can detect the expression of Id3, its expression level along with chronic hepatitis to the conversion of liver cirrhosis and improve, the expression of Id3 in WD liver cancer cell will be higher than PD liver cancer cell.Therefore, Id3 has complicated expression pattern and regulatory pathway in tumour cell.
The proteic expression of Id3, function and regulatory mechanism are also lacked further investigation both at home and abroad at present, the conclusion that some research is not agreed as yet.The preparation of relevant reorganization proteic prokaryotic expression of hId3 and purifying thereof, anti-hId3 antibody there is no report at present both at home and abroad.
Reference:
[1]Ruzinova?M?B，Benezra?R.Id?proteins?in?development，cell?cycle?and?cancer[J].Trends?Cell?Biol，2003，13(8)：410-418.
[2]Norton?J?D.ID?helix-loop-helix?proteins?in?cell?growth，differentiation?andtumorigenesis[J].J?Cell?Sci，2000，113(Pt.22)：3897-3905.
[3] Li Xiaojun, the Qin Jun river.Differentiation inhibiting factor progress [J].Biological chemistry and biophysics progress, 2004,31 (10): 865-869.
[4]Yokota?Y，Mori?S.Role?of?Id?family?proteins?in?growth?control[J].J?Cell?Physiol，2002，190(1)：21-28.
[5]Kee?BL.，Rivera?RR.，Murre?C.Id3?inhibits?B?lymphocyte?progenitor?growth?andsurvival?in?response?to?TGF-beta[J]，Nat?Immunol，2001，2(3)：242-247.
[6]Kee?BL.Id3?induces?growth?arrest?and?caspase-2-dependent?apoptosis?in?Blymphocyte?progenitors[J].J?Immunol，2005，175(7)：4518-4527.
[7]Forrest?ST，Barringhaus?KG，Perlegas?D，et?al.Intron?retention?generates?a?novel?Id3isoform?that?inhibits?vascular?lesion?formantion[J].J?BiolChem，2004，279(3)：32897-32903.
[8]Maeda?Y，Tsuji?K，Nifuji?A，et?al.Inhibitory?helix-loop-helix?transcription?factorsId1/Id3?promote?bone?formation?in?vivo[J]，J?Cell?Biol?Chem，2004，93(2)：337-344.
[9]Damdinsuren?B，Nagano?H，Kondo?M，et?al.Expression?of?Id?proteins?in?humanhepatocellular?carcinoma：relevance?to?tumor?dedifferatiation[J]，Int?JOncol，2005，26(2)：319-321.
[10]Tsuchiya?T，Okaji?Y，Tsuno?NH，et?al.Targeting?Id1?and?Id3?inhibits?peritonealmetastasis?of?gastric?cancer[J].Cancer?Sci?2005，96(11)：784-790.
[11]Coppe?JP，Itahana?Y，Moore?DH，et?al.Id-1?and?Id-2?proteins?as?molecular?markersfor?human?prostate?cancer?progression[J].Clin?Cancer?Res，2004，10(6)：2044-2051.
[12] Jia Li, Li Xiaojun. the expression analysis [J] of Id3 in the cisplatin induction Daudi cell inhibitory effect. Clinical Laboratory magazine, 2007,25 (3): 111-113.
Summary of the invention
The purpose of this invention is to provide a kind of reorganization hId3 prokaryotic expression carrier.
Another object of the present invention provides a kind of reorganization hId3 fusion rotein.
Another object of the present invention provides a kind of anti-hId3 polyclonal antibody.
A further object of the invention provides above-mentioned expression vector, fusion rotein and anti-hId3 Polyclonal Antibody Preparation method.
The mode of employing of the present invention and sulphur hydrogen reduction albumen (Trx)-Histidine (His) amalgamation and expression efficiently expresses hId3 fusion rotein and separation and purification recombinant protein in intestinal bacteria DE3, make it to prepare anti-hId3 polyclonal antibody, lay the foundation for further studying hId3 protein biology function and setting up the immunological method that detects hId3 as the immunogen immune rabbit.
Technology contents of the present invention comprises: the structure of segmental pcr amplification of 1.hId3 gene coding region cDNA and recombinant cloning vector Id3/pGEM-T.2.pET32a/Trx-His-hId3 construction of prokaryotic expression vector.3.hId3 expression and separation and purification and the evaluation of fusion rotein in intestinal bacteria DE3.4. the hId3 immunizing rabbit with fusion protein is prepared the anti-hId3 polyclonal antibody of specificity.
