CN104694572A - Expression vector capable of efficiently expressing cow menin in eukaryotic cells - Google Patents

Abstract

The invention relates to the animal genetics and the animal molecular cell biology, and provides an expression vector capable of efficiently expressing cow menin in eukaryotic cells. The expression vector capable of efficiently expressing cow menin in the eukaryotic cells provides necessary tools and means for the further research on the functions of the gene in the cells and the functional mechanism of the gene. The gene has expression in multiple organs in an organism and plays an important regulating role in regulating organism metabolism. According to the research on the functions and metabolic regulating and controlling pathways of the gene in a particular tissue or cell type, the fact that the gene is made to be excessively expressed under a specified condition by constructing the eukaryotic expression vector is an essential technological mean. The constructed efficient eukaryotic cell expression vector of the cow MEN1 gene (hereinafter referred to as bMEN1) can be used for providing necessary earlier research for the organism metabolic diseases of each tissue and organ of a cow, and even provides important tools and research means of a mode type research for the treatment of human diseases.

Description

A kind of can at the expression vector of eukaryotic cell high expression ox menin

Technical field

The present invention relates to zoogenetics and animal molecular cytobiology, provide a kind of can at the expression vector of eukaryotic cell high expression ox menin.

Background technology

The MEN1 very high homology of ox MEN1 gene and people or mouse, the homology of its proteins encoded and people or mouse menin is respectively up to 98.69% and 96.73%.The MEN1/menin of the achievement in research hint ox of people or mouse, regulating the important regulative in ox (milk cow) body eubolism, infers that the outbreak of the metabolic disturbance diseases (ketoacidosis, oxypathy, fatty liver etc.) of its unconventionality expression and ox has substantial connection.People menin albumen is the expression product of MEN1 (multiple endocrine neoplasiatype 1, MEN1) gene, 610 amino acid of encoding, and is a nucleoprotein of expressing in many senses of organization.Its pathogenic key gene MEN1 gene is positioned at human chromosome 11q13 site, and people MEN1 gene is in first identifieds in 1997 and clone.People MEN1 is a kind of familial autosomal dominant disorder being feature with endocrine organ's tumours such as parathyroid gland, pancreas islet and prepituitary gland tumours, usually also known as wermer syndromes.MEN1 gene knock-out mice model, owing to having caused serious dysplasia, comprises the developmental defect of the vitals such as neural system, heart, liver, dead 11-13 days embryonic stage, the keying action of prompting MEN1 gene in Growth of Cells and growth course.On the other hand, MEN1 genetic heterozygosis depleted mice model can be survived, but the later stage have also discovered the parathyroidoma similar with the mankind, and shows the symptom being similar to mankind MEN1 syndromes, the crucial restraining effect of prompting menin in pathogenic process.Research in recent years shows, menin abnormal expression and leukemia, diabetes, (non-alcoholic) fatty liver diseases are also closely related.Simultaneously, the vitals of cancerous organ's normally hormone secretion that MEN1 causes, as islet β cell Regular Insulin, prepituitary gland Prolactin Secretion etc., thus more highlight the critical function of menin in the organism metabolism process of the normal development of regulation and control body, balance stable state.

Current confirmation, menin can participate in genome albumen epigenetics and modifies and then affect the transcribing of downstream gene, also can make adjustments genomic stability mutually with genome protein, participate in regulating the regulatory factor of cell cycle and the division growth of regulating cell.Many investigators have started to look forward to treatment menin/MEN1 being used for some diseases.Certainly, before this, also need to carry out large quantifier elimination and demonstration in many ways.Study the effect of menin in various physiology and pathologic event, or use it for treatment, all need can in eukaryotic cell great expression menin.In research process, the cellular environment of high expression level menin can make the external exploration of investigator disclose the regulatable concrete biologic activity of menin and function, for treatment menin process LAN in pathological cells being used for disease provides necessary Research foundation.The domestic research to MEN1 at present, many relations also resting on discussion MEN1 gene pleiomorphism and tumor development, or the impact of a certain section of MEN1 gene pairs cell, by the restriction of research material, also there is no the concrete mechanism that investigator's further investigated MEN1 gene pairs cytobiology affects.

