CN103333244B - hZimp10 N-terminal high titer antibody prepared by using GST expression system and applications thereof - Google Patents
hZimp10 N-terminal high titer antibody prepared by using GST expression system and applications thereof Download PDFInfo
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Abstract
The invention belongs to the field of bioengineering technology, and relates to a hZimp10 N-terminal high titer antibody prepared by using GST expression system and applications thereof. The preparation method comprises following steps: the N-terminal of hZimp10 gene is cloned into a prokaryotic?expression?vector by using genetic engineering technologies; after restriction analysis, the vector is transformed into escherichia coli by a recombinant?plasmid; a GST- hZimp10 fusion protein is generated through IPTG induction; and a New Zealand rabbit is immunized with the purified fusion protein so as to prepare an antiserum, and the antiserum is purified by proteinA/G. Titer and specificity of the antibody are detected by ELISA and Western blot. The anti-hZimp10 polyclonal?antibody possesses excellent titer and specificity; can be used in the detection of hZimp10 protein in cells and can be used for effective detection of differential expression of hZimp10 protein in different cells; and has great significance for systemic study of hZimp10 protein functions and the action mechanism of hZimp10 protein in tumor?cell?apoptosis.
Description
Technical field
The invention belongs to the DNA recombinant technology field in biotechnology, be specifically related to utilize GST prokaryotic expression system to prepare polyclonal antibody and the application of GST-hZimp10 fusion rotein.
Background technology
HZimp10 albumen (being defined as the PIAS albuminoid mankind's No. 10 karyomit(e)s comprising the Miz1 of zinc fingers) is selectively expressed in the ovary of the mankind, testis and prostata tissue.As a kind of new PIAS albuminoid, hZimp10 has been proved to be has important regulating effect to the transcriptional activity of some important albumen.Such as: AR, Smad3/Smad4 mixture and P53 etc.
Going deep into us more thinking created for hZimp10 protein function and mechanism of action along with research, as it whether with the growth of reproductive system and function closely related, whether it plays restraining effect to tumour, and whether it can promote cancer cell-apoptosis etc.And study a kind of function of new protein, antibody is one of the strongest instrument, the immunohistochemistry of widespread use in the detection and functional study of protein, a series of technology such as immunoblotting and immunoprecipitation, all grow up based on antigen-antibody interaction.Therefore our the hZimp10 albumen high-titer antibody prepared is significant for the mechanism of action of systematic study hZimp10 in tumor cell proliferation and apoptosis.
Summary of the invention
The object of the invention is to set up a kind of model that can be used in the detection of hZimp10 albumen, for providing necessary experimental tool from protein level further investigation hZimp10 protein function and relative disease.
Utilize genetic engineering technique, hZimp10 albumen n end 128 amino acid corresponding DNA fragments are cloned in prokaryotic expression carrier pGEX-4T-1.Through enzyme cut with sequential analysis after, use recombinant plasmid transformed e. coli bl21, and produce GST-hZimp10 fusion rotein through isopropyl-β-D-thiogalactoside(IPTG) (IPTG) induction.Prepare antiserum(antisera) with the fusion protein immunization new zealand rabbit of purifying, and apply ProteinA/G and be purified, obtain polyclonal antibody.Tiring and specificity employing ELISA and Western blot detection of antibody.
