CN115044585A

CN115044585A - Eukaryotic cell promoter CF1 and application thereof in cell gene expression

Info

Publication number: CN115044585A
Application number: CN202210710505.1A
Authority: CN
Inventors: 黄浩; 杜君卿; 邱猛生
Original assignee: Zhejiang Ousaisi Biotechnology Co ltd
Current assignee: Zhejiang Ousaisi Biotechnology Co ltd
Priority date: 2022-06-22
Filing date: 2022-06-22
Publication date: 2022-09-13
Anticipated expiration: 2042-06-22
Also published as: CN115044585B

Abstract

The invention discloses a eukaryotic cell promoter CF1 and application thereof in cell gene expression, comprising a nucleotide sequence shown as SEQ ID No. 1; the promoter designed by the invention has shorter sequence length and stronger capability of driving the target gene positioned at the downstream of the promoter to express, and can be used for improving the expression level of target protein in various eukaryotic cell expression systems; particularly when used in gene delivery systems for various viral vectors, which have a restriction on the length of the nucleic acid sequences to be accommodated, the CF1 promoter can accommodate the insertion of longer foreign gene fragments while increasing the expression level of the foreign gene. The invention can be applied to the improvement of the expression process of various proteins including monoclonal antibodies and cytokines.

Description

Eukaryotic cell promoter CF1 and application thereof in cell gene expression

Technical Field

The invention relates to the technical field of biological gene expression regulation, in particular to a eukaryotic cell promoter CF1 and application thereof in cell gene expression.

Background

Genes generally consist of a coding region and an expression control region (i.e., a promoter). The promoter determines the temporal and spatial characteristics and the expression strength of the expression of the gene. Life science, medical research and industrial production all need to use different promoters to realize the expression of protein products of target genes in various systems. Different promoters may have species specificity, cell type preference, and the ability to drive gene expression also vary. Eukaryotic cells, particularly mammalian cells, are important systems for protein expression, production, and functional validation thereof. Protein expression products, such as growth factors, enzymes, antibodies, etc., from eukaryotic cells, particularly mammalian cells, often have activities not available for prokaryotic expression. However, eukaryotic, and in particular mammalian, expression systems are much weaker than prokaryotic cells. Therefore, increasing the expression level of target genes in eukaryotic cells is an important issue in eukaryotic cell expression systems. The strength of gene expression in eukaryotic cells is the most important factor, in addition to factors such as culture conditions and cell types. Among the common promoters capable of driving strong expression of a target gene, the CMV promoter and CAG promoter are commonly used.

CMV is a mammalian strong expression promoter derived from human cytomegalovirus, but its expression level fluctuates greatly depending on host cells and is also easily silenced. The CAG promoter consists of an enhancer region of a CMV promoter, a chicken beta-actin promoter and an intron region, and an intron fragment of rabbit beta-globin. The CAG promoter is a strong promoter commonly used at present, has higher activity than other promoters such as CMV, EF1 (infection factor-1a), PGK and the like, and is widely used for expression of recombinant proteins including antibodies. However, the CAG promoter sequence is too long, reaching 1.7 kb. This feature makes CAG promoters less useful in many viral infection-dependent systems because of the limited length of genetic material that can be packaged by the viral particle, and too long a promoter often results in a limited length of target gene that can be expressed. For this reason, there is a need to develop a new promoter having a shorter sequence length, which is capable of driving the expression of a foreign gene in mammalian cells more efficiently. The CAG promoter achieves a strategy to increase promoter activity by combining several promoter elements, suggesting that the combination of different promoters may be a way to obtain a more potent promoter.

Disclosure of Invention

The invention provides a eukaryotic cell promoter CF1 and application thereof in cell gene expression, so as to solve the problems in the prior art.

The scheme of the invention is as follows:

a eukaryotic cell promoter CF1, comprising a nucleotide sequence shown in SEQ ID No. 1.

SEQ ID No.1：

Preferably, the promoter CF1 comprises a strong CMV promoter at the 5 '-end and a medium strength EF1 promoter at the 3' -end, and further comprises a connecting region.

Preferably, the CF1 promoter directly drives a plasmid or a vector for expressing a target gene.

As a preferred technical solution, the plasmid is used for preparing cells or cell lines expressing protein products; the vector is used for preparing cells or cell lines expressing protein products.

The invention also discloses an expression application of the exogenous gene driven by the CF1 promoter, including growth factor and antibody application.

The invention also discloses a pCF1 recombinant plasmid, wherein the skeleton of the plasmid is a pCAGGS plasmid, the CAG promoter of the pCAGGS plasmid is replaced by the CF1 promoter, and NheI and BsrGI are introduced behind the promoter to be used as multiple cloning sites.

The eukaryotic cell promoter CF1 and the application thereof in cell gene expression, which adopt the technical scheme, comprise a nucleotide sequence shown as SEQ ID No. 1.

The invention has the advantages that:

the CF1 promoter has gene expression regulation capability which is several times higher than that of common CMV and EF1, and particularly can obviously improve the expression of protein with larger molecular weight.

