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

Abstract

The present invention relates to animal genetics and animal molecular cell biology, provides an expression vector capable of efficiently expressing bovine menin in eukaryotic cells, and provides necessary tools and tools for further research on the function of the gene in cells and its mechanism of action means. The gene is expressed in many organs of the body, and plays an important regulatory role in regulating the metabolism of the body. To study the function and regulatory metabolic pathway of this gene in specific tissues or cell types, it is an essential technical means to construct a eukaryotic expression vector to overexpress it under specific conditions. The construction of the bovine MEN1 gene (hereinafter referred to as bMEN1) The high-efficiency eukaryotic cell expression vector can be used in the future to provide necessary preliminary research on metabolic diseases of bovine tissues and organs, and even provide important tools and research means for model research for the treatment of human diseases.

Description

An expression vector capable of highly expressing bovine menin in eukaryotic cells

technical field

The invention relates to animal genetics and animal molecular cell biology, and provides an expression vector capable of efficiently expressing bovine menin in eukaryotic cells.

Background technique

The bovine MEN1 gene is highly homologous to human or mouse MEN1, and the homology of its encoded protein to human or mouse menin is as high as 98.69% and 96.73%, respectively. The results of human or mouse research imply that bovine MEN1/menin plays an important role in regulating the normal metabolism of bovine (dairy cows), and it is speculated that its abnormal expression is related to bovine metabolic disorders (ketosis, acidosis, fatty liver, etc.) seizures are closely related. Human menin protein is the expression product of multiple endocrine neoplasia type 1 (MEN1) gene, which encodes 610 amino acids and is a nuclear protein expressed in multiple tissues. The key pathogenic gene MEN1 gene is located at the 11q13 locus of human chromosome, and the human MEN1 gene was first identified and cloned in 1997. Human MEN1 is a familial autosomal dominant disease characterized by tumors of endocrine organs such as parathyroid glands, pancreatic islets, and anterior pituitary tumors, commonly known as Wermer syndrome. The MEN1 gene knockout mouse model caused severe developmental disorders, including developmental defects of the nervous system, heart, liver and other important organs, and died at the embryonic day 11-13, suggesting that the MEN1 gene plays a key role in cell growth and development. effect. On the other hand, the heterozygous deletion mouse model of the MEN1 gene can survive, but a parathyroid tumor similar to that of a human is also found later, and exhibits symptoms similar to human MEN1 syndrome, suggesting that menin plays a key role in the pathogenesis inhibition. Studies in recent years have shown that abnormal expression of menin is also closely related to leukemia, diabetes, (non-alcoholic) fatty liver and other diseases. At the same time, the cancerous organs caused by MEN1 are usually important organs for hormone secretion, such as the secretion of insulin by the pancreatic beta cells and the secretion of prolactin by the anterior pituitary gland, etc., thus highlighting the importance of menin in regulating the normal development of the body and balancing the metabolic process of the homeostasis Function.

At present, it has been confirmed that menin can participate in the epigenetic modification of genomic proteins, thereby affecting the transcription of downstream genes, can also interact with genomic proteins to regulate the stability of the genome, and participate in the regulation of cell cycle regulatory factors to regulate cell division and proliferation. Many researchers have begun to look forward to the use of menin/MEN1 in the treatment of some diseases. Of course, before that, a lot of research and multi-party demonstrations are needed. In order to study the role of menin in various physiological and pathological activities, or to use it in therapy, it is necessary to be able to express menin in eukaryotic cells in large quantities. During the research process, the cell environment with high expression of menin can enable researchers to explore and reveal the specific biological activities and functions that menin can regulate in vitro, and provide the necessary research basis for the overexpression of menin in pathological cells for the treatment of diseases. At present, most of the research on MEN1 in China is still discussing the relationship between MEN1 gene polymorphism and tumor development, or the influence of a certain section of MEN1 gene on cells. Specific mechanisms of cell biology impact.