GST-hZimp10 fusion rotein of the present invention to utilize special sequence (hZimp10 albumen n end 128 amino acid corresponding DNA fragments) to build GST-hZimp10 fusion protein prokaryotic expression carrier, transform the specific GST-hZimp10 fusion rotein of high expression that obtains of BL21, this section of N holds 128 aminoacid sequences to be:
Met Asn Ser Met Asp Arg His Ile Gln Gln Thr Asn Asp Arg Leu Gln Cys Ile Lys Gln
1 5 10 15 20
His Leu Gln Asn Pro Ala Asn Phe His Asn Ala Ala Thr Glu Leu Leu Asp Trp Cys Gly
25 30 35 40
Asp Pro Arg Ala Phe Gln Arg Pro Phe Glu Gln Ser Leu Met Gly Cys Leu Thr Val Val
45 50 55 60
Ser Arg Val Ala Ala Gln Gln Gly Phe Asp Leu Asp Leu Gly Tyr Arg Leu Leu Ala Val
65 70 75 80
Cys Ala Ala Asn Arg Asp Lys Phe Thr Pro Lys Ser Ala Ala Leu Leu Ser Ser Trp Cys
85 90 95 100
Glu Glu Leu Gly Arg Leu Leu Leu Leu Arg His Gln Lys Ser Arg Gln Ser Asp Pro Pro
105 110 115 120
Gly Lys Leu Pro Met Gln Pro Pro
125
Its corresponding nucleotides sequence is classified as:
1 atgaattcta tggacaggca catccagcag accaatgacc gactgcagtg catcaagcag
61 cacttacaga atcctgccaa cttccacaat gccgccacgg agctgctgga ctggtgcgga
121 gacccacggg ccttccagcg gcccttcgag cagagcctga tgggctgttt gacggtggtc
181 agtcgggtgg cagcccagca aggctttgac ctggacctcg gctacagact gctggctgtg
241 tgtgctgcaa accgagacaa gttcaccccg aagtctgccg ccttgttgtc ctcctggtgc
301 gaagagctcg gccgcctgct gctgctccga catcagaaga gccgccagag cgatccccct
361 gggaaactcc ccatgcagcc ccct
This GST-hZimp10 fusion rotein and polyclonal antibody preparation thereof comprise the following steps: the first step, the structure of cloning vector
Be template from people hZimp10 full length gene application round pcr with pcCDNA3.1-Flag-hZimp10, the object fragment obtained that increases is connected with PMD18-T carrier, and after the steps such as conversion, extraction obtain recombinant plasmid, enzyme is cut and identified and check order.Second step, the structure of prokaryotic expression carrier pG`EX-4T-1
After cloned plasmids and plasmid pGEX-4T-1 double digestion, utilize recovery test kit to obtain this district's fragment of hZimp10 gene and be connected with carrier.The expression vector of restructuring is obtained through steps such as conversion, extractions.Enzyme cuts qualification recombinant chou, and order-checking is determined further.
3rd step, the expression and purity of GST-hZimp10 fusion rotein
Recombinant plasmid PGEX-4T-1/hZimp10 transformation of E. coli BL21, utilizes IPTG to induce, the expression of GST-hZimp10 fusion rotein.Identify with SDS-PAGE, and optimization expression condition, induction of increasing in a large number.Use ultrasonic degradation bacterium, the albumen Glutathione-Sepharose4B column purification that obtains, SDS-PAGE purification Identification product.
4th step, the sero-fast preparation of the anti-hZimp10 of rabbit and purifying
With the hZimp10 fusion protein immunization Male New Zealand White Rabbit of purifying, initial immunity 500 μ g fusion roteins, fully mix the subcutaneous multi-point injection of emulsification back part with isopyknic complete Freund's adjuvant.Ear vein blood separation of serum is got, as the serum control before immunity before immunity.Carry out the 1st booster immunization after 2wk, GST-hZimp10 fusion rotein and the incomplete Freund's adjuvant equal-volume of 500 μ g purifying mix, front and back four sole intramuscular injection.Afterwards every 2wk booster immunization 1 time.After final immunization, 1wk gets ear blood, measures tiring of antibody by ELISA method, and when antibody titer reaches 1: 100000, carotid artery bloodletting, collects serum.Purify anti-hZimp10 serum with ProteinA/G, prepare albumin A sepharose CL-4B affinity column, affinity chromatography makes antibodies on pillar, after 2 washings, by antibody elution, reach purifying object, SDS-PAGE purification Identification product, obtains the polyclonal antibody of this invention.Application panimmunity method detects it and tires and specificity, and result shows this antibody can be specific with hZimp10 protein binding.。
HZimp10 albumen n end 128 amino acid gene orders are utilized successfully to build GST-hZimp10 fusion protein prokaryotic expression carrier, can the specific GST-hZimp10 fusion rotein of high expression after transforming BL21.The high-titer antibody obtaining anti-GST-hZimp10 fusion rotein with this fusion protein immunization rabbit detects antibody titer and specificity through panimmunity method, and result shows that antibody titer is up to 1:100000 and specificity is good.Antibody can be applicable to the detection of hZimp10 albumen in cell and effectively can detect the differential expression of hZimp10 albumen in different cell strain, for helpful in the dependency of probing into the expression intensity of hZimp10 albumen in cancer cells and malignancy further, for systematic study hZimp10 protein function and the mechanism of action in tumor cell proliferation and apoptosis significant.In addition, hZimp10 protein expression has different spliced bodies (isoform), comprise total length and N-128 absence type etc., the hZimp10-N-128aa antibody that we prepare is that under detecting various disease condition, different hZimp10isoform expresses and judges that the relation of hZimp10 protein expression and disease is laid a good foundation with this.The detection model setting up a kind of malignancy for applying this antibody provides condition.