The CF1 promoter has the ability to drive the expression of foreign genes comparable to the widely used CAG promoter, but is shorter in length and more suitable for virus-dependent gene delivery systems. The limited length of foreign gene fragments contained in various viral vectors, with the shorter CF1 promoter, allows for the expression of proteins of greater molecular weight through a viral delivery system.

3. The CF1 promoter designed by the invention can be used as an exogenous gene expression regulation element and is used for being inserted into various eukaryotic cell expression vectors or being recombined into the genome of a host cell, thereby driving the high-efficiency expression of a downstream target gene;

the use of host cells and organisms having a desired trait or characteristic controlled by a promoter comprising the invention is also part of the invention. Compared with other promoters, the promoter designed by the invention has shorter sequence length and stronger capability of driving the target gene located at the downstream, and can be used for improving the expression level of target protein in various eukaryotic cell expression systems. The invention can be applied to the improvement of the expression process of various proteins including monoclonal antibodies and cytokines.

Drawings

FIG. 1 is a graph comparing the effect of EGFP expression driven by the CF1 promoter with other promoters;

FIG. 2 is a graph showing the effect of the CF1 promoter-driven MYRF-N, MYRF-FL expression compared to other promoters by Western Blot;

FIG. 3 shows the respective plasmid maps of the modified CF1, EF1, CMV, CAG promoters.

Detailed Description

In order to make up for the above deficiencies, the present invention provides a eukaryotic promoter CF1 and its application in cell gene expression to solve the above problems in the background art.

SEQ ID No.1：

The promoter CF1 comprises a strong CMV promoter at the 5 '-end and a medium strength EF1 promoter at the 3' -end, and also comprises a connecting region.

The CF1 promoter directly drives a plasmid or a vector for expressing a target gene.

The use of said plasmid to prepare a cell or cell line expressing a protein product; the vector is used for preparing cells or cell lines expressing protein products.

In order to make the technical means, the creation characteristics, the achievement purposes and the effects of the invention easy to understand, the invention is further described with the specific embodiments.

Example 1: vector construction method

Acquisition of the CF1 promoter fragment:

the CF1 promoter is formed by combining a CMV promoter and an EF1a promoter, and two DNA fragments are spliced together by the overlapping extension PCR technology. Wherein the CMV promoter fragment is derived from pCDH-CMV plasmid, and the EF1a promoter fragment is derived from pCDH-EF1s plasmid. Wherein the CMV promoter DNA sequence is shown as SEQ ID No. 2; the EF1 promoter DNA sequence is shown in SEQ ID No. 3;

the primers were designed as follows:

CMV-F:5-GGACTAGTattatgcccagtacatgac-3

CF1-F:

5-ctagtcatcattgatgtcacatcaacgcaggtacgaaggatctgcgatcgctccggtgc-3

CF1-R:5-gttgatgtgacatcaatgatgactagaatcttctatggaggtcaaaacagcg-3

EF-R:

5-gcgaattctgtacaggacatctggatgatggctagctggcgtctagcgtaggcgccggtcacagcttggat-3

PCR-1# was performed using CMV-F and CF1-R primers as a template for pCDH-CMV (Addgene, #72265) and the target product was about 390 bp. PCR-2# amplification was performed using primers CF1-F and EF-R as primers and plasmid pCDH-EF1s (Addgene, #72484) to obtain a target product of about 630 bp. The 2 times of PCR are 30uL systems and 5 cycles. The enzyme was PrimeSTAR GXL DNA Polymerase from TAKARA, and the amplification system was described in the DNA Polymerase instruction. And after the PCR-1# and the PCR-2# are finished, performing a third PCR reaction by using the two PCR products as templates (the dosage is 1uL/30uL system) and CMV-F and EF1-R as primers to obtain the PCR-3 #. The PCR reaction system was as above, but the amplification was 60uL, the number of cycles was set to 30, and the target fragment size was about 980 bp. After the amplification was completed, the PCR-3# product was subjected to 1% agarose gel electrophoresis, and then the objective fragment was purified by a gel recovery kit (50uL water eluted product). The target fragment is a CF1 promoter DNA fragment formed by tandem splicing CMV and EF1 promoters. The target fragment was digested with SpeI and EcoRI (enzymes from Thermo, # FD1254, # FD0274), the system was 50uL, the product was recovered from gel (43 uL, Buffer 5 uL), and each of the two endonucleases was incubated at 37 ℃ overnight. And recovering the PCR product subjected to enzyme digestion to 30uL deionized water by using the DNA Clean-Up kit for later use.