Summary of the invention

The invention provides a kind of can the carrier for expression of eukaryon of high expression ox menin albumen, for studying function in this gene cell further and the mechanism of action provides necessary instrument and means.This gene has expression at the multiple organ of body, serves important regulating effect for adjustment organism metabolism.Study the function of this gene in particular organization or cell type and regulation and control pathways metabolism, by build carrier for expression of eukaryon make its under given conditions overexpression be a kind of requisite technique means, the efficient eukaryotic expression vector building ox MEN1 gene (hereinafter referred to as bMEN1) can be in the future provides necessary early-stage Study, even for the treatment of human disease supplies a pattern the important tool of type research and research means for ox each histoorgan organism metabolism disease.

Provided by the present inventionly the carrier for expression of eukaryon of high expression ox menin albumen can be specially pcDNA3.1-mycHis (-) A/bMEN, its nucleotide sequence of MEN1 gene fragment wherein inserted is as shown in Seq ID No:8;

Contriver in the present invention adopted concrete grammar first obtains ox wild type full-length MEN1 gene, built afterwards and enter in pcDNA3.1-mycHis (-) A, forms recombinant plasmid, finally at eukaryotic cell expression pattern of fusion menin albumen; More specifically step is as follows:

1, the acquisition of ox wild type full-length MEN1 gene cDNA:

Gather the china holstein cows mammary tissue being in lactation mid-term, frozen in liquid nitrogen immediately, use Trizol (Invetrogen, US.) to extract total serum IgE afterwards, Superstcript III test kit (Invitrogen, US.) synthesizes cDNA;

Primer is designed according to the complete mRNA sequence (NM_001076161.2) of the bMEN1 gene announced in Genebank, and add protection base g respectively at 5 ' end of upstream primer, the enzyme of EcoRI cuts recognition site and kozak sequence gccacc, obtain brand-new upstream primer sequence following (5 '-ggaattcgccaccatggggctgaaggctgcccagaaaacg-3 ', its nucleotide sequence is as shown in Seq ID No:1), downstream primer adds that the enzyme of protection base cg and HindIII cuts recognition site, obtain brand-new downstream primer sequence following (5 '-cgaagctttcagagacccttgcgctgccgcttgag-3 ', its nucleotide sequence is as shown in Seq ID No:2),

PCR condition is as follows:

With the cDNA of above-mentioned synthesis for template, at high-fidelity pfu enzyme (KP201-01, TIANGEN) under effect, through 94 DEG C of 5min denaturations, 94 DEG C of 45sec, 54 DEG C of 50sec, 72 DEG C of 2min, through 35 circulations, 72 DEG C of 10min total elongations, the object band length amplified is 1851bp, and its nucleotide sequence is as shown in Seq ID No:3;

2, the enzyme of MEN1 gene is cut

The increase bMEN1 gene fragment that obtains of upper one step RT-PCR carries out the single endonuclease digestion of EcoRI and HindIIII respectively, digestion products through 1% agarose gel electrophoresis purifying and can be used for the ligation of pcDNA3.1-mycHis (-) the A carrier after cutting with enzyme after reclaiming (DP209, TIANGEN);

3, the extraction of pcDNA3.1-mycHis (-) A plasmid, enzyme are cut

Extract after plasmid, carry out the double digestion of EcoRI and HindIIII, digestion products, after the agarose gel electrophoresis of 1%, carries out the recovery of large fragment with recovery test kit, and be stored in-20 DEG C for subsequent use;

4, connect

Enzyme cut after MEN1 gene fragment and pcDNA3.1-mycHis (-) A carrier segments mix with 8-10: 1 molar ratio, set up T ₄the ligation (20 μ L system) of DNA ligase (Promega, US.), spends the night 16 DEG C of connections;

5, transform

10 μ L connect product conversion 200 μ L bacillus coli DH 5 alpha competent cell, getting 300ul converted product, to coat containing final concentration be on the LB solid medium of 100 μ g/mL ammonia benzyl mycins, being inverted for 37 DEG C cultivates after 16-18 hour, single bacterium colony is chosen, add the LB liquid nutrient medium containing 100 μ g/mL ammonia benzyl mycins, cultivate in 37 DEG C of shaking tables after 16 hours and extract plasmid, be target product of the present invention: the expression vector of recombination bMEN1: pcDNA3.1-mycHis (-) A/bMEN.