Accompanying drawing illustrates:
Fig. 1 is the hZimp10 gene N-128 sheet segment DNA agarose gel electrophoresis figure that pcr amplification goes out;
Fig. 2 is double digestion plasmid pGEX-4T-1 and restructuring carrier T agarose gel electrophoresis figure;
Fig. 3 is that pGEX-4T-1/hZimp10 recombinant enzyme cuts qualification result;
Fig. 4 is that GST and the GST-hZimp10 fusion protein S DS-PAGE after purifying schemes;
Fig. 5 is that the hZimp10 antibody after ProteinA/G purifying and the anti-hZimp10 serum SDS-PAGE before purifying scheme;
Fig. 6 is that indirect elisa method measures tiring of antibody;
Fig. 7 is that the specific Western blot of antiserum(antisera) analyzes.
Embodiment:
Embodiment 1: preparing of anti-GST-hZimp10 serum
The structure of 1.PCR-PMD18-T/hZimp10 recombinant plasmid
HZimp10 full length gene obtains from Genebank, and gene order number is AY235683.Take pcCDNA3.1-Flag-hZimp10 as template, upstream primer is 5 '-GGATCC ATGAAT TCTATG GACAGG (containing BamHI, EcoRI restriction enzyme site); Downstream primer is 5 '-CTCGAG AGGGGG CTGCAT GGGGAG (containing XhoI restriction enzyme site).Application PCR Successful amplification has gone out hZimp10 gene N and has held 128 amino acid corresponding DNA sequence length 384bp[Fig. 1].The fragment obtained that increases is connected with PMD18-T carrier, is proceeded to by connection product in competence bacillus coli DH 5 alpha, containing selected clone on Amp+ agar plate, after carrying recombinant plasmid so that alkaline lysis is little, with BamH I and the qualification of Xhol I double digestion.
2. the structure of prokaryotic expression carrier pGEX-4T-1
By the pMD18-T plasmid containing hZimp10 gene N-128 fragment after BamH I and Xho1 double digestion (fragment is about 0.4kbp), utilize and reclaim this district's fragment of test kit acquisition hZimp10 gene, simultaneously with identical ferment treatment plasmid pGEX-4T-1 (about 4.9kbp) [Fig. 2].Then spend the night in 16 DEG C of connections under the effect of T4DNA ligase enzyme by the hZimp10 gene N-128 fragment of recovery with through the vector pGEX-4T-1 that enzyme is cut.Enzyme cuts qualification recombinant chou, and result shows this hZimp10 gene N-128 fragment correct [Fig. 3].