Construction of the pCF1 plasmid:

the pCAGGS plasmid 5ug, 50uL system, was digested with SpeI and EcoRI in two enzymes, each 2.5uL, incubated overnight at 37 ℃. The 1% agarose gel was run, the 1.7kb CAG promoter fragment was discarded, and the target fragment of about 3.1kb was purified by a gel recovery kit (50uL water elution product). This fragment was ligated with the CF1 fragment described previously: 1uL of 3.1kb double-restriction enzyme fragment, 5uL of CF1 double-restriction enzyme fragment, 2.5uL of deionized water, 1uL of Ligase Buffer, and 0.5uL of T4 Ligase. Ligation was performed at room temperature for 30min to transform E.coli DH5a competent cells. The next day, monoclonal colonies were picked for sequencing. The clone with correct sequencing can be inoculated and cultured, and then the plasmid is extracted. The pCF1 plasmid is obtained, the DNA sequence of the pCF1 plasmid is shown in SEQ ID No.5, the plasmid replaces the CAG promoter of pCAGGS with a new CF1 promoter, and a multiple cloning site is introduced behind the CAG promoter, and the pCF1 plasmid contains NheI and BsrGI restriction enzyme recognition sites and can be used for inserting a subsequent exogenous gene fragment. Wherein the DNA sequence of the CAG promoter is shown as SEQ ID No. 4.

Construction of pCMV, pEF1, pCAG plasmids

In order to compare the ability of several different promoters to express foreign genes, we used the same method, after PCR of DNA fragments of CMV promoter, EF1 promoter, and CAG promoter, after SpeI and EcoRI double digestion and recovery, they were ligated to the 3.1kb plasmid backbone after SpeI and EcoRI double digestion, respectively, to obtain 3 plasmids, pCMV, pEF1, and pCAG. These plasmids all have the same NheI and BsrGI multiple cloning sites as pCF1 plasmid, and can be used for inserting exogenous gene fragments. The PCR template of CAG promoter is plasmid pCAGGS. The primers required for PCR of each promoter fragment were as follows:

CMV-F:5-GGACTAGTattatgcccagtacatgac-3

CMV-R:

5-GCGAATTCtgtacaggacatctggatgatgGCTAGCactagaatcttctatggaggtcaaaacag-3

EF1-F:5-GGACTAGTTGTCACATCAACGCAgGTACGaaggatctgcgatcgctccggtgc-3

EF-R:

5-GCGAATTCgtacgggacatctggatgatgtctagatggcgtctagcgtaggcgccggtcacagcttggat-3

CAG-F:5-GGACTAGTtattaatagtaatcaattacggggtc-3

CAG-R:

5-GCGAATTCtgtacaggacatctggatgatgGCTAGCtttgccaaaatgatgagacagcacaataacc-3。

example two:

to verify the ability of the CF1 promoter to drive expression of foreign genes, we cloned EGFP gene fragments into pEF1, pCMV, pCAG, and pCF1 plasmids, respectively. HEK293T cells were transfected after plasmid purification (1 ug of plasmid was transfected in one well of a 12-well plate) and EGFP expression was visualized by photographing 24 hours later. The results show that CMV is slightly more active than the EF1 promoter, while the expression level of EGFP driven by the CAG and CF1 promoters is significantly higher (about 2.5 times) than the EF1 and CMV promoters (as shown in FIG. 1).

EGFP PCR primers were as follows (XbaI, Acc65I double digestion, NheI, BsrGI for isocaudare):

EGFP-F-XbaI:5-GCTCTAGAgccaccATGGTGAGCAAGGGCGAGGAGCTG-3

EGFP-R-Acc65I:5-GCGCTGGTACCttaCTTGTAgAGCTCGTCCATGCCGAG-3

because the EGFP protein has a smaller molecular weight, the expression of the protein with a larger molecular weight driven by CF1 and CMV promoters is further tested. To this end, we cloned the mouse MYRF gene and its partial fragments into the pCF1 plasmid, obtaining two plasmids, pCF1-MYRF-N and pCF 1-MYRF-FL. Wherein the pCF1-MYRF-FL plasmid expresses the full length of MYRF (NCBI ID: XP _006526992.1, containing K592R mutation), contains 1138 amino acid residues and belongs to protein with larger molecular weight; pCF1-MYRF-N expresses a fragment of MYRF comprising the N-terminal 587 amino acid residues, belonging to a medium molecular weight protein. The N ends of MYRF-FL and MYRF-N are fused with Flag tags for detecting and verifying the expression of the MYRF-FL and the MYRF-N. The same fragment was ligated to the pCMV vector. HEK293T cells were transfected after plasmid purification, and after 36 hours, Western Blot analysis was performed on lysed cells (the expression level of the target protein was represented by the detection result of Flag-tagged antibody). The results show that for a MYRF-N protein of about 80kDa, more than 3-fold expression of the CMV promoter alone can be obtained with protein expression driven by the CF1 promoter. Compared with the CMV promoter, the CF1 promoter can also increase the expression level of the protein to 3 times for MYRF-FL protein with the molecular weight of 160kDa, and the expression level is similar to that of the CAG promoter (as shown in figure 2).

