CN1570106A - Transcription related zinc belt protein , monoclonal antibody, preparation method and its uses - Google Patents
Transcription related zinc belt protein , monoclonal antibody, preparation method and its uses Download PDFInfo
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- CN1570106A CN1570106A CN 200410034145 CN200410034145A CN1570106A CN 1570106 A CN1570106 A CN 1570106A CN 200410034145 CN200410034145 CN 200410034145 CN 200410034145 A CN200410034145 A CN 200410034145A CN 1570106 A CN1570106 A CN 1570106A
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Abstract
The invention discloses a transcription related zinc-ribbon protein, its monoclonal antibody and its preparation method and uses, belongs to gene engineering field. The protein is coded by ZNRD1 gene open reading frame nucleotides, its amino acid sequence is indicated in No.1 sequence list. The monoclonal antibody is transcription related zinc-ribbon protein obtained using hybridoma technology. The advantages of the invention: the preparation method is simple, antibody has strong specificity, and sensitivity is high. The invention can be used in gastric carcinoma cell drug tolerance identification, diagnosis and prognosis.
Description
Technical field
The present invention relates to transcription related zinc-ribbon protein, its monoclonal antibody, its preparation method and application, more specifically say so and utilize the zinc ribbon protein and the monoclonal antibody thereof of transcription related zinc-ribbon protein gene ZNRD1 preparation, and be used to detect the drug-fast application of stomach cancer cell, belong to gene engineering technology field.
Background technology
Cancer of the stomach is the modal malignant tumour of China, and chemotherapy is one of main method of treatment cancer of the stomach.But after contacting certain medicine repeatedly, tumour cell can obtain the tolerance of multiple medicine that this medicine and other structures and mechanism of action are not quite similar, promptly multidrug resistance (Multidrug Resistance, MDR).Cancer cells after the chemotherapy has produced resistance, and chemotherapy is not only invalid once more, and constantly breeds, and finally captures patient's life.Simultaneously, because cancer cells has the multidrug resistance of intersection,, also be difficult to cancer cells is thoroughly killed so change the kind of cancer therapy drug in any case.Therefore, resistance is the one of the main reasons that causes the chemotherapy of tumors failure, is the present oncotherapy most thorny issue.The resistance that solves cancer cells is a worldwide difficult problem.
The present invention uses vincristine(VCR) and induces stomach cancer cell SGC7901 (drawing from Military Medical Science Institute) to obtain resistant Gastric Cancer subbreed SGC7901/VCR, it is not only to vincristine(VCR), and resistance all taken place in Zorubicin, 5 FU 5 fluorouracil and mitomycin, prompting SGC7901/VCR is a better model in the in vitro study Multidrug Resistance of Gastric Cancer.Transcription related zinc-ribbon protein gene ZNRD1 is that the inventor uses the inhibition difference and subtracts hybridization (suppression subjectivehybridization, SSH) method screening obtains the gene of differential expression in SGC7901/VCR.Further the result shows that there is differential expression in this gene in resistant Gastric Cancer, and relevant with the multidrug resistance of cell.At present, only stay in laboratory stage, do not have any report to show that it is developed to the chemical sproof diagnostic tool of diagnosis of gastric cancer into auxiliary curing gastric cancer as yet at the research of this gene.
Summary of the invention
Main purpose of the present invention provides transcription related zinc-ribbon protein.
Second purpose of the present invention provides the monoclonal antibody of this transcription related zinc-ribbon protein.
The 3rd purpose of the present invention provides the MONOCLONAL ANTIBODIES SPECIFIC FOR method of transcription related zinc-ribbon protein.
The 4th purpose of the present invention provides the application of the monoclonal antibody of this transcription related zinc-ribbon protein.
For achieving the above object, the present invention takes following technical scheme:
Transcription related zinc-ribbon protein is the nucleotide sequence coded albumen of ZNRD1 gene open reading frame.
Described transcription related zinc-ribbon protein has the aminoacid sequence shown in the sequence table No.1.