Contents of the invention

The invention provides a eukaryotic expression vector capable of highly expressing the bovine menin protein, and provides necessary tools and means for further researching the function of the gene in cells and its mechanism of action. The gene is expressed in many organs of the body, and plays an important regulatory role in regulating the metabolism of the body. To study the function and regulatory metabolic pathway of this gene in specific tissues or cell types, it is an essential technical means to construct a eukaryotic expression vector to overexpress it under specific conditions. The construction of the bovine MEN1 gene (hereinafter referred to as bMEN1) The high-efficiency eukaryotic cell expression vector can provide the necessary preliminary research for the metabolic diseases of bovine tissues and organs in the future, and even provide an important tool and research means for model research for the treatment of human diseases.

The eukaryotic expression vector that can efficiently express bovine menin protein provided by the present invention is specifically pcDNA3.1-mycHis(-)A/bMEN, wherein the nucleotide sequence of the inserted MEN1 gene fragment is as shown in Seq ID No:8;

The specific method adopted by the inventors in the present invention is to first obtain the bovine wild-type full-length MEN1 gene, then construct it into pcDNA3.1-mycHis(-)A to form a recombinant plasmid, and finally express the fusion-type menin in eukaryotic cells protein; more specific steps are as follows:

1. Obtaining the full-length MEN1 gene cDNA of bovine wild type:

The mammary gland tissues of Chinese Holstein dairy cows in mid-lactation were collected and immediately frozen in liquid nitrogen, and then the total RNA was extracted with Trizol (Invetrogen, US.), and cDNA was synthesized with the Superstcript III kit (Invitrogen, US.);

Primers were designed according to the complete mRNA sequence of the bMEN1 gene (NM_001076161.2) published in Genebank, and the protection base g, EcoRI enzyme recognition site and kozak sequence gccacc were added to the 5' end of the upstream primer to obtain a brand new upstream The primer sequence is as follows (5'-ggaattcgccaccatggggctgaaggctgcccagaaaacg-3', its nucleotide sequence is shown in Seq ID No: 1), the downstream primer plus the protection base cg and HindIII enzyme digestion recognition site, to obtain a new downstream primer sequence As follows (5'-cgaagctttcagagacccttgcgctgccgcttgag-3', its nucleotide sequence is shown in Seq ID No:2);

The PCR conditions are as follows:

Using the cDNA synthesized above as a template, under the action of high-fidelity pfu enzyme (KP201-01, TIANGEN), it was pre-denatured at 94°C for 5min, followed by 35 cycles at 94°C for 45sec, 54°C for 50sec, and 72°C for 2min. 10min total extension, the length of the amplified target band is 1851bp, and its nucleotide sequence is shown in Seq ID No:3;

2. Restriction of MEN1 gene

The bMEN1 gene fragments amplified by RT-PCR in the previous step were subjected to single-enzyme digestion with EcoRI and HindIII, respectively, and the digested products were purified by 1% agarose gel electrophoresis and recovered (DP209, TIANGEN), which can be used for digestion with the digested Ligation reaction of pcDNA3.1-mycHis(-)A vector;

3. Extraction and digestion of pcDNA3.1-mycHis(-)A plasmid

After extracting the plasmid, perform double enzyme digestion with EcoRI and HindIIII, and after electrophoresis on 1% agarose gel, use the recovery kit to recover large fragments and store them at -20°C for later use;

4. Connection

The digested MEN1 gene fragment and the pcDNA3.1-mycHis(-)A carrier fragment were mixed at a molar ratio of 8-10:1 to establish a ligation reaction (20 μL system) with T ₄ DNA ligase (Promega, US.) in Connect at 16°C overnight;

5. Conversion

Transform 10 μL of the ligation product into 200 μL E. coli DH5α competent cells, take 300ul of the transformation product and spread it on the LB solid medium containing ampicillin with a final concentration of 100μg/mL, culture it upside down at 37°C for 16-18 hours, and pick a single colony out, add LB liquid medium containing 100 μg/mL ampicillin, and extract the plasmid after cultivating in a shaker at 37°C for 16 hours, which is the target product of the present invention: the expression vector of recombinant gene bMEN1: pcDNA3.1-mycHis(- )A/bMEN.