Mouse muscle-forming cell C2C12 recombinant gene expression vector pcDNA3.1-mycHis (-) A/bMEN1 finally obtained is distinguished hamster ovary cell CHO the most frequently used in the mammary epithelial cell MAC-T of transfection ox, vitro expression systems, growing in vitro study muscle growth, can detect the high expression of mRNA and the menin albumen of MEN1 respectively after 24h hour.This shows that this recombinant plasmid can at eukaryotic expression MEN1 gene and pattern of fusion menin albumen.The biological characteristics and function of furtheing investigate MEN1 gene for numerous scientific research personnel are further provided an instrument easily and effectively and means by this invention.

The present invention preferably have employed pcDNA3.1/myc-His A plasmid vector in numerous carrier, and this carrier is a kind of carrier being applicable to mammal cell with high efficient expressing protein; This carrier optimizes the expression efficiency of gene using people CMV (cytomegalovirus) as promotor, the detection being convenient to whether to express containing myc and His two kinds of expression vector labels, carrier resistance are the screening that ammonia benzyl (ampicillin) resistance is convenient to positive colony; The screening that the selection markers that carrier contains G418 (Neomycin) contributes to stable expression cell line is set up.

In sum, what the present invention obtained can the carrier for expression of eukaryon of high expression ox menin albumen, for studying function in this gene cell further and the mechanism of action provides necessary instrument and means.This gene has expression at the multiple organ of body, serves important regulating effect for adjustment organism metabolism.Study the function of this gene in particular organization or cell type and regulation and control pathways metabolism, by build carrier for expression of eukaryon make its under given conditions overexpression be a kind of requisite technique means, the efficient eukaryotic expression vector building ox MEN1 gene (hereinafter referred to as bMEN1) can be in the future provides necessary early-stage Study, even for the treatment of human disease supplies a pattern the important tool of type research and research means for ox each histoorgan organism metabolism disease.

Accompanying drawing explanation

Fig. 1 is that pcDNA3.1-mycHis (-) A-bMEN1 plasmid cuts rear electrophoresis figure through EcoRI and HindIII enzyme,

In figure, 2 is pcDNA3.1-mycHis (-) A-bMEN1 plasmid result after EcoRI and HindIII enzyme is cut, wherein form two bar segment of 5.5kb and 1.8kb, M is Marker, and stripe size indicates 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb and 0.5kb respectively;

Fig. 2 is three kinds of dissimilar healthy cell schematic diagram in cultivating, and empirical tests pcDNA3.1-mycHis (-) A/bMEN can express wherein normally and efficiently, and three kinds of cells have representative widely;

In figure, A is bovine mammary epithelial cell MAC-T, is used to detect the expression efficiency of ox MEN1 gene recombination plasmid in this cell; B is Chinese hamster ovary cell CHO, and this cell is the most frequently used expression of biotech drug or production system, is widely used in research and development and the suitability for industrialized production of the biological technology products such as antibody, gene recombinant protein medicine, virus vaccines; C is mouse muscle-forming cell C2C12, is the clone being used to vitro system research muscle development, differentiation;

Fig. 3 is that pcDNA3.1-mycHis (-) A/bMEN expresses MEN1RNA result schematic diagram normally and efficiently in different cell; In figure after Transfected Recombinant Plasmid MAC-T cell (A), Chinese hamster ovary celI (B) and C2C12 cell (C) 24h, after 48h and after 72h, extract total serum IgE, the rna expression detection of MEN1 and β-actin is carried out with quantitative fluorescent PCR, the internal reference using the expression amount of β-actin as MEN1 expression amount in sample after reverse transcription.The expression amount of bMEN1 in each time point transfection empty carrier cell is corrected to the amount obtaining corresponding time point recombinant plasmid cell expressing bMEN1 after 1; The result of three kinds of dissimilar cells all shows: after transit cell contaminates this recombinant plasmid 24h, the relative expression quantity of (24hpt) bMEN1 reaches the highest, 48hpt and 72hpt expression efficiency reduces gradually;

Fig. 4 is pcDNA3.1-mycHis (-) A/bMEN expressing protein menin result schematic diagram normally and efficiently in different cell;