The expression and purity of 3.GST-hZimp10 fusion rotein
In recombinant plasmid PGEX-4T-1/hZimp10 transformation of E. coli BL21, picking list bacterium colony access LB/Ampr substratum, 37 DEG C of jolting overnight incubation.Next day, by culture in 1: 50 ratio transfer in containing Amp+ LB substratum in, continue to be cultured to mid log phase at 37 DEG C of shaking tables.When the A600 of nutrient solution is 0.5 ~ 0.6, adding IPTG to final concentration is 0.08mmol/L, does not add IPTG person for negative control, puts 25 DEG C and continues cultivation 4 ~ 5h.Collected by centrifugation thalline, carries out with SDS-PAGE the expression identifying GST-hZimp10 fusion rotein, and optimization expression condition, induction of increasing in a large number.With 5000r/min in 4 DEG C of centrifugal 5min, collect thalline, by the precipitation of the NETN suspension 1L bacterium liquid of 60mL ice precooling.Use ultrasonic degradation bacterium, again with 9600rpm, in 4 DEG C of centrifugal 15min, get supernatant, cross Glutathione-Sepharose4B post, first with equal-volume elution buffer 1 (containing 20mM gsh, 50mM Triscl, pH=8.0) wash-out, collects elutriant, then washes twice with equal-volume elution buffer 2 (containing 100mM gsh), collect elutriant, SDS-PAGE purification Identification product [Fig. 4].
4. the sero-fast preparation of the anti-hZimp10 of rabbit
With the GST-hZimp10 fusion protein immunization Male New Zealand White Rabbit of purifying, initial immunity 500 μ g fusion roteins, fully mix the subcutaneous multi-point injection of emulsification back part with isopyknic complete Freund's adjuvant.Ear vein blood separation of serum is got, as the serum control before immunity before immunity.Carry out the 1st booster immunization after 2wk, GST-hZimp10 fusion rotein and the incomplete Freund's adjuvant equal-volume of 500 μ g purifying mix, front and back four sole intramuscular injection.Afterwards every 2wk booster immunization 1 time.After final immunization, 1wk gets ear blood, measures tiring of antibody by ELISA method, and when antibody titer reaches 1: 100000, carotid artery bloodletting, collects serum.
The anti-hZimp10 serum of 5.ProteinA/G purifying
ProteinA/G sepharose CL-4B filler is slowly filled post, the antiserum(antisera) through dilution is added after balance pillar, coutroi velocity ensures the combination of antiserum(antisera) and filler, namely affinity chromatography makes antibodies on pillar, after 2 washings, add pH2.7 elution buffer solution by antibody elution, collect elutriant and measure the 280nm optical density(OD) of each collection tube, mixed by collection tube containing antibody, the antibody after purifying and the antiserum(antisera) SDS-PAGE before purifying and coomassie brilliant blue staining are identified.Coloration result display purification effect is obvious, and the sample after purifying has light, heavy chain band [Fig. 5] clearly.
The Analyzes and nurses of embodiment 2:GST-hZimp10 antibody
The mensuration of 1.GST-hZimp10 antibody titer
With the new zealand white rabbit serum before GST-hZimp10 fusion protein immunization in contrast, the GST-hZimp10 antibody after purifying is first diluted 10 times again after doubling dilution, measures tiring of antibody with indirect ELISA.Result shows, and the rabbit anteserum before immunity does not measure the antibody of anti-fusion rotein GST-hZimp10, and the titre of GST-hZimp10 antibody is up to more than 1: 100000 [Fig. 6].
2.GST-hZimp10 antibody is applied to the detection of hZimp10 albumen in cell
Collect prostate cancer cell LNCaP and PC3 cultivated, the ultrasonic rear BCA protein quantification test kit of cracking carries out protein quantification to it, afterwards the protein contents such as sample are carried out SDS-PAGE, then electrotransfer is on nitrocellulose filter.Close 1h with 5% skim-milk, drip the anti-hZimp10 antibody of rabbit (room temperature 2h, PBS wash 3 times) and goat anti-rabbit igg 2HRP (room temperature reaction 1h, PBS wash 3 times) successively, finally add substrate DAB and develop the color, and take pictures.Result display [Fig. 7], hZimp10 albumen expressed in the hZimp10 antibody that we prepare and LNCaP and PC3 cell strain all there occurs antigen-antibody reaction, a special protein band has been there is at the 123KD place of Marker instruction, and the expression intensity of specific protein band is different in different cell strains, the differential protein band strength of LNCaP cell is high, proves LNCaP cell high expression level hZimp10.