The finally obtained recombinant gene expression vector pcDNA3.1-mycHis(-)A/bMEN1 was respectively transfected into bovine mammary epithelial cells MAC-T and the most commonly used hamster ovary cell CHO in vitro expression system for in vitro research on muscle growth and development In the mouse myoblast C2C12, the high expression of MEN1 mRNA and menin protein could be detected after 24 hours. This indicates that the recombinant plasmid can express MEN1 gene and fusion menin protein in eukaryotic cells. The invention will provide a convenient and effective tool and means for researchers to further study the biological characteristics and functions of the MEN1 gene.

The present invention preferably adopts pcDNA3.1/myc-His A plasmid carrier among numerous carriers, and this carrier is a kind of carrier that is applicable to mammalian cell high-efficiency expression protein; This carrier optimizes gene with human CMV (cytomegalovirus) as promoter The expression efficiency is high, the two expression vector tags containing myc and His are convenient for the detection of expression, the resistance of the vector is ampicillin (ampicillin) resistance, which is convenient for the screening of positive clones; the selection marker of G418 (Neomycin) in the vector is helpful for stable expression Screening of cell lines established.

In summary, the eukaryotic expression vector obtained by the present invention, which can efficiently express bovine menin protein, provides necessary tools and means for further research on the function and mechanism of this gene in cells. The gene is expressed in many organs of the body, and plays an important regulatory role in regulating the metabolism of the body. To study the function and regulatory metabolic pathway of this gene in specific tissues or cell types, it is an essential technical means to construct a eukaryotic expression vector to overexpress it under specific conditions. The construction of the bovine MEN1 gene (hereinafter referred to as bMEN1) The high-efficiency eukaryotic cell expression vector can provide the necessary preliminary research for the metabolic diseases of bovine tissues and organs in the future, and even provide an important tool and research means for model research for the treatment of human diseases.

Description of drawings

Figure 1 is the electrophoresis diagram of the pcDNA3.1-mycHis(-)A-bMEN1 plasmid after EcoRI and HindIII digestion,

Figure 2 is the result of the pcDNA3.1-mycHis(-)A-bMEN1 plasmid digested with EcoRI and HindIII, in which two fragments of 5.5kb and 1.8kb are formed, M is Marker, and the band sizes indicate 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb and 0.5kb;

Figure 2 is a schematic diagram of three different types of healthy cells in culture. It has been verified that pcDNA3.1-mycHis(-)A/bMEN can be expressed normally and efficiently in it, and the three types of cells are widely representative;

In the figure A is bovine mammary gland epithelial cell MAC-T, which is used to detect the expression efficiency of bovine MEN1 gene recombinant plasmid in this cell; B is Chinese hamster ovary cell CHO, which is the most commonly used expression or production system for biotech drugs , is widely used in the research and development and industrial production of biotechnology products such as antibodies, gene recombinant protein drugs, virus vaccines, etc.; C is mouse myoblast C2C12, which is a cell line used to study muscle development and differentiation in vitro;

Figure 3 is a schematic diagram of the results of pcDNA3.1-mycHis(-)A/bMEN expressing MEN1RNA normally and efficiently in different cells; in the figure, the recombinant plasmid was transfected into MAC-T cells (A), CHO cells (B) and C2C12 cells (C) After 24h, 48h and 72h, total RNA was extracted, and after reverse transcription, quantitative PCR was used to detect the RNA expression of MEN1 and β-actin, and the expression of β-actin was used as the internal reference for the expression of MEN1 in the sample. The expression level of bMEN1 in cells transfected with empty vector at each time point was corrected to 1 to obtain the amount of bMEN1 expressed by recombinant plasmid cells at corresponding time points; the results of three different types of cells all showed that: after 24 hours of transfection of the recombinant plasmid in cells ( 24hpt) the relative expression of bMEN1 reached the highest, and the expression efficiency of 48hpt and 72hpt decreased gradually;

Figure 4 is a schematic diagram of the results of pcDNA3.1-mycHis(-)A/bMEN normally and highly expressing the protein menin in different cells;

Total protein was extracted 24h, 48h and 72h after the recombinant plasmid pcDNA3.1-mycHis(-)A/bMEN was transfected into MAC-T cells (A), CHO cells (B) and C2C12 cells (C), and carried out the detection of menin For expression detection, the expression level of β-actin was used as the internal reference for the protein loading amount of each sample. Mock indicates cells without transfection, Vector indicates cells transfected with empty vector, MEN1 indicates cells transfected with MEN1 recombinant plasmid; the results show that there is endogenous menin expression in MAC-T cells, but only cells transfected with recombinant plasmid The cells can express two menin fusion proteins that are slightly larger than the endogenous protein, and the highest transfection efficiency can be achieved at 24 hours, and then the expression efficiency decreases.