By after recombinant plasmid pcDNA3.1-mycHis (-) A/bMEN transfection MAC-T cell (A), Chinese hamster ovary celI (B) and C2C12 cell (C) 24h, extract total protein after 48h and after 72h, carry out the detection of expression of menin, the internal reference using the expression amount of β-actin as each sample albumen applied sample amount.Mock represents the cell not having transfection, and Vector represents the cell of transfection empty carrier, and MEN1 represents the cell of transfection MEN1 recombinant plasmid; Result shows: the expression that there is endogenous menin in MAC-T cell, but only have the cell of transfection recombinant plasmid can give expression to the two outer menin fusion roteins being a bit larger tham intrinsic protein, and most high transfection efficiency can be reached when 24h, expression efficiency reduces subsequently.

Embodiment

Embodiment 1

Can at an expression vector of eukaryotic cell high expression ox menin, the acquisition of pcDNA3.1-mycHis (-) A/bMEN, specifically comprises the steps:

1, the cDNA clone of ox wild type full-length MEN1 gene

Gather the china holstein cows mammary tissue being in lactation mid-term, frozen in liquid nitrogen immediately, use Trizol (Invetrogen, US.) to extract total serum IgE afterwards, Superstcript III test kit (Invitrogen, US.) synthesizes cDNA;

Primer is designed according to the complete mRNA sequence NM_001076161.2 of the ox MEN1 gene announced in Genebank, wherein, the sequence of upstream primer (5 '-atggggctgaaggctgcccagaaaacg-3 '); In addition, need to add protection base g successively at 5 ' end, the enzyme of EcoRI cuts recognition site and the kozak sequence gccacc of enhancing eukaryotic cell expression efficiency, the sequence of such upstream primer is 5 '-g gaattcgccaccatggggctgaaggctgcccagaaaacg-3 ', its nucleotide sequence is as shown in such as Seq IDNo:1; The design of downstream primer should be noted removing terminator codon TGA (being positioned at the downstream of fusion rotein owing to expressing label in expression vector); the matching sequence selected is other sequence (5 '-gagacccttgcgctgccgcttgag-3 ') of TGA upstream; also need to add that the enzyme of protection base cg and HindIII cuts recognition site at 5 ' end, such downstream primer is 5 '-cg aagcttgagacccttgcgctgccgcttgag-3 ', its nucleotide sequence is as shown in such as Seq ID No:2;

PCR condition is as follows:

At high-fidelity pfu enzyme (KP201-01, TIANGEN) under effect, with the cDNA synthesized for template, dNTP and damping fluid is added, through 94 DEG C of 5min denaturations, 94 DEG C of 45sec, 54 DEG C of 50sec, 72 DEG C of 2min, through 35 circulations, 72 DEG C of 10min total elongations, the object band length amplified is 1851bp, and its nucleotide sequence is as shown in such as Seq ID No:3.

2, the enzyme of MEN1 gene is cut

MEN1 gene PCR product carries out the single endonuclease digestion of EcoRI and HindIII successively, method reference enzyme specification sheets and " molecular cloning " (third edition) (Sambrook, J.and Russell, DW.2002), enzyme cut after product through 1% agarose gel electrophoresis purifying and reclaim object fragment (DP209, TIANGEN), the fragment that second time enzyme reclaims after cutting and terminating can be used for the ligation with pcDNA3.1-mycHis (-) A digested plasmid;

3, the extraction of pcDNA3.1-mycHis (-) A plasmid, enzyme are cut

The extraction step of pEGFP-N2 plasmid is with reference to TIANprep Rapid N96 plasmid extraction kit (TIANGEN) product description, extract the enzyme that then pcDNA3.1-mycHis (-) A plasmid carry out EcoRI and HindIII respectively to cut, enzyme cut after product through 1% agarose gel electrophoresis purifying and reclaim object fragment (DP209, TIANGEN), second time enzyme cut terminate after reclaim fragment can be used for the connection with MEN1 endonuclease bamhi, can be stored in-20 DEG C for subsequent use;