The monoclonal antibody of transcription related zinc-ribbon protein, this monoclonal antibody are the monoclonal antibody with the transcription related zinc-ribbon protein of hybridoma technology acquisition.
Described monoclonal antibody is the IgG1 subclass, and molecular weight is about 20kD.
The method for preparing monoclonal antibody of transcription related zinc-ribbon protein, this method may further comprise the steps:
(1) albumen of preparation transcription related zinc-ribbon protein gene ZNRD1:
Use the inhibition difference and subtract hybridizing method obtains gene ZNRD1 open reading frame in resistant Gastric Cancer subbreed SGC7901/VCR nucleotide sequence, be connected among the cloning vector pUCm-T, be built into pUCm-T-ZNRD1; With NcoI and HindIII cutting plasmid pUCm-T-ZNRD1, obtain the encoding sequence of zinc ribbon protein ZNRD1, under the effect of T4DNA ligase enzyme, the directed protein expression vector pRSETA that inserts through identical double digestion, make its T7 promotor that places pRSETA control down, be built into pRSETA-ZNRD1; PRSETA-ZNRD1 is transformed into e. coli bl21, at expression in escherichia coli; Adopt Ni-NTA Spin kit purifying protein;
(2) preparation transcription related zinc-ribbon protein gene ZNRD1 coded protein specific monoclonal antibody:
Balb/c mouse with subcutaneous 6～8 weeks of routine immunization of ZNRD1 albumen back of purifying, getting immune mouse spleen cell and SP2/0 murine myeloma cell merges under 50% polyoxyethylene glycol effect, with the HAT selective medium that contains 20% calf serum with the cell cultures that merges 10～14 days, it is produced and the proliferative cell clone, be replaced by the HT substratum then; The ELISA indirect method detects the antibody in the culture supernatant, carries out cloning with limiting dilution assay, is 100% up to cloning cell antibody positive rate; Give every Balb/c mouse peritoneal injecting fluid paraffin 0.5ml; After 1～2 week, with the positive monoclonal cell of enlarged culturing with 5 * 10
5Cell/mouse is injected the mouse peritoneal of whiteruss sensitization, 1～2 week back collection ascites; Adopt affinity chromatography-Protein A-Sepharose purifying, obtain the IgG protein molecular that molecular weight is about 20kD.
The monoclonal antibody of transcription related zinc-ribbon protein is used to prepare the application that detects the chemical sproof reagent of stomach cancer cell.
Utilizing molecule clone technology to be structured in the carrier of expression in escherichia coli transcription related zinc-ribbon protein ZNRD1, adopt Ni-NTA Spin kit purifying protein, is antigen with the ZNRD1 albumen of purifying, and the application hybridoma cell technology prepares monoclonal antibody.By immunohistochemical methods, ELISA and Western blot monoclonal antibody being used for the stomach cancer cell resistance detects.
Advantage of the present invention is: it is simple to the invention provides the MONOCLONAL ANTIBODIES SPECIFIC FOR method, the height of tiring; The monoclonal antibody high specificity, highly sensitive; The present invention can be used for the judgement of the drug-fast discriminating of stomach cancer cell, diagnosis and prognosis, thereby helps people to solve the drug-fast problem of cancer of the stomach.
The present invention will be further described below in conjunction with the drawings and specific embodiments, all this areas of having done according to the disclosure of invention be equal to replacement, all belong to protection scope of the present invention.
Description of drawings
Fig. 1 analyzes the expression figure of zinc ribbon protein ZNRD1 for SDS-PAGE.
Embodiment
The protein Preparation of embodiment 1, transcription related zinc-ribbon protein gene ZNRD1
One, the amplification of ZNRD1 open reading frame nucleotide sequence
The present invention uses vincristine(VCR) and induces stomach cancer cell SGC7901 to obtain resistant Gastric Cancer subbreed SGC7901/VCR, use the inhibition difference and subtract hybridizing method (Zhao Z, J Biotechnol, 1999,73:35～41), in SGC7901/VCR, obtain the nucleotide sequence of gene ZNRD1 open reading frame, be connected among the cloning vector pUCm-T (Shanghai bio-engineering corporation), be built into pUCm-T-ZNRD1, order-checking is identified, base " a " difference in (Genomics, 2000) ZNRD1 open reading frame nucleotide sequence of the 168th base " g " and Fan report.