Below be concrete technical measures of the present invention:
1. according to the hId3 cDNA encoding sequence that provides among the GeneBank, design a pair of special primer (SEQ ID No.2, SEQ ID No.3), with the pGEM-T/Id3 carrier is template (gene segment that contains hId3 that also can ordinary method obtains is a template), carries out pcr amplification (the results are shown in Figure 1) with above-mentioned primer.Adopt pGEM-T Easy Vector System that the PCR product hId3 cDNA of purifying is inserted pGEM-T Easy carrier, make up recombinant cloning vector Id3/pGEM-T (make up flow process and see Fig. 2).Sequencing result is seen SEQ ID No.1.
2. the hId3 encoding gene behind the pcr amplification is cloned between the EcoR I and Sal I site of the expression vector pET-32a that has T7 promotor, 6 Histidines and sulphur hydrogen reduction protein coding gene, obtains hId3 Prokaryotic Expression carrier pET32a/Trx-His-hId3.Because the hId3 upstream region of gene has merged 6 Histidines and sulphur hydrogen reduction protein gene fragment, utilize His label and Ni ionic affinity interaction, the protein product that amalgamation and expression goes out can be further purified with the Ni-NAT resin, the amalgamation and expression of hId3 and Trx increases the solubility of expression product, the hId3/pET32a that carries the Trx-His-hId3 fusion gene is under the T7 promotor control of pET32a, the recognition site that also has enteropeptidase and zymoplasm between foreign protein genes in the antigen-4 fusion protein gene and the Trx is convenient to downcut Trx from fusion rotein.
3. recombinant vectors pET32a/Trx-His-hId3 transformed into escherichia coli BL21 (DE3) after IPTG induces, obtains efficient expression strain.Transformed bacteria is carried out the basis at 37 ℃ cultivate, as bacterial density OD
600Value is 0.4～0.6 o'clock, induces 4h for 37 ℃, collects bacterium, be suspended in 1 * Binding Buffer (20mM Tris-HCl, pH 7.9,500mMNaCl, 10mM beta-mercaptoethanol, 1mmol/L PMSF), the ultrasonication cell, centrifugal after, get supernatant liquor and be used for Ni-NTA affinity column (Novagen company), separation and purification obtains the hId3 fusion rotein, confirms that through Western blot this fusion rotein has immunogenic protein (Western blotting adopts anti-Id3 antibody when identifying).
In purge process, Washing buffer I is: 20mM Tris-HCl, and pH 7.9,500mM NaCl, 10mM beta-mercaptoethanol, 30mmol/L imidazoles; Washing Buffer II is: 20mM Tris-HCl, and pH 7.9,500mMNaCl, the 10mM beta-mercaptoethanol, the 45mmol/L imidazoles can improve purification effect greatly.
The recombinant expression vector that makes up is by the method for the invention transformed into escherichia coli, induces that solubility target protein concentration is about 0.41g/L in the bacterium liquid.The albumen yield is the wet bacterium of 0.76mg/g.
HId3 fusion rotein by separation purification method provided by the invention obtains from transformed bacteria detects through SDS-PAGE and coomassie brilliant blue staining, is the band of the about 34KD of molecular weight, and purity is greater than 90%.
4. the hId3 immunizing rabbit with fusion protein is prepared specific anti-human Id3 antibody: the recombinant protein of purifying is cut the Trx-His-hId3 fusion rotein with zymoplasm, also separate once more again with the Ni-NTA affinity column, obtain to downcut the hId3 albumen of Trx-His, with this albumen is antigen, immunity New Zealand white rabbit in 8 age in week, multiple spot intracutaneous, subcutaneous injection.During first immunisation, the recombinant protein 1mg (volume 1ml) that gets purifying is fully emulsified with the 1ml Freund's complete adjuvant, in the intradermal injection of every rabbit nape portion; After 15 days, booster immunization once, dosage is the same but fully emulsified with Freund's incomplete adjuvant, it is subcutaneous to be injected in nape portion; Booster immunization is once once more after two weeks; Booster immunization detected antibody titer from ear edge vein exploitating blood after 10 days for the second time.Recording sero-fast the tiring of rabbit source property with the agarose single diffusion test is 1: 8; Collect antiserum(antisera), packing, frozen in-70 ℃.