PCR primers corresponding to MYRF-N and MYRF-FL are respectively as follows:

MYRF-F:

5-GCTCTAGAgccaccATGGACTACAAAGACGACGATGACAAGatggaggtggtggacgagaccgaag-3

MYRF-N-R:5-GCGCTGGTACCTTAagggtgcataagagaacccatgac-3

MYRF-FL-R:5-GCGCTGGTACCTTAgtcacacaggcggtagaagtggaag-3

the template used for PCR is cDNA obtained by reverse transcription of total RNA extracted from adult mouse brain. Primers MYRF-F and MYRF-N-R are used to obtain MYRF-N fragments, and the combination of primers MYRF-F and MYRF-FL-R is used to obtain MYRF-FL fragments. The DNA sequence of the full-length MYRF protein is shown in SEQ ID No. 6.

The foregoing shows and describes the general principles, essential features, and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, which are described in the specification and illustrated only to illustrate the principle of the present invention, but that various changes and modifications may be made therein without departing from the spirit and scope of the present invention, which fall within the scope of the invention as claimed. The scope of the invention is defined by the appended claims and equivalents thereof.

Sequence listing

<110> Zhejiang Oseisi Biotechnology Ltd

<120> eukaryotic cell promoter CF1 and application thereof in cell gene expression

<130> /

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 944

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

actagtatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 60

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 120

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 180

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 240

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc 300

gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga TTCTAGTCAT 360

CATTGATGTC ACATCAACGC AGGTACGaag gatctgcgat cgctccggtg cccgtcagtg 420

ggcagagcgc acatcgccca cagtccccga gaagttgggg ggaggggtcg gcaattgaac 480

gggtgcctag agaaggtggc gcggggtaaa ctgggaaagt gatgtcgtgt actggctccg 540

cctttttccc gagggtgggg gagaaccgta tataagtgca gtagtcgccg tgaacgttct 600

ttttcgcaac gggtttgccg ccagaacaca gctgaagctt cgaggggctc gcatctctcc 660

ttcacgcgcc cgccgcccta cctgaggccg ccatccacgc cggttgagtc gcgttctgcc 720

gcctcccgcc tgtggtgcct cctgaactgc gtccgccgtc taggtaagtt taaagctcag 780

gtcgagaccg ggcctttgtc cggcgctccc ttggagccta cctagactca gccggctctc 840

cacgctttgc ctgaccctgc ttgctcaact ctacgtcttt gtttcgtttt ctgttctgcg 900

ccgttacaga tccaagctgt gaccggcgcc tacgctagac gcca 944

<210> 2

<211> 350

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

actagtatta tgcccagtac atgaccttat gggactttcc tacttggcag tacatctacg 60

tattagtcat cgctattacc atggtgatgc ggttttggca gtacatcaat gggcgtggat 120

agcggtttga ctcacgggga tttccaagtc tccaccccat tgacgtcaat gggagtttgt 180

tttggcacca aaatcaacgg gactttccaa aatgtcgtaa caactccgcc ccattgacgc 240

aaatgggcgg taggcgtgta cggtgggagg tctatataag cagagctcgt ttagtgaacc 300

gtcagatcgc ctggagacgc catccacgct gttttgacct ccatagaaga 350

<210> 3

<211> 557

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

aaggatctgc gatcgctccg gtgcccgtca gtgggcagag cgcacatcgc ccacagtccc 60

cgagaagttg gggggagggg tcggcaattg aacgggtgcc tagagaaggt ggcgcggggt 120

aaactgggaa agtgatgtcg tgtactggct ccgccttttt cccgagggtg ggggagaacc 180

gtatataagt gcagtagtcg ccgtgaacgt tctttttcgc aacgggtttg ccgccagaac 240

acagctgaag cttcgagggg ctcgcatctc tccttcacgc gcccgccgcc ctacctgagg 300

ccgccatcca cgccggttga gtcgcgttct gccgcctccc gcctgtggtg cctcctgaac 360

tgcgtccgcc gtctaggtaa gtttaaagct caggtcgaga ccgggccttt gtccggcgct 420

cccttggagc ctacctagac tcagccggct ctccacgctt tgcctgaccc tgcttgctca 480

actctacgtc tttgtttcgt tttctgttct gcgccgttac agatccaagc tgtgaccggc 540

gcctacgcta gacgcca 557

<210> 4

<211> 1762

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

TATTAATAGT AATCAATTAC GGGGTCATTA GTTCATAGCC CATATATGGA GTTCCGCGTT 60

ACATAACTTA CGGTAAATGG CCCGCCTGGC TGACCGCCCA ACGACCCCCG CCCATTGACG 120

TCAATAATGA CGTATGTTCC CATAGTAACG CCAATAGGGA CTTTCCATTG ACGTCAATGG 180

GTGGACTATT TACGGTAAAC TGCCCACTTG GCAGTACATC AAGTGTATCA TATGCCAAGT 240

ACGCCCCCTA TTGACGTCAA TGACGGTAAA TGGCCCGCCT GGCATTATGC CCAGTACATG 300

ACCTTATGGG ACTTTCCTAC TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG 360