The cDNA sequence of transcription related zinc-ribbon protein gene ZNRD1 is as follows:
1?atgtctgtca?tggacctcgc?caatacttgc?tccagctttc?agtcggacct?ggatttctgt
61?tcagattgcg?gctcggtcct?gcctctgccc?ggggctcagg?atacggtcac?ctgtattcgc
121?tgtggcttca?acatcaacgt?tcgggacttt?gaggggaagg?ttgtgaagac?ttcggttgtg
181?ttccaccaac?tggggacagc?catgcctatg?tcggtggagg?aagggcctga?gtgccaggga
241?cctgtggttg?acaggcgctg?ccctcgatgt?ggtcatgaag?gaatggcata?ccacaccaga
301?cagatgcgtt?cagccgatga?agggcaaact?gttttttaca?cctgtaccaa?ctgcaagttc
361?caggagaagg?aagactcttg?a
The aminoacid sequence of transcription related zinc-ribbon protein gene ZNRD1 proteins encoded is as follows:
1?msvmdlantc?ssfqsdldfc?sdcgsvlplp?gaqdtvtcir?cgfninvrdf?egkvvktsvv
61?mqlgtampm?sveegpecqg?pvvdrrcprc?ghegmayhtr?qmrsadegqt?Vfytctnckf
121?qekeds
Two, the protein Preparation of ZNRD1
Adopt conventional Protocols in Molecular Biology (molecular cloning experiment guide, the third edition).
With Nco and Hind (Takara company) cutting plasmid pUCm-T-ZNRD1, obtain the encoding sequence of zinc ribbon protein ZNRD1, under T4DNA ligase enzyme (Takara company) effect, the directed insertion through the protein expression vector pRSETA of identical double digestion (Invitrogen company), make its T7 promotor that places pRSETA control down, be built into pRSETA-ZNRD1.
PRSETA-ZNRD1 is transformed into e. coli bl21 (Invitrogen company), at expression in escherichia coli.The e. coli bl21 that will contain ZNRD1 cDNA recombinant plasmid is inoculated in LB substratum (containing penbritin 50ug/mL), 37 ℃ of shaken overnight, be diluted to the LB substratum at 1: 100, it is about 0.6～0.8 to be cultured to OD600 at 37 ℃, and IPTG induces through the 1mm isopropylthio-.Cell adopts ultrasonic degradation ,-20 ℃ of preservations.
Get a certain amount of total bacterial protein sample, analyze the expression of zinc ribbon protein ZNRD1 with SDS-PAGE.Detect the albumen of suitable big or small 20kD in through inductive sample cell lysate, detection limit is 32% of a total protein.The expression electrophoretic analysis of zinc ribbon protein ZNRD1 as shown in Figure 1,1:protein marker wherein; 2:pRSETA; 3:pRSETA-ZNRD1/BL21 (uninduced); 4-8:pRSETA-ZNRD1/BL21 induced by IPTG of 2～6h.
Three, the proteic evaluation of ZNRD1
PRSETA carrier N end His-Tag is the little peptide of 6 series connection Histidines, when the fusion of the N of expressed exogenous gene product end has the polypeptide label of His-tag, can be used for detecting proteic expression, adopt Western blot and anti-His antibody (Santa Cruz product) to identify the expression of zinc ribbon protein ZNRD1.Occur the specific staining district band with anti-His antibody response at the albumen place of suitable size, prove that expression product has the ability with anti-His antibodies, ZNRD1 correctly expression in intestinal bacteria.