4, connect

MEN1 endonuclease bamhi and pcDNA3.1-mycHis (-) A carrier endonuclease bamhi with 8-10: 1 molar ratio mixing, set up T4DNA ligase enzyme (Promega, US.) ligation (20 μ L system), put and carry out spending the night ligation in 16 DEG C hours, step is with reference to product description;

5, transform

10 μ L connect product conversion 200 μ L bacillus coli DH 5 alpha competent cell, and (method is with reference to " molecular cloning " (third edition) (Sambrook, J.and Russell, DW.2002), getting 300ul converted product, to coat containing final concentration be on the LB solid medium of 100 μ g/mL ammonia benzyl mycins, and be coated with two other solid medium is contrast simultaneously: the competent cell of untransfected as negative control, transform the competent cell of pcDNA3.1-mycHis (-) A empty plasmid as positive control; Be inverted for 37 DEG C and cultivate after 16-18 hour, single bacterium colony is chosen, adds the LB liquid nutrient medium containing 100 μ g/mL ammonia benzyl mycins, cultivate in 37 DEG C of shaking tables after 16 hours and extract plasmid;

6, the enzyme of recombinant plasmid cuts qualification

After connecting product conversion competent cell, the above-mentioned recombinant plasmid extracted is identified after carrying out double digestion with EcoRI and HindIII respectively, enzyme cut after product on 0.7% sepharose, carry out electrophoresis; PcDNA3.1-mycHis (-) the A plasmid length that we select is 5522bp (about 5.5kb), the pcr amplification product of ox MEN1cDNA is about 1851bp (about 1.8kb), and therefore recombinant plasmid carries out respectively there is a band (qualification result is shown in accompanying drawing 1) in 5Kb position and about 2Kb position after double digestion electrophoresis through EcoRI and HindIII.Visible the present invention have successfully been obtained target product: the expression vector of recombination bMEN1: pcDNA3.1-mycHis (-) A/bMEN;

7, the order-checking qualification of recombinant plasmid

Enzyme cuts the recombinant plasmid after qualification, serve marine life Engineering Co., Ltd and carry out sequencing, sequencing primer is T7promoter primer (5 '-TAATACGACTCACTATAGGG-3 ') and BGH sequencing primer (5 '-TAGAAGGCACAGTCGAGG-3 ').Sequence in the sequence that sequencing result display is inserted and Genbank is completely the same, demonstrates the expression vector that recombinant expression vector that the present invention obtains is exactly recombination bMEN1: pcDNA3.1-mycHis (-) A/bMEN.

The detection of expression of embodiment 2pcDNA3.1-mycHis (-) A/bMEN recombinant plasmid respectively in bovine mammary epithelial cell MAC-T cell, Chinese hamster ovary cell CHO and mouse muscle-forming cell C2C12

Cell culture condition and transfection: bovine mammary epithelial cell MAC-T, Chinese hamster ovary cell CHO and mouse muscle-forming cell C2C12 are respectively at the DMEM (Gibico being added with dual anti-(1%) and 10%FBS (Gibico, US.) ^tM, US.) and in substratum, 37 DEG C, 5%CO ₂condition under cultivate two weeks after, before transfection, 24h is with not being coated with 6 orifice plates containing dual anti-substratum with the density of 100 cells/well, every hole adds the DMEM substratum of 2ml containing 10%FBS, after 24 hours later cell are paved with 50-80%, with liposome transfection (lipofectamine 2000 ^tM, Invitrogen, US.) method pcDNA3.1-mycHis (-) A/bMEN recombinant plasmid and pcDNA3.1-mycHis (-) A empty plasmid are each separately transfected into three kinds of cells.

Total serum IgE and the albumen of cell is extracted respectively, respectively with the expression efficiency of the cell of empty carrier transfection for control test recombinant plasmid after cell cultures 24h after transfection, 48h and 72h.Need to illustrate: recombinant plasmid transfection after 96 hours in specific cells, according to the needs of research, the screening and foundation that proceed stably express bMEN1 clone in case can be deposited: empty plasmid pcDNA3.1-mycHis (-) A transfectional cell group and recombinant plasmid pcDNA3.1-mycHis (-) A/bMEN1 transfectional cell group at G418, go down to posterity at 1: 1, G418 (Geneticin418, Invitrogen, US) screening concentration is 400ug/ml, selecting resistant cell colonies kind enters in 96 orifice plates, Secondary Culture after picking monoclonal colonies progressively increases, cell is maintained with the G418 of 300ug/m1 concentration.