Beneficial effect of the present invention:
The expression vector that the present invention makes up is owing to merged 6 Histidines and sulphur hydrogen reduction protein gene fragment at the hId3 upstream region of gene, utilize His label and Ni ionic affinity interaction, the protein product that amalgamation and expression goes out can be further purified with the Ni-NAT resin, the amalgamation and expression of hId3 and Trx increases the solubility of expression product, the Id3/pET32a that carries the Trx-His-hId3 fusion gene is under the T7 promotor control of pET32a, the recognition site that also has enteropeptidase and zymoplasm between foreign protein genes in the antigen-4 fusion protein gene and the Trx is convenient to downcut Trx from fusion rotein.
The present invention adopts round pcr to obtain the hId3 encoding gene, use restriction enzyme digestion and interconnection technique, with hId3 encoding gene directed cloning to prokaryotic expression carrier, obtain the hId3 fusion protein expression vector, realized hId3 efficiently expressing in escherichia coli, expression amount accounts for 27% of bacterial protein, and a whole set of engineering bacteria abduction delivering and purifying process also is provided simultaneously.The present invention uses gene recombination technology, make up the pET32a/Trx-His-hId3 fusion expression vector, and bacterium is expressed in conversion, through induce, protein purification obtains the hId3 fusion rotein, with the hId3 fusion rotein is the anti-hId3 polyclonal antibody of immunogen preparing rabbit, for further research proteic biological function of hId3 and the application in clinical diagnose thereof lay the foundation.
Description of drawings
Fig. 1: the agarose gel electrophoresis figure of hId3 gene fragment PCR product.
Wherein: 1.DNA molecular weight standard (DL2000); 2.PCR product
Fig. 2: recombinant cloning vector hId3/pGEM-T makes up flow process.
Fig. 3: recombinant expression vector pET32a/Trx-His-hId3 double digestion is identified electrophorogram.
Wherein: 1,150bp dna molecular amount standard;
2, pET32a/Trx-His-hId3: cut through EcoR I and Sal I enzyme;
3, plasmid pET32a
Fig. 4: the SDS-PAGE of recombinant protein analyzes under the different induction times.
Wherein: M. low molecular weight protein standard;
1. without IPTG inductive pET32a/Trx-His-hId3;
2～7. induce 1,2,4,6,8 and the hId3/pET32a of 12h through IPTG;
Fig. 5: the SDS-PAGE of nickel ion affinity chromatograph purifying hId3 fusion rotein analyzes.
Wherein: M. low molecular weight protein standard;
1.pET32a/Trx-His-hId3 plasmid transformed bacteria lysate supernatant;
2. wash assorted liquid; 3. the recombinant protein of purifying.
Fig. 6: Western blot trace is identified the specificity of recombinant protein.
Wherein: M. low molecular weight protein standard; 1. recombinant protein
Embodiment
Below in conjunction with accompanying drawing embodiments of the invention are specifically described.
Embodiment 1:
1. according to the hId3 cDNA encoding sequence that provides among the GeneBank, design a pair of special primer, introduce EcoR I restriction enzyme site at upstream primer 5 ' end; Add Sal I restriction enzyme site at downstream primer 5 ' end, wherein also comprise terminator codon TAA.
Upstream primer: 5 '-CG
GAATTCAAGGCGCTGAGCCCGGTG-3 ' (SEQ ID No.2)
(underscore is an EcoR I restriction enzyme site)
Downstream primer: 5 '-ACGC
GTCGACTTAGTGGCAAAAGCTCCTTTTG-3 ' (SEQ ID No.3)
(underscore partly is a Sal I restriction enzyme site).
(see Li Xiao army etc. with the pGEM-T/Id3 carrier, the structure of Id3-EGFP integrative gene expression vector and the expression in the human lung cancer cell A549 thereof, the 29th the 12nd phase of volume of " Chinese laboratory medicine magazine " December in 2006) for template (gene segment that contains hId3 that also can ordinary method obtains is a template), carries out pcr amplification (Fig. 1) with above-mentioned primer.Adopt pGEM-T Easy Vector System that the PCR product hId3 cDNA of purifying is inserted pGEM-T Easy carrier, make up recombinant cloning vector hId3/pGEM-T (make up flow process and see Fig. 2).Sequencing result is seen SEQ ID No.1.。
2. from the hId3/pGEM-T carrier, cut out the hId3 gene clone in pET32a with EcoR I and Sal I, obtain expression vector hId3/pET32a (pET32a/Trx-His-hId3).With hId3/pET32a expression plasmid transformed into escherichia coli, the host bacterium is e. coli bl21 (DE3), and plasmid hId3/pET32a transforms and adopts calcium chloride transformation.The screening of transformed bacteria: transform bacterium colony from the dull and stereotyped picking of going up of fresh conversion, be inoculated in the LB substratum that contains penbritin (100mg/L) respectively, 37 ℃ of shaking culture are spent the night, and collect thalline, extract plasmid enzyme restriction and identify (Fig. 3).