GGTCGAGGTG AGCCCCACGT TCTGCTTCAC TCTCCCCATC TCCCCCCCCT CCCCACCCCC 420

AATTTTGTAT TTATTTATTT TTTAATTATT TTGTGCAGCG ATGGGGGCGG GGGGGGGGGG 480

GGCGCGCGCC AGGCGGGGCG GGGCGGGGCG AGGGGCGGGG CGGGGCGAGG CGGAGAGGTG 540

CGGCGGCAGC CAATCAGAGC GGCGCGCTCC GAAAGTTTCC TTTTATGGCG AGGCGGCGGC 600

GGCGGCGGCC CTATAAAAAG CGAAGCGCGC GGCGGGCGGG AGTCGCTGCG TTGCCTTCGC 660

CCCGTGCCCC GCTCCGCGCC GCCTCGCGCC GCCCGCCCCG GCTCTGACTG ACCGCGTTAC 720

TCCCACAGGT GAGCGGGCGG GACGGCCCTT CTCCTCCGGG CTGTAATTAG CGCTTGGTTT 780

AATGACGGCT CGTTTCTTTT CTGTGGCTGC GTGAAAGCCT TAAAGGGCTC CGGGAGGGCC 840

CTTTGTGCGG GGGGGAGCGG CTCGGGGGGT GCGTGCGTGT GTGTGTGCGT GGGGAGCGCC 900

GCGTGCGGCC CGCGCTGCCC GGCGGCTGTG AGCGCTGCGG GCGCGGCGCG GGGCTTTGTG 960

CGCTCCGCGT GTGCGCGAGG GGAGCGCGGC CGGGGGCGGT GCCCCGCGGT GCGGGGGGGC 1020

TGCGAGGGGA ACAAAGGCTG CGTGCGGGGT GTGTGCGTGG GGGGGTGAGC AGGGGGTGTG 1080

GGCGCGGCGG TCGGGCTGTA ACCCCCCCCT GCACCCCCCT CCCCGAGTTG CTGAGCACGG 1140

CCCGGCTTCG GGTGCGGGGC TCCGTACGGG GCGTGGCGCG GGGCTCGCCG TGCCGGGCGG 1200

GGGGTGCCGG CAGGTGGGGG TGCCGGGCGG GGCGGGGCCG CCTCGGGCCT GGGAGGGCTC 1260

GGGGGAGGGG CGCGGCGGCC CCCGGAGCGC CGGCGGCTGT CGAGGCGCGG CGAGCCGCAG 1320

CCATTGCCTT TTATGGTAAT CGTGCGAGAG GGCGCAGGGA CTTCCTTTGT CCCAAATCTG 1380

TGCGGAGCCG AAATCTGGGA GGCGCCGCCG CACCCCCTCT AGCGGGCGCG GGGCGAAGCG 1440

GTGCGGCGCC GGCAGGAAGG AAATGGGCGG GGAGGGCCTT CGTGCGTCGC CGCGCCGCCG 1500

TCCCCTTCTC CCTCTCCAGC CTCGGGGCTG TCCGCGGGGG GACGGCTGCC TTCGGGGGGG 1560

ACGGGGCAGG GCGGGGTTCG GCTTCTGGCG TGTGACCGGC GGCTCTAGAG CCTCTGCTAA 1620

CCATGTTCAT GCCTTCTTCT TTTTCCTACA GCTCCTGGGC AACGTGCTGG TTATTGTGCT 1680

GTCTCATCAT TTTGGCAAAG AATTCCTCGA GATCTCGAGC TCAAGCTTCG AATTCTGCAG 1740

TCGACGGTAC CGCGGGCCCG GG 1762

<210> 5

<211> 4049

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

GTCGACATTG ATTATTGACT AGTATTATGC CCAGTACATG ACCTTATGGG ACTTTCCTAC 60

TTGGCAGTAC ATCTACGTAT TAGTCATCGC TATTACCATG GTGATGCGGT TTTGGCAGTA 120

CATCAATGGG CGTGGATAGC GGTTTGACTC ACGGGGATTT CCAAGTCTCC ACCCCATTGA 180

CGTCAATGGG AGTTTGTTTT GGCACCAAAA TCAACGGGAC TTTCCAAAAT GTCGTAACAA 240

CTCCGCCCCA TTGACGCAAA TGGGCGGTAG GCGTGTACGG TGGGAGGTCT ATATAAGCAG 300

AGCTCGTTTA GTGAACCGTC AGATCGCCTG GAGACGCCAT CCACGCTGTT TTGACCTCCA 360

TAGAAGATTC TAGTCATCAT TGATGTCACA TCAACGCAGG TACGAAGGAT CTGCGATCGC 420

TCCGGTGCCC GTCAGTGGGC AGAGCGCACA TCGCCCACAG TCCCCGAGAA GTTGGGGGGA 480

GGGGTCGGCA ATTGAACGGG TGCCTAGAGA AGGTGGCGCG GGGTAAACTG GGAAAGTGAT 540

GTCGTGTACT GGCTCCGCCT TTTTCCCGAG GGTGGGGGAG AACCGTATAT AAGTGCAGTA 600

GTCGCCGTGA ACGTTCTTTT TCGCAACGGG TTTGCCGCCA GAACACAGCT GAAGCTTCGA 660

GGGGCTCGCA TCTCTCCTTC ACGCGCCCGC CGCCCTACCT GAGGCCGCCA TCCACGCCGG 720

TTGAGTCGCG TTCTGCCGCC TCCCGCCTGT GGTGCCTCCT GAACTGCGTC CGCCGTCTAG 780