Four, the proteic purifying of ZNRD1
Adopt Ni-NTA Spin kit (QIAGEN company) purifying protein.Bacterium is induced in centrifugal collection, adds Buffer B (8M urea; 0.1M NaH2PO4; 0.01MTris-HCl; PH8.0), room temperature is shaken 1h, centrifugal 20～the 30min of 10 000g, collect supernatant, use the Ni-NTA column purification, (prescription is with Buffer B for Buffer C, pH6.3) wash twice, (prescription is with Buffer B, and pH4.5) wash-out twice, and SDS-PAGE analyzes purified product can see single purifying band for Buffer E.
Embodiment 2, transcription related zinc-ribbon protein prepare ZNRD1 coded protein specific monoclonal antibody
ZNRD1 albumen with purifying is antigen, with hybrid cell knurl technology (Kohler, the Milstein.Nature.1975 of Kohler and Milstein foundation; 256:495～497), prepare monoclonal antibody of the present invention.
Balb/c mouse (The Fourth Military Medical University's Experimental Animal Center) with subcutaneous 6～8 weeks of routine immunization of ZNRD1 albumen back of purifying, first immunisation is with equivalent Freund's complete adjuvant (Sigma company) emulsification antigen, every mouse injection 0.2ml (containing protein 20 μ g), two weeks of every interval with Freund's incomplete adjuvant (Sigma company) emulsification antigen with the method duplicate injection, booster immunization twice merges first three sky and strengthens once.Getting immune mouse spleen cell and SP2/0 murine myeloma cell (The Fourth Military Medical University's Experimental Animal Center) merges under 50% polyoxyethylene glycol effect, with the HAT selective medium that contains 20% calf serum (Sigma company) with the cell cultures that merges 10～14 days, it is produced and the proliferative cell clone, be replaced by HT substratum (Sigma company) then.ZNRD1 albumen with purifying is that antigen is made ELISA indirect method (Xu Zhikai, practical monoclonal antibody technique, first version, 1992) antibody in the detection culture supernatant, the hybridoma of choosing the maximum secretion antibody of specific antibody OD value carries out cloning with limiting dilution assay, subclone 3～4 times is 100% up to cloning cell antibody positive rate.
Give every Balb/c mouse peritoneal injecting fluid paraffin 0.5ml.After 1～2 week, with the positive monoclonal cell (5 * 10 of enlarged culturing
5Cell/mouse) mouse peritoneal of injection whiteruss sensitization, 1～2 week back collection ascites.
Adopt affinity chromatography-Protein A-Sepharose purifying (fine works molecular biology experiment guide, first version), identify that through SDS-PAGE ascites IgG molecular weight of albumen is about 20kD, gel scanning analysis content reaches more than 95%.On the Hybridoma Cell Culture in the cleer and peaceful ascites ELISA of monoclonal antibody tire and be respectively 10
4With 10
5Get the Hybridoma Cell Culture supernatant, press the operation of Mouse Monoclonal Antibody Isotyping Kit (Gibco company) specification sheets and identify that the ZNRD1 monoclonal antibody that the present invention obtains is the IgG1 subclass.
The ZNRD1 albumen of prokaryotic expression is made Western blot with the ZNRD1 monoclonal antibody, compare with other fusion roteins simultaneously with belt transect His-tag polypeptide label, the ZNRD1 monoclonal antibody is only discerned ZNRD1 albumen as a result, the reaction and other fusion rotein bands all are negative, show the ZNRD1 monoclonal antibody at the only anti-ZNRD1 albumen of protokaryon level, not anti-His-tag polypeptide label.From stomach cancer cell be SGC7901, stomach organization and nearby healthy tissues extract total protein, make Western blot with the ZNRD1 monoclonal antibody, the result is a positive reaction band in about 20kD place, be contrast with anti-His antibody simultaneously, the result all is negative at the ZNRD1 albumen at histocyte 20kD place, show that the ZNRD1 monoclonal antibody has the specificity of height in the eucaryon level, only anti-ZNRD1 albumen, with other histocyte albumen no cross reactions, can be used for detecting the ZNRD1 albumen of expressing in cell and the tissue.