With the quantitative fluorescent PCR analysis of the total serum IgE of said extracted cell to bMEN1 expression level in transfectional cell:

Cell after empty carrier and Transfected Recombinant Plasmid extracts total serum IgE respectively, and with ox β-actin for internal reference, the RT-PCR carrying out bMEN analyzes.

The primer sequence of bMEN1 is just 5'-gatggaggtggcatttatgg-3 ', and its nucleotide sequence is as shown in such as Seq ID No:4 and antisense 5 '-gatgtgctcatcccggtagt-3 ', and its nucleotide sequence is as shown in such as Seq ID No:5.Ox β-actin primer sequence is just 5'-cccagcacaatgaagatcaa-3', and its nucleotide sequence is as shown in such as Seq ID No:6 and antisense 5'-tagaagcatttgcggtggac-3', and its nucleotide sequence is as shown in such as Seq ID No:7.

PCR reaction is carried out in 96 orifice plates, and reaction system is that 2 μ L cDNA templates add 18 μ LPCR reaction solutions.Reaction conditions is as follows: 95 DEG C, 10s; 95 DEG C, 5s, 40 circulations; 60 DEG C, 34s.For avoiding the difference of amplification system, amplification β-actin while amplification bMEN1 gene, ABI-7500 real-time quantitative Sequence Detection Software reads Ct value automatically.Each template does three repetitions, obtains three Ct values, gets its mean value and compares analysis as final Ct value, uses " average relative content=2 ^{-△ △ Ct}" method calculates the relative expression quantity of this gene.By cells different for the transfection conditions transfection three kinds that recombinant plasmid is same respectively, then carry out RT-PCR detection respectively.In often kind of cell, carry out the transfection experiment of three times, the result of comprehensive three times obtains the expression efficiency of recombinant plasmid pcDNA3.1-mycHis (-) A/bMEN1 in three kinds of different cells respectively.As shown in Figure 3, result after detection is shown: pcDNA3.1-mycHis (-) A/bMEN can express MEN1RNA normally and efficiently in different cell, and transit cell contaminate this recombinant plasmid 24h after the relative expression quantity of (24hpt) bMEN1 reach the highest, 48hpt and 72hpt expression efficiency reduces gradually.

The detection of bMEN1 proteins encoded expression level in transfectional cell

Cell after empty carrier and Transfected Recombinant Plasmid extracts gross protein respectively, and Western-Blot detects the relative expression quantity of bMEN1 proteins encoded menin, using ox β-actin as internal reference albumen.Mouse source β-actin antibody (AA128, the green skies) is purchased from the green skies, Beijing biotechnology research institute, and rabbit source menin polyclonal antibody (A300-105A) is purchased from BETHYL, USA.HRP marks mountain sheep anti-mouse igg (A0216), goat anti-rabbit igg (A0208) purchased from the green skies, Beijing biotechnology research institute.The albumen extracted carries out SDS-PAGE electrophoresis with the metaprotein applied sample amount of 15 μ g after quantitatively, transferring film and closing subsequently, primary antibodie are hatched and anti-ly with two hatched immune response (the green skies).Required object band can be obtained after development, fixing (P0018, the green skies).Scan film (color scanner, BenQ SCANNER 5560), and with the gray-scale value of ImageJ software analysis object band, the ratio of the gray-scale value of target protein and internal reference albumen (β-actin), be the albumen relative expression quantity of each treatment group, its result as shown in Figure 4.

Result shows:

1, Niu Quanchang MEN1 gene has accurately been cloned

We build MEN1 carrier for expression of eukaryon and pcDNA3.1-mycHis (-) A/bMEN recombinant plasmid, through enzyme cut qualification with expected results completely the same, sequencing result also confirms consistent with the sequence that GeneBank announces;

2, pcDNA3.1-mycHis (-) A/bMEN recombinant plasmid can in eukaryotic cell normal expression fusion rotein