GTAAGTTTAA AGCTCAGGTC GAGACCGGGC CTTTGTCCGG CGCTCCCTTG GAGCCTACCT 840

AGACTCAGCC GGCTCTCCAC GCTTTGCCTG ACCCTGCTTG CTCAACTCTA CGTCTTTGTT 900

TCGTTTTCTG TTCTGCGCCG TTACAGATCC AAGCTGTGAC CGGCGCCTAC GCTAGACGCC 960

AGCTAGCCAT CATCCAGATG TCCTGTACAG AATTCACTCC TCAGGTGCAG GCTGCCTATC 1020

AGAAGGTGGT GGCTGGTGTG GCCAATGCCC TGGCTCACAA ATACCACTGA GATCTTTTTC 1080

CCTCTGCCAA AAATTATGGG GACATCATGA AGCCCCTTGA GCATCTGACT TCTGGCTAAT 1140

AAAGGAAATT TATTTTCATT GCAATAGTGT GTTGGAATTT TTTGTGTCTC TCACTCGGAA 1200

GGACATATGG GAGGGCAAAT CATTTAAAAC ATCAGAATGA GTATTTGGTT TAGAGTTTGG 1260

CAACATATGC CATATGCTGG CTGCCATGAA CAAAGGTGGC TATAAAGAGG TCATCAGTAT 1320

ATGAAACAGC CCCCTGCTGT CCATTCCTTA TTCCATAGAA AAGCCTTGAC TTGAGGTTAG 1380

ATTTTTTTTA TATTTTGTTT TGTGTTATTT TTTTCTTTAA CATCCCTAAA ATTTTCCTTA 1440

CATGTTTTAC TAGCCAGATT TTTCCTCCTC TCCTGACTAC TCCCAGTCAT AGCTGTCCCT 1500

CTTCTCTTAT GAAGATCCCT CGACCTGCAG CCCAAGCTTG GCGTAATCAT GGTCATAGCT 1500

GTTTCCTGTG TGAAATTGTT ATCCGCTCAC AATTCCACAC AACATACGAG CCGGAAGCAT 1560

AAAGTGTAAA GCCTGGGGTG CCTAATGAGT GAGCTAACTC ACATTAATTG CGTTGCGCTC 1620

ACTGCCCGCT TTCCAGTCGG GAAACCTGTC GTGCCAGCGG ATCCGCATCT CAATTAGTCA 1740

GCAACCATAG TCCCGCCCCT AACTCCGCCC ATCCCGCCCC TAACTCCGCC CAGTTCCGCC 1800

CATTCTCCGC CCCATGGCTG ACTAATTTTT TTTATTTATG CAGAGGCCGA GGCCGCCTCG 1860

GCCTCTGAGC TATTCCAGAA GTAGTGAGGA GGCTTTTTTG GAGGCCTAGG CTTTTGCAAA 1920

AAGCTAACTT GTTTATTGCA GCTTATAATG GTTACAAATA AAGCAATAGC ATCACAAATT 1980

TCACAAATAA AGCATTTTTT TCACTGCATT CTAGTTGTGG TTTGTCCAAA CTCATCAATG 2040

TATCTTATCA TGTCTGGATC CGCTGCATTA ATGAATCGGC CAACGCGCGG GGAGAGGCGG 2100

TTTGCGTATT GGGCGCTCTT CCGCTTCCTC GCTCACTGAC TCGCTGCGCT CGGTCGTTCG 2160

GCTGCGGCGA GCGGTATCAG CTCACTCAAA GGCGGTAATA CGGTTATCCA CAGAATCAGG 2220

GGATAACGCA GGAAAGAACA TGTGAGCAAA AGGCCAGCAA AAGGCCAGGA ACCGTAAAAA 2280

GGCCGCGTTG CTGGCGTTTT TCCATAGGCT CCGCCCCCCT GACGAGCATC ACAAAAATCG 2340

ACGCTCAAGT CAGAGGTGGC GAAACCCGAC AGGACTATAA AGATACCAGG CGTTTCCCCC 2400

TGGAAGCTCC CTCGTGCGCT CTCCTGTTCC GACCCTGCCG CTTACCGGAT ACCTGTCCGC 2460

CTTTCTCCCT TCGGGAAGCG TGGCGCTTTC TCAATGCTCA CGCTGTAGGT ATCTCAGTTC 2520

GGTGTAGGTC GTTCGCTCCA AGCTGGGCTG TGTGCACGAA CCCCCCGTTC AGCCCGACCG 2580

CTGCGCCTTA TCCGGTAACT ATCGTCTTGA GTCCAACCCG GTAAGACACG ACTTATCGCC 2640

ACTGGCAGCA GCCACTGGTA ACAGGATTAG CAGAGCGAGG TATGTAGGCG GTGCTACAGA 2700

GTTCTTGAAG TGGTGGCCTA ACTACGGCTA CACTAGAAGG ACAGTATTTG GTATCTGCGC 2760

TCTGCTGAAG CCAGTTACCT TCGGAAAAAG AGTTGGTAGC TCTTGATCCG GCAAACAAAC 2820

CACCGCTGGT AGCGGTGGTT TTTTTGTTTG CAAGCAGCAG ATTACGCGCA GAAAAAAAGG 2880

ATCTCAAGAA GATCCTTTGA TCTTTTCTAC GGGGTCTGAC GCTCAGTGGA ACGAAAACTC 2940

ACGTTAAGGG ATTTTGGTCA TGAGATTATC AAAAAGGATC TTCACCTAGA TCCTTTTAAA 3000