The application of embodiment 3.ZNRD1 monoclonal antibody
One, the location of ZNRD1 albumen in stomach cancer cell system
The ZNRD1 monoclonal antibody is made immunocytochemistry and is detected, and it is painted that the result shows that the karyon of SGC7901 culturing cell is the specificity positive, and the positive is painted to be evenly distributed in nucleus, illustrates that ZNRD1 albumen is the nucleus protein molecular.
Two, the expression of ZNRD1 albumen in stomach cancer cell system
With the ZNRD1 monoclonal antibody by immunocytochemistry and Western blot, analyze the expression of ZNRD1 protein level among stomach cancer cell SGC7901 and the resistant Gastric Cancer subbreed SGC7901/VCR, as seen ZNRD1 albumen is weak positive expression at the karyon of SGC7901 cell, karyon at the SGC7901/VCR cell is high expression level, and the result shows that the multidrug resistance of ZNRD1 and stomach cancer cell has certain dependency.
Three, ZNRD1 monoclonal antibody application in animal body
With 6～8 all Balb/c nude mouses (The Fourth Military Medical University's Experimental Animal Center) is research object, sets up cancer of the stomach resistance model by abdominal cavity chemotherapy induced tumor at intermittence resistance.At first with stomach cancer cell SGC7901 suspension with 5 * 10
6It is subcutaneous that cell count is inoculated in the nude mice back, sets up the subcutaneous model of nude mice cancer of the stomach.
Behave by the dosage of people's kg body weight conversion dosage nude mouse 50 times.An about week after the one-tenth knurl; at mouse peritoneal injection chemotherapeutics mitomycin (1mg/kg; Shanghai Xinya Pharmaceutical Industry Co. Ltd.), Zorubicin (0.8mg/kg; the production of Shantou Mingzhi medicine company limited), vincristine(VCR) (0.15mg/kg; Hualian Pharmaceutical Co., Ltd., Shanghai); 2 times/week, in totally 6 weeks, set up nude mice cancer of the stomach resistance model.Use the ZNRD1 monoclonal antibody, detect in vincristine(VCR) inductive nude mice drug-resistant tumor tissue by immunohistochemical methods, ELISA and Westernblot, the protein expression level of ZNRD1 is variant.
Facts have proved that it is simple to the invention provides the MONOCLONAL ANTIBODIES SPECIFIC FOR method, the height of tiring; The monoclonal antibody high specificity, highly sensitive; The present invention can be used for the judgement of the drug-fast discriminating of stomach cancer cell, diagnosis and prognosis, thereby helps people to solve the drug-fast problem of cancer of the stomach.
Sequence table
＜110〉word plum opens
Dai Ming, Fan
＜120〉transcription related zinc-ribbon protein, monoclonal antibody, preparation method and application
<130>
<160>1
<170>PatentIn?version?3.1
<210>1
<211>126
<212>PRT
＜213〉Genus Homo, and ethnic group (Homo sapiens, human)
<400>1
Met?Ser?Val?Met?Asp?Leu?Ala?Asn?Thr?Cys?Ser?Ser?Phe?Gln?Ser?Asp
1 5 10 15
Leu?Asp?Phe?Cys?Ser?Asp?Cys?Gly?Ser?Val?Leu?Pro?Leu?Pro?Gly?Ala
20 25 30
Gln?Asp?Thr?Val?Thr?Cys?Ile?Arg?Cys?Gly?Phe?Asn?Ile?Asn?Val?Arg
35 40 45
Asp?Phe?Glu?Gly?Lys?Val?Val?Lys?Thr?Ser?Val?Val?Phe?His?Gln?Leu
50 55 60
Gly?Thr?Ala?Met?Pro?Met?Ser?Val?Glu?Glu?Gly?Pro?Glu?Cys?Gln?Gly
65 70 75 80
Pro?Val?Val?Asp?Arg?Arg?Cys?Pro?Arg?Cys?Gly?His?Glu?Gly?Met?Ala
85 90 95
Tyr?His?Thr?Arg?Gln?Met?Arg?Ser?Ala?Asp?Glu?Gly?Gln?Thr?Val?Phe
100 105 110
Tyr?Thr?Cys?Thr?Asn?Cys?Lys?Phe?Gln?Glu?Lys?Glu?Asp?Ser
115 120 125
Claims (6)
1, transcription related zinc-ribbon protein is characterized in that: this albumen is the nucleotide sequence coded albumen of ZNRD1 gene open reading frame.