Detailed ways

Example 1

An expression vector capable of efficiently expressing bovine menin in eukaryotic cells, the acquisition of pcDNA3.1-mycHis(-)A/bMEN, specifically comprises the following steps:

1. cDNA cloning of bovine wild-type full-length MEN1 gene

The mammary gland tissues of Chinese Holstein dairy cows in mid-lactation were collected and immediately frozen in liquid nitrogen, and then the total RNA was extracted with Trizol (Invetrogen, US.), and cDNA was synthesized with the Superstcript III kit (Invitrogen, US.);

Primers were designed according to the complete mRNA sequence NM_001076161.2 of the bovine MEN1 gene published in Genebank, wherein, the sequence of the upstream primer (5'-atggggctgaaggctgcccagaaaacg-3'); in addition, protection bases g, EcoRI enzyme recognition site and the kozak sequence gccacc that enhances the expression efficiency of eukaryotic cells, so the sequence of the upstream primer is 5'-g gaattc gccaccatggggctgaaggctgcccagaaaacg-3', and its nucleotide sequence is as shown in Seq IDNo: 1; downstream The design of primers should pay attention to remove the stop codon TGA (because the expression tag in the expression vector is located downstream of the fusion protein), the selected matching sequence is other sequences upstream of TGA (5'-gagacccttgcgctgccgcttgag-3'), and the 5' end Add the restriction recognition sites of the protection bases cg and HindIII, so that the downstream primer is 5'-cg aagctt gagacccttgcgctgccgcttgag-3', and its nucleotide sequence is as shown in Seq ID No:2;

The PCR conditions are as follows:

Under the action of high-fidelity pfu enzyme (KP201-01, TIANGEN), using the synthesized cDNA as a template, add dNTP and buffer, pre-denaturation at 94°C for 5min, 94°C for 45sec, 54°C for 50sec, 72°C for 2min, after 35 cycle, 10min total extension at 72°C, the length of the amplified target band is 1851bp, and its nucleotide sequence is shown in Seq ID No:3.

2. Restriction of MEN1 gene

The MEN1 gene PCR product was subjected to EcoRI and HindIII single-enzyme digestion in turn. The method was referred to the enzyme manual and "Molecular Cloning" (third edition) (Sambrook, J. and Russell, DW.2002). Purify and recover the target fragment (DP209, TIANGEN) by agarose gel electrophoresis, and the recovered fragment after the second digestion can be used for the ligation reaction with the pcDNA3.1-mycHis(-)A restriction plasmid;

3. Extraction and digestion of pcDNA3.1-mycHis(-)A plasmid

The extraction steps of pEGFP-N2 plasmid refer to the TIANprep Rapid N96 Plasmid Extraction Kit (TIANGEN) product manual, extract the pcDNA3.1-mycHis(-)A plasmid and then perform EcoRI and HindIII enzyme digestion respectively. % agarose gel electrophoresis to purify and recover the target fragment (DP209, TIANGEN). The fragment recovered after the second digestion can be used for connection with the MEN1 digestion fragment and can be stored at -20°C for later use;

4. Connection

MEN1 digested fragments and pcDNA3.1-mycHis(-)A vector digested fragments were mixed at a molar ratio of 8-10:1, and a T4DNA ligase (Promega, US.) ligation reaction (20 μL system) was established, and placed at 16°C. Hours for the overnight ligation reaction, the steps refer to the product manual;