TTAAAAATGA AGTTTTAAAT CAATCTAAAG TATATATGAG TAAACTTGGT CTGACAGTTA 3060

CCAATGCTTA ATCAGTGAGG CACCTATCTC AGCGATCTGT CTATTTCGTT CATCCATAGT 3120

TGCCTGACTC CCCGTCGTGT AGATAACTAC GATACGGGAG GGCTTACCAT CTGGCCCCAG 3180

TGCTGCAATG ATACCGCGAG ACCCACGCTC ACCGGCTCCA GATTTATCAG CAATAAACCA 3240

GCCAGCCGGA AGGGCCGAGC GCAGAAGTGG TCCTGCAACT TTATCCGCCT CCATCCAGTC 3300

TATTAATTGT TGCCGGGAAG CTAGAGTAAG TAGTTCGCCA GTTAATAGTT TGCGCAACGT 3360

TGTTGCCATT GCTACAGGCA TCGTGGTGTC ACGCTCGTCG TTTGGTATGG CTTCATTCAG 3420

CTCCGGTTCC CAACGATCAA GGCGAGTTAC ATGATCCCCC ATGTTGTGCA AAAAAGCGGT 3480

TAGCTCCTTC GGTCCTCCGA TCGTTGTCAG AAGTAAGTTG GCCGCAGTGT TATCACTCAT 3540

GGTTATGGCA GCACTGCATA ATTCTCTTAC TGTCATGCCA TCCGTAAGAT GCTTTTCTGT 3600

GACTGGTGAG TACTCAACCA AGTCATTCTG AGAATAGTGT ATGCGGCGAC CGAGTTGCTC 3660

TTGCCCGGCG TCAATACGGG ATAATACCGC GCCACATAGC AGAACTTTAA AAGTGCTCAT 3720

CATTGGAAAA CGTTCTTCGG GGCGAAAACT CTCAAGGATC TTACCGCTGT TGAGATCCAG 3780

TTCGATGTAA CCCACTCGTG CACCCAACTG ATCTTCAGCA TCTTTTACTT TCACCAGCGT 3840

TTCTGGGTGA GCAAAAACAG GAAGGCAAAA TGCCGCAAAA AAGGGAATAA GGGCGACACG 3900

GAAATGTTGA ATACTCATAC TCTTCCTTTT TCAATATTAT TGAAGCATTT ATCAGGGTTA 3960

TTGTCTCATG AGCGGATACA TATTTGAATG TATTTAGAAA AATAAACAAA TAGGGGTTCC 4020

GCGCACATTT CCCCGAAAAG TGCCACCTG 4049

<210> 6

<211> 3414

<212> DNA

<213> Mus musculus

<400> 6

atggaggtgg tggacgagac cgaagcgctg cagcgcttct tcgaaggcca tgacatcagc 60

ggtgccctgg agccctccaa tatagacacc agtatcctgg aggagtacat tggcaaagag 120

gacgcctctg atctctgctt ccctgagatc tctgcaccag ccagcactgc ctccttcccc 180

cacgggccac cggccattcc cggctccagc gggctccacc atctgagccc ccctgggagc 240

ggaccatccc ctgggcgcca tggccccctc ccacccccga cctacggcac cccactcaac 300

tgcaacaaca acaacggcat gggcaccgcc cctaagccct tcctgggggg ctctgggcct 360

cccatcaaag cagagccaaa ggccccctat gccccaggca cactgccaga ctcgccccca 420

gactcaggct ctgaggccta ctcccctcag caggtgaatg acccccatct tctacgcacc 480

ataaccccgg agactctatg ccacgtggga gtttcttccc gcctggagca cccgccccca 540

cctccagccc acctgccagg cccaccacca ccgccacctc ccccacctca ctaccctgtc 600

ctgcaacggg acctctacat gaaggctgag ccccctgtac ccccttatgc tgccatgggg 660

ccgggtctgg tgccccccga gctccatcac acccagcaga cccaggtgct acaccagctg 720

ctgcaacagc atggagctga actccctcca cacccctcta agaagaggaa gcactctgaa 780

tcacccccca acaccctcaa tgcccaaatg ctgaacggaa tgatcaagca ggaacctggg 840

actgtcacag ccttgcctcc acaccctgcc agagccccat cccctccatg gcctcctcaa 900

ggcccactgt cacctggcac tggatccttg cccctcagca ttgcccgagc ccagactcca 960

ccctggcacc cgccaggtgc accctcccca ggcctcctgc aggacagtga cagcctcagt 1020

ggctcctatt tggaccccaa ctaccaatcc atcaaatggc agccgcatca gcagaacaag 1080

tgggcgaccc tgtatgacgc taactacaag gagctgccta tgctcaccta tcgtgtggac 1140

gctgacaagg gcttcaactt ttccgtgggc gacgatgctt ttgtgtgcca gaagaagaac 1200