3. transformed bacteria is inoculated in the LB nutrient solution that contains penbritin (100mg/L), 37 ℃ of shaking culture are spent the night.Be inoculated in the fresh LB nutrient solution that contains penbritin (100mg/L) with bacterium liquid with 1: 100 ratio next day, and 37 ℃ are cultured to OD
600Value is 0.4～0.6, add IPTG to final concentration be 1mmol/L, continue to cultivate about 4h in 37 ℃.Get respectively and induce preceding and induce the thalline of 1～12h to do the SDS-PAGE electrophoretic analysis.The new band that a treaty 34kD on electrophoretogram, occurs.The densitometric scan result shows that the band of the transformed bacteria 34kD of abduction delivering 4h accounts for 27% (Fig. 4) of total bacterial protein.
4. collect IPTG in a large number and induce transformed bacteria E.coli BL21, be resuspended in 1 * Binding Buffer (20mMTris-HCl, pH 7.9,500mM NaCl, 10mM beta-mercaptoethanol), adding final concentration is the PMSF of 1mmol/L, puts 30min in the ice bath.Carry out ultrasonication with ultrasonic cell disruptor.After the ultrasonic end, adding Triton X-100 is 1% (v/v) to final concentration, and the centrifugal 10min of 6000r/min collects that sample is to the Ni-NTAHis-Bind chromatography column of 1mL on the centrifugal supernatant liquor afterwards, and chromatography column is used 1 * Binding Buffer balance in advance.Behind the last sample,, then use 20ml Washing buffer I (20mM Tris-HCl earlier with 1 * Binding Buffer wash-out twice, pH 7.9,500mM NaCl, 10mM beta-mercaptoethanol, 30mmol/L imidazoles) wash post, use Washing Buffer II (20mM Tris-HCl again, pH7.9,500mM NaCl, 10mM beta-mercaptoethanol, the 45mmol/L imidazoles) washes post, collect elutriant; With 4mlElution buffer wash-out, collect elutriant.The effluent liquid of collecting of respectively managing is carried out the SDS-PAGE analysis, and the reorganization Id3 of purifying merges a band that is the about 35KD of molecular weight, and purity is greater than 90% (Fig. 5).Immunoblotting (Westernblotting) qualification result consistent with it (Fig. 6).Western blotting adopts anti-Id3 antibody when identifying.
5. the recombinant protein with purifying cuts the Trx-His-hId3 fusion rotein with zymoplasm, also separating once more with the Ni-NTA affinity column, obtain to downcut the hId3 albumen of Trx-His, is antigen with this albumen, immunity New Zealand white rabbit in 8 age in week, multiple spot intracutaneous, subcutaneous injection.During first immunisation, the recombinant protein 1mg (volume 1ml) that gets purifying is fully emulsified with the 1ml Freund's complete adjuvant, in the intradermal injection of every rabbit nape portion; After 15 days, booster immunization once, dosage is the same but fully emulsified with Freund's incomplete adjuvant, it is subcutaneous to be injected in nape portion; Booster immunization is once once more after two weeks; Booster immunization detected antibody titer from ear edge vein exploitating blood after 10 days for the second time.Recording sero-fast the tiring of rabbit source property with the agarose single diffusion test is 1: 8; Collect antiserum(antisera), packing, frozen in-70 ℃.