2, transcription related zinc-ribbon protein according to claim 1 is characterized in that: described transcription related zinc-ribbon protein has the aminoacid sequence shown in the sequence table No.1.
3, the monoclonal antibody of transcription related zinc-ribbon protein is characterized in that: this monoclonal antibody is with the claim 1 of hybridoma technology acquisition or the monoclonal antibody of 2 described transcription related zinc-ribbon protein.
4, the monoclonal antibody of transcription related zinc-ribbon protein according to claim 3 is characterized in that: described monoclonal antibody is the IgG1 subclass, and molecular weight is about 20kD.
5, the method for preparing monoclonal antibody of transcription related zinc-ribbon protein, this method may further comprise the steps:
(1) albumen of preparation transcription related zinc-ribbon protein gene ZNRD1:
Use the inhibition difference and subtract hybridizing method obtains gene ZNRD1 open reading frame in resistant Gastric Cancer subbreed SGC7901/VCR nucleotide sequence, be connected among the cloning vector pUCm-T, be built into pUCm-T-ZNRD1; With Nco I and HindIII cutting plasmid pUCm-T-ZNRD1, obtain the encoding sequence of zinc ribbon protein ZNRD1, under the effect of T4DNA ligase enzyme, the directed protein expression vector pRSETA that inserts through identical double digestion, make its T7 promotor that places pRSETA control down, be built into pRSETA-ZNRD1; PRSETA-ZNRD1 is transformed into e. coli bl21, at expression in escherichia coli; Adopt Ni-NTA Spinkit purifying protein;
(2) preparation transcription related zinc-ribbon protein gene ZNRD1 coded protein specific monoclonal antibody:
Balb/c mouse with subcutaneous 6～8 weeks of routine immunization of ZNRD1 albumen back of purifying, getting immune mouse spleen cell and SP2/0 murine myeloma cell merges under 50% polyoxyethylene glycol effect, with the HAT selective medium that contains 20% calf serum with the cell cultures that merges 10～14 days, it is produced and the proliferative cell clone, be replaced by the HT substratum then; The ELISA indirect method detects the antibody in the culture supernatant, carries out cloning with limiting dilution assay, is 100% up to cloning cell antibody positive rate; Give every Balb/c mouse peritoneal injecting fluid paraffin 0.5ml; After 1～2 week, with the positive monoclonal cell of enlarged culturing with 5 * 10
5Cell/mouse is injected the mouse peritoneal of whiteruss sensitization, 1～2 week back collection ascites; Adopt affinity chromatography-Protein A-Sepharose purifying, obtain the IgG protein molecular that molecular weight is about 20kD.
6, the monoclonal antibody of transcription related zinc-ribbon protein is used to prepare the application that detects the chemical sproof reagent of stomach cancer cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881770A (en) * | 2009-05-08 | 2010-11-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN105316417A (en) * | 2015-11-27 | 2016-02-10 | 北京泱深生物信息技术有限公司 | ZNRD1 gene and expression products thereof serving as diagnosis and treatment targets of intracranial aneurysm |
-
2004
- 2004-04-26 CN CN 200410034145 patent/CN1570106A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101881770A (en) * | 2009-05-08 | 2010-11-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN101881770B (en) * | 2009-05-08 | 2013-07-10 | 青岛农业大学 | Method for preparing porcine circovirus type 2 colloidal gold antibody fast test strip |
| CN105316417A (en) * | 2015-11-27 | 2016-02-10 | 北京泱深生物信息技术有限公司 | ZNRD1 gene and expression products thereof serving as diagnosis and treatment targets of intracranial aneurysm |
| CN105316417B (en) * | 2015-11-27 | 2018-11-30 | 北京泱深生物信息技术有限公司 | The diagnosis and treatment target of ZNRD1 gene and its expression product as intracranial aneurysm |
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