Claims (2)
1. the hZimp10-N utilizing GST expression system to prepare holds high-titer antibody, it is characterized in that the GST-hZimp10 fusion rotein utilizing GST expression system to prepare using hZimp10 albumen n end 128 amino acid corresponding DNA fragments as distinguished sequence produces antiserum(antisera) as antigen-immunized animal and obtains, in this antiserum(antisera), the titre of anti-GST-hZimp10 antibody is up to 1: 100 000, and identify that this anti-GST-hZimp10 serum has good specificity through Western blot, wherein, N holds 128 aminoacid sequences to be:
Met Asn Ser Met Asp Arg His Ile Gln Gln Thr Asn Asp Arg Leu Gln Cys Ile Lys Gln
1 5 10 15 20
His Leu Gln Asn Pro Ala Asn Phe His Asn Ala Ala Thr Glu Leu Leu Asp Trp Cys Gly
25 30 35 40
Asp Pro Arg Ala Phe Gln Arg Pro Phe Glu Gln Ser Leu Met Gly Cys Leu Thr Val Val
45 50 55 60
Ser Arg Val Ala Ala Gln Gln Gly Phe Asp Leu Asp Leu Gly Tyr Arg Leu Leu Ala Val
65 70 75 80
Cys Ala Ala Asn Arg Asp Lys Phe Thr Pro Lys Ser Ala Ala Leu Leu Ser Ser Trp Cys
85 90 95 100
Glu Glu Leu Gly Arg Leu Leu Leu Leu Arg His Gln Lys Ser Arg Gln Ser Asp Pro Pro
105 110 115 120
Gly Lys Leu Pro Met Gln Pro Pro
125
Its corresponding nucleotides sequence is classified as:
1 atgaattcta tggacaggca catccagcag accaatgacc gactgcagtg catcaagcag
61 cacttacaga atcctgccaa cttccacaat gccgccacgg agctgctgga ctggtgcgga
121 gacccacggg ccttccagcg gcccttcgag cagagcctga tgggctgttt gacggtggtc
181 agtcgggtgg cagcccagca aggctttgac ctggacctcg gctacagact gctggctgtg
241 tgtgctgcaa accgagacaa gttcaccccg aagtctgccg ccttgttgtc ctcctggtgc
301 gaagagctcg gccgcctgct gctgctccga catcagaaga gccgccagag cgatccccct
361 gggaaactcc ccatgcagcc ccct 。
2. the preparation method of the polyclonal antibody of prokaryotic expression GST-hZimp10 fusion rotein as claimed in claim 1, it is characterized in that utilizing GST expression system to obtain GST-hZimp10 fusion rotein, obtain anti-GST-hZimp10 antiserum(antisera) through immunize rabbit again, specifically comprise five steps:
The first step, the structure of PCR-PMD18-T/hZimp10 recombinant plasmid, hZimp10 gene N-128 fragment obtains from Genebank, gene order number is AY235683, take pcCDNA3.1-Flag-hZimp10 as template, upstream primer is 5 '-GGATCC ATGAAT TCTATG GACAGG, comprises BamHI and EcoRI restriction enzyme site; Downstream primer is 5 '-CTAGAG AGGGGG CTGCAT GGGGAG, comprise XhoI restriction enzyme site, the fragment obtained that increases is connected with PMD18-T carrier, connection product is proceeded in competence bacillus coli DH 5 alpha, containing selected clone on Amp+ agar plate, after carrying recombinant plasmid so that alkaline lysis is little, with the mono-double digestion qualification of EcoR I and BamHI, Xhol I;
Second step, the structure of prokaryotic expression carrier pGEX-4T-1, by the pMD18-T plasmid containing hZimp10 gene N-128 fragment after BamHI and Xho1 double digestion, utilize and reclaim test kit acquisition hZimp10 gene N-128 fragment, simultaneously with identical ferment treatment plasmid pGEX-4T-1, then spend the night in 16 DEG C of connections under the effect of T4DNA ligase enzyme by the hZimp10 gene N-128 fragment of recovery with through the vector pGEX-4T-1 that enzyme is cut, enzyme cuts qualification recombinant chou, and order-checking is determined further;