Sequence table
Claims (2)
1. anti-hId3 polyclonal antibody, it is characterized in that it is the polyclonal antibody of rabbit source property, prepare by following method: with zymoplasm cleavage of fusion proteins Trx-His-hId3, separate once more with the Ni-NTA affinity column, obtain to downcut the hId3 albumen of Trx-His, with this albumen be antigen as immunizing antigen, immunity have immunocompetent 8 the week age new zealand male rabbit, the subcutaneous minute multi-point injection in back; During first immunisation, fully emulsified with the recombinant protein 1mg and the 1ml Freund's complete adjuvant of purifying, in every rabbit back intradermal injection; After 15 days, booster immunization, dosage is the same but use Freund's incomplete adjuvant, and it is subcutaneous to be injected in the back; The method of two weeks back use booster immunization is booster immunization once more; Booster immunization began to get ear edge vein exploitating blood and detects antibody titer after 10 days for the second time, recorded sero-fast the tiring of rabbit source property with the agarose single diffusion test, collected antiserum(antisera), and packing is frozen in-70 ℃;
Wherein, fusion rotein Trx-His-hId3 adopts following method to prepare:
The recombinant expression vector that makes up: with the hId3 gene clone behind the pcr amplification between the EcoR of fusion expression vector pET32a I and Sal I site, obtain recombinant expression vector hId3/pET32a, wherein the hId3 upstream region of gene has merged 6 Histidines and sulphur hydrogen reduction protein gene fragment, utilize His label and Ni ionic affinity interaction, the protein product that amalgamation and expression goes out is further purified with the Ni-NAT resin, the amalgamation and expression of hId3 and Trx increases the solubility of expression product, the hId3/pET32a that carries the Trx-His-hId3 fusion gene is under the T7 promotor control of pET32a, the recognition site that also has enteropeptidase and zymoplasm between foreign protein genes in the antigen-4 fusion protein gene and the Trx is convenient to downcut Trx from fusion rotein;
With constructed recombinant expression vector transformed into escherichia coli BL21, carry out the basis at 37 ℃ and cultivate, as bacterial density OD
600Value is 0.4～0.6 o'clock, expresses 3～4 hours for 37 ℃; Collect express cell, be suspended in and contain 20mM Tris-HCl, pH 7.9,500mM NaCl, 10mM beta-mercaptoethanol, 1 * Binding Buffer of 1mmol/L PMSF, the ultrasonication cell, after centrifugal, get supernatant liquor and be used for Ni-NTA affinity chromatography column separating purification, obtain fusion rotein Trx-His-hId3.
2. the described anti-hId3 Polyclonal Antibody Preparation method of claim 1, it is characterized in that described immunologic process is for using zymoplasm cleavage of fusion proteins Trx-His-hId3, separate once more with the Ni-NTA affinity column, obtain to downcut the hId3 albumen of Trx-His, with this recombinant protein is immunizing antigen, immunity new zealand male rabbit in 8 age in week, the subcutaneous minute multi-point injection in back; During first immunisation, fully emulsified with the recombinant protein 1mg and the 1ml Freund's complete adjuvant of purifying, in every rabbit back intradermal injection; After 15 days, booster immunization, dosage is the same but use Freund's incomplete adjuvant, and it is subcutaneous to be injected in the back; The method of two weeks back use booster immunization is booster immunization once more; Booster immunization began to get ear edge vein exploitating blood and detects antibody titer after 10 days for the second time, recorded sero-fast the tiring of rabbit source property with the agarose single diffusion test, collected antiserum(antisera), and packing is frozen in-70 ℃;
Wherein, fusion rotein Trx-His-hId3 adopts following method to prepare:
The structure of recombinant expression vector: with the hId3 gene clone behind the pcr amplification between the EcoR of fusion expression vector pET32a I and Sal I site, obtain recombinant expression vector hId3/pET32a, wherein the hId3 upstream region of gene has merged 6 Histidines and sulphur hydrogen reduction protein gene fragment, utilize His label and Ni ionic affinity interaction, the protein product that amalgamation and expression goes out is further purified with the Ni-NAT resin, the amalgamation and expression of hId3 and Trx increases the solubility of expression product, the hId3/pET32a that carries the Trx-His-hId3 fusion gene is under the T7 promotor control of pET32a, the recognition site that also has enteropeptidase and zymoplasm between foreign protein genes in the antigen-4 fusion protein gene and the Trx is convenient to downcut Trx from fusion rotein;
With the recombinant expression vector transformed into escherichia coli BL21 (DE3) that makes up, carry out the basis at 37 ℃ and cultivate, as bacterial density OD
600Value is 0.4～0.6 o'clock, expresses 4 hours for 37 ℃; Collect express cell, be suspended in and contain 20mM Tris-HCl, pH 7.9,500mM NaCl, 10mM beta-mercaptoethanol, 1 * Binding Buffer of 1mmol/L PMSF, the ultrasonication cell after centrifugal, got supernatant liquor and is used for Ni-NTA affinity chromatography column separating purification, in the purge process, Washingbuffer I is: 20mM Tris-HCl, and pH 7.9,500mM NaCI, the 10mM beta-mercaptoethanol, the 30mmol/L imidazoles; Washing Buffer II is: 20mM Tris-HCl, and pH 7.9,500mM NaCI, the 10mM beta-mercaptoethanol, the 45mmol/L imidazoles obtains fusion rotein Trx-His-hId3.
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