Because carrier physique grain pcDNA3.1-mycHis (-) A selected by us after its multiple clone site with label protein expressing gene, if the gene order that we insert causes the reading frame displacement of plasmid own, then can not normal expression outgoing label albumen.We can detect two band of expression with menin antibody simultaneously: endogenous menin albumen and the menin albumen having merged expression label, and the latter is owing to carrying expression label, and molecular weight is greater than endogenous menin;

3, the bMEN1 gene on recombinant plasmid can in eukaryotic cell normal expression

Respectively RT-PCR analysis is carried out to the cell of transfection empty plasmid and the cell of transfection recombinant plasmid, relative to the expression level (being corrected to 1) of transfection zero load, in transfection recombinant plasmid cell, the relative transcript levels of MEN1 is significantly higher than not unloaded transfectional cell, this illustrates that we successfully construct the eukaryotic expression system of bMEN1, recombinant plasmid with bMEN1 gene can in different eukaryotic cells normal expression.In this research: namely MAC-T cell, Chinese hamster ovary celI and C2C12 cell all reach higher expression efficiency after transfection recombinant plasmid 24h, decrease subsequently.

Claims (3)

1. at an expression vector of eukaryotic cell high expression ox menin, can it is characterized in that: be specially pcDNA3.1-mycHis (-) A/bMEN, its nucleotide sequence of MEN1 gene fragment wherein inserted is as shown in Seq ID No:8.

2. at a preparation method for the expression vector of eukaryotic cell high expression ox menin, can it is characterized in that:

Concrete step is as follows:

(1), the acquisition of ox wild type full-length MEN1 gene cDNA:

Gather the china holstein cows mammary tissue being in lactation mid-term, frozen in liquid nitrogen immediately, extract total serum IgE with Trizol afterwards, Superstcript III test kit synthesis cDNA;

According to the complete mRNA primers of the bMEN1 gene announced in Genebank, and upstream primer 5 ' end add respectively protection base g, EcoRI enzyme cut recognition site and kozak sequence gccacc, obtain brand-new upstream primer sequence, its nucleotide sequence as shown in Seq ID No:1, downstream primer add protection base cg and HindIII enzyme cut recognition site, obtain brand-new downstream primer sequence, its nucleotide sequence is as shown in Seq ID No:2;

PCR condition is as follows:

With the cDNA of above-mentioned synthesis for template, under the effect of high-fidelity pfu enzyme, through 94 DEG C of 5min denaturations, 94 DEG C of 45sec, 54 DEG C of 50sec, 72 DEG C of 2min, through 35 circulations, 72 DEG C of 10min total elongations, the object band length amplified is 1851bp, and its nucleotide sequence is as shown in Seq ID No:3;

(2), the enzyme of MEN1 gene is cut

The increase bMEN1 gene fragment that obtains of upper one step RT-PCR carries out the single endonuclease digestion of EcoRI and HindIIII respectively, digestion products through 1% agarose gel electrophoresis purifying and can be used for the ligation of pcDNA3.1-mycHis (-) the A carrier after cutting with enzyme after reclaiming;

(3), the extraction of pcDNA3.1-mycHis (-) A plasmid, enzyme are cut

Extract after plasmid, carry out the double digestion of EcoRI and HindIIII, digestion products, after the agarose gel electrophoresis of 1%, carries out the recovery of large fragment with recovery test kit, and be stored in-20 DEG C for subsequent use;

(4), connect

Enzyme cut after MEN1 gene fragment and pcDNA3.1-mycHis (-) A carrier segments mix with 8-10: 1 molar ratio, set up the ligation of T4DNA ligase enzyme, spend the night 16 DEG C of connections;

(5), transform

10 μ L connect product conversion 200 μ L bacillus coli DH 5 alpha competent cell, getting 300ul converted product, to coat containing final concentration be on the LB solid medium of 100 μ g/mL ammonia benzyl mycins, being inverted for 37 DEG C cultivates after 16-18 hour, single bacterium colony is chosen, add the LB liquid nutrient medium containing 100 μ g/mL ammonia benzyl mycins, cultivate in 37 DEG C of shaking tables after 16 hours and extract plasmid, be target product of the present invention: the expression vector of recombination bMEN1: pcDNA3.1-mycHis (-) A/bMEN.

3. one kind can in the application of expression vector in ox body or tissue metabolism's Mechanism Study of eukaryotic cell high expression ox menin.