3rd step, the expression and purity of GST-hZimp10 fusion rotein, recombinant plasmid PGEX-4T-1/hZimp10 transformation of E. coli BL21, in picking list bacterium colony access LB/Ampr substratum, 37 DEG C of jolting overnight incubation, next day, by culture in 1: 50 ratio transfer in containing Amp+ LB substratum in, continue to be cultured to mid log phase at 37 DEG C of shaking tables, when the A600 of nutrient solution is 0.5 ~ 0.6, adding IPTG to final concentration is 0.08mmol/L, do not add IPTG person for negative control, put 25 DEG C and continue cultivation 4 ~ 5h, collected by centrifugation thalline, the expression identifying GST-hZimp10 fusion rotein is carried out with SDS-PAGE, and optimization expression condition, to increase in a large number induction, with 5000r/min in 4 DEG C of centrifugal 5min, collect thalline, by the precipitation of the NETN suspension 1L bacterium liquid of 60mL ice precooling, use ultrasonic degradation bacterium, again with 9600rpm, in 4 DEG C of centrifugal 15min, get supernatant, cross Glutathione-Sepharose4B post, first with equal-volume elution buffer 1, containing 20mM gsh, 50mM Triscl, pH=8.0, wash-out, collect elutriant, again with equal-volume elution buffer 2, containing 100mM gsh, wash twice, collect elutriant, SDS-PAGE purification Identification product,
4th step, the sero-fast preparation of the anti-hZimp10 of rabbit and purifying
With the hZimp10 fusion protein immunization Male New Zealand White Rabbit of purifying, initial immunity 500 μ g fusion roteins, the subcutaneous multi-point injection of emulsification back part is fully mixed with isopyknic complete Freund's adjuvant, ear vein blood separation of serum is got before immunity, as the serum control before immunity, the 1st booster immunization is carried out after 2wk, GST-hZimp10 fusion rotein and the incomplete Freund's adjuvant equal-volume of 500 μ g purifying mix, front and back four sole intramuscular injection, afterwards every 2wk booster immunization 1 time, after final immunization, 1wk gets ear blood, tiring of antibody is measured by ELISA method, when antibody titer reaches 1: 100 000, carotid artery bloodletting, collect serum, ProteinA/G sepharose CL-4B filler is slowly filled post, the antiserum(antisera) through dilution is added after balance pillar, coutroi velocity ensures the combination of antiserum(antisera) and filler, namely affinity chromatography makes antibodies on pillar, after 2 washings, add pH2.7 elution buffer solution by antibody elution, collect elutriant and measure the 280nm optical density(OD) of each collection tube, collection tube containing antibody is mixed, antibody after purifying and the antiserum(antisera) SDS-PAGE before purifying and coomassie brilliant blue staining are identified,
5th step, the immunology detection of hZimp10 antibody
With the new zealand white rabbit serum before GST-hZimp10 fusion protein immunization in contrast, GST-hZimp10 antibody after purifying is first diluted 10 times again after doubling dilution, indirect ELISA method is adopted to measure tiring of antibody, and adopt the anti-hZimp10 serological specificity of Western blot methods analyst, by the fusion rotein GST-hZimp10 sample of purifying, through SDS-PAGE be separated after again electrotransfer on nitrocellulose filter, 1h is closed with 5% skim-milk, drip rabbit against murine hZimp10 antiserum(antisera) successively, room temperature 2h, PBS washes 3 times, goat anti-rabbit igg 2HRP, room temperature reaction 1h, PBS washs 3 times, finally add substrate DAB to develop the color, and take pictures.
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