5. Conversion

10 μL of the ligation product was transformed into 200 μL Escherichia coli DH5α competent cells (for the method, refer to "Molecular Cloning" (third edition) (Sambrook, J. and Russell, DW.2002), and 300 ul of the transformation product was applied to a solution containing a final concentration of 100 μg/mL On the LB solid medium of ampicillin, two other solid mediums were used as controls at the same time: untransfected competent cells were used as negative controls, and competent cells transformed with pcDNA3.1-mycHis(-)A empty plasmid As a positive control; after inverting at 37°C for 16-18 hours, pick out a single colony, add LB liquid medium containing 100 μg/mL ampicillin, and culture in a shaker at 37°C for 16 hours to extract the plasmid;

6. Enzyme digestion and identification of recombinant plasmids

After the ligation product was transformed into competent cells, the extracted recombinant plasmids were identified by double digestion with EcoRI and HindIII, respectively, and the digested products were electrophoresed on 0.7% agarose gel; the pcDNA3.1-mycHis we chose (-) The length of the A plasmid is 5522bp (about 5.5kb), and the PCR amplification product of bovine MEN1cDNA is about 1851bp (about 1.8kb). Therefore, after double-digestion and electrophoresis of the recombinant plasmid by EcoRI and HindIII, the 5Kb position and the about 2Kb position respectively There is a band (see Figure 1 for identification results). It can be seen that the present invention has successfully obtained the target product: the expression vector of the recombinant gene bMEN1: pcDNA3.1-mycHis(-)A/bMEN;

7. Sequencing identification of recombinant plasmids

The recombinant plasmid identified by enzyme digestion was sent to Shanghai Bioengineering Co., Ltd. for sequence determination. The sequencing primers were T7promoter primer (5'-TAATACGACTCACTATAGGG-3') and BGH sequencing primer (5'-TAGAAGGCACAGTCGAGG-3'). Sequencing results showed that the inserted sequence was completely consistent with the sequence in Genbank, which verified that the recombinant expression vector obtained in the present invention is the expression vector of recombinant gene bMEN1: pcDNA3.1-mycHis(-)A/bMEN.

Example 2 Expression detection of pcDNA3.1-mycHis(-)A/bMEN recombinant plasmid in bovine mammary gland epithelial cell MAC-T cell, Chinese hamster ovary cell CHO and mouse myoblast C2C12

Cell culture conditions and transfection: Bovine mammary gland epithelial cells MAC-T, Chinese hamster ovary cells CHO and mouse myoblast C2C12 cells were added with double antibody (1%) and 10% FBS (Gibico, US.) in DMEM ( Gibico ^TM , US.) culture medium, 37 ° C, 5% CO ₂ conditions after two weeks, 24 hours before transfection with medium without double antibody at a density of 100 cells/well coated with 6 wells In the plate, add 2ml of DMEM medium containing 10% FBS to ^each well, and after 24 hours, the cells are confluent to 50-80%, and the pcDNA3. 1-mycHis(-)A/bMEN recombinant plasmid and pcDNA3.1-mycHis(-)A empty plasmid were transfected into three kinds of cells respectively.

After the transfected cells were cultured for 24h, 48h and 72h, the total RNA and protein of the cells were extracted respectively, and the expression efficiency of the recombinant plasmid was detected with the cells transfected with the empty vector as a control. It should be noted: After the recombinant plasmid is transfected in specific cells for 96 hours, according to the needs of the research, the screening and establishment of stable bMEN1 cell lines can be continued in the presence of G418: Empty plasmid pcDNA3.1-mycHis(- ) A transfected cell group and the recombinant plasmid pcDNA3.1-mycHis(-)A/bMEN1 transfected cell group, 1:1 subculture, G418 (Geneticin418, Invitrogen, US) selection concentration is 400ug/ml, select resistant cell clone Planted in a 96-well plate, picked a single clone colony to gradually expand and then subcultured, and maintained the cells with G418 at a concentration of 300ug/m1.

Fluorescent quantitative PCR analysis of the expression level of bMEN1 in the transfected cells with the total RNA extracted from the cells above:

The total RNA was extracted from the cells transfected with the empty vector and the recombinant plasmid, respectively, and the RT-PCR analysis of bMEN was carried out with bovine β-actin as the internal control.

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

The PCR reaction was carried out in a 96-well plate, and the reaction system was 2 μL cDNA template plus 18 μL PCR reaction solution. The reaction conditions are as follows: 95°C, 10s; 95°C, 5s, 40 cycles; 60°C, 34s. In order to avoid differences in amplification systems, β-actin was amplified while bMEN1 gene was amplified, and the ABI-7500 real-time quantitative sequence detection software automatically read the Ct value. Each template was repeated three times to obtain three Ct values, and the average value was taken as the final Ct value for comparative analysis, and the relative expression of the gene was calculated using the "average relative content = 2 ^{- △△Ct} " method. The recombinant plasmids were transfected into three different cells under the same transfection conditions, and then RT-PCR detection was performed respectively. Three transfection experiments were carried out in each cell, and the results of the three times were combined to obtain the expression efficiency of the recombinant plasmid pcDNA3.1-mycHis(-)A/bMEN1 in three different cells. As shown in Figure 3, the results after detection show that: pcDNA3.1-mycHis(-)A/bMEN can normally and highly express MEN1RNA in different cells, and the relative expression of bMEN1 (24hpt) in cells transfected with the recombinant plasmid 24h The expression efficiency of 48hpt and 72hpt decreased gradually.

Detection of bMEN1-encoded protein expression level in transfected cells

Total protein was extracted from cells transfected with empty vector and recombinant plasmid, and the relative expression of bMEN1-encoded protein menin was detected by Western-Blot. Bovine β-actin was used as an internal reference protein. Mouse-derived β-actin antibody (AA128, Beyotime) was purchased from Beijing Beyotime Institute of Biotechnology, and rabbit-derived menin polyclonal antibody (A300-105A) was purchased from BETHYL, USA. HRP-labeled goat anti-mouse IgG (A0216) and goat anti-rabbit IgG (A0208) were purchased from Beijing Beyontien Institute of Biotechnology. After the extracted protein was quantified, 15 μg of denatured protein was used for SDS-PAGE electrophoresis, membrane transfer, subsequent blocking, primary antibody incubation, and secondary antibody incubation for immune reaction (Beiyuntian). After developing and fixing (P0018, Biyuntian), the desired target band can be obtained. Scan the film (color scanner, BenQ SCANNER 5560), and use ImageJ software to analyze the gray value of the target band. The ratio of the gray value of the target protein to the internal reference protein (β-actin) is the relative expression of the protein in each treatment group , and the results are shown in Figure 4.

The results show:

1. Accurately cloned the bovine full-length MEN1 gene

The MEN1 eukaryotic expression vector we constructed, namely the pcDNA3.1-mycHis(-)A/bMEN recombinant plasmid, was completely consistent with the expected result after digestion and identification, and the sequencing results also confirmed that it was consistent with the sequence published on GeneBank;

2. The pcDNA3.1-mycHis(-)A/bMEN recombinant plasmid can normally express the fusion protein in eukaryotic cells

Because the vector plasmid pcDNA3.1-mycHis(-)A we selected has a tag protein expression gene behind its multiple cloning site, if the gene sequence we insert causes the reading frame of the plasmid itself to shift, it will not be expressed normally tagged protein. We can detect two expression bands at the same time with the menin antibody: the endogenous menin protein and the menin protein fused with the expression tag, the latter has a larger molecular weight than the endogenous menin because of the expression tag;

3. The bMEN1 gene on the recombinant plasmid can be expressed normally in eukaryotic cells

RT-PCR analysis was performed on the cells transfected with the empty plasmid and the cells transfected with the recombinant plasmid, and the relative transcription level of MEN1 in the cells transfected with the recombinant plasmid was significantly higher than the expression level of the transfected empty load (corrected to 1). Since the cells were not transfected with no load, this shows that we have successfully constructed the eukaryotic expression system of bMEN1, and the bMEN1 gene carried by the recombinant plasmid can be expressed normally in different eukaryotic cells. In this study: MAC-T cells, CHO cells and C2C12 cells all achieved high expression efficiency 24 hours after transfection with the recombinant plasmid, and then decreased.

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.