cacttccagg tgaccgtgta catcggcatg ctgggggagc ccaagtacgt caagacaccg 1260

gaaggcctca agcccctgga ctgcttctat ctgaagctgc atggtgtgaa gctagaggcc 1320

ctgaaccagt ctatcaacat tgagcagtca cagtcagaca gaagcaagag gcccttcaac 1380

cccgtcacgg tcaatcttcc ccctgagcag gtcacaaaag tgaccgtggg gcggctccat 1440

ttcagtgaga ccactgccaa caacatgcgc aagaagggca agcccaaccc tgaccagagg 1500

tatttcatgc tggtggtggc cctccaggca catgcacaga accagaacta cacactggca 1560

gcccagatct cagagcgtat cattgtgcgg gcctctaacc caggccagtt tgaaagtgac 1620

agtgacgtgc tgtggcaacg ggcgcagctg ccagatacgg tcttccacca tggccgtgtg 1680

ggcatcaaca ccgaccggcc agatgaggca ttggtcgtcc acggcaacgt gaaggtcatg 1740

ggttctctta tgcacccttc cgatctgcgg gccaaggagc acgtgcagga ggtggacacc 1800

accgagcagc tgaagaggat ctctcggatg cggctggtgc actacagata caagcctgag 1860

ttcgctgcta gcgcaggcat tgaagccacc gcaccggaga caggtgtcat cgcccaggaa 1920

gtgaaggaga tcctgcctga agctgtgaag gacacagggg atgtagtctt tgccaatggg 1980

aaaaccatag agaacttcct tgtagtgaac aaggagcgaa tcttcatgga gaatgtgggg 2040

gctgtgaagg agttatgcaa actgacagac aacctagaga ctcgcattga tgagttggag 2100

cgatggagcc acaagctggc caagctgcgg cgccttgaca gcctcaagtc aactggcagc 2160

tcaggggctt tcagccatgc agggagccag tttagccggg caggcagtgt cccccacaaa 2220

aagaggcccc ctaaactggc caataagtca tcgccagcgg tcccagacca ggcctgcatc 2280

agtcagcgtt ttctgcaggg aactatcata gctctggtgg tggtcatggc cttcagcgtg 2340

gtgtctatgt ccacactata tgtgctgagc ctgcgctctg aagaggacct ggtggatgct 2400

gatggctctc ttgctgtgtc tacttcctgt cttctggccc tgcttcggcc ccaggaccct 2460

ggggggagtg aggccatgtg cccatggtcc agccagagct tcgggaccac acagctccga 2520

cagtcctcca tgaccaccgg actaccaggc acacagccct ctttgctgct ggttaccaag 2580

tcagcctcgg gtccagctct ccgtgccttg gacctgtgct ccagccagcc ctgccccatc 2640

gtctgctgct ctcctcccgt ctccagtcct gctacagatc ctgcccttgg ccccactctt 2700

actcctactc caagccccag ctctaacccc aagcactcag gccctggcca gatggcccca 2760

ctgccagtca ccaacatcag agccaaatcc tggggcatct cagctaatgg catcagctat 2820

tccaagcatt ccaagagcct ggaacctctg gccagtcctg tggtcccctt tcctggaggg 2880

cagagcaaga ccaagaacag ccccagcttc aatctccaaa gtcgagcccg cagaggagcc 2940

ccgcaaccca gcccaagccc tgcccaattc acacagaccc agggccagct agacccagcg 3000

ccatccctga cctccatcca gctgctggag aattccatgc ctatcacttc tcagtactgt 3060

gtgccagaag gtgcttgcag gcttggcaac ttcacctacc acatccctgt cagcagcagc 3120

acaccactgc acctcagcct gaccctgcag atgaattcct ccacccctgt gtccgtggta 3180

ctgtgcagcc tgacatcgga ggaggagccc tgtgaggagg gaggcttttt gcagaggttc 3240

cacccgcatc aggacaccca gggcacctct catcagtggc cagtaaccat cctgtccttc 3300

cgtgaattca cataccactt ccgggtgaca ttgctgggtc aggccaactg cagctcagag 3360

gccatcgttc agccagccac cgactactac ttccacttct accgcctgtg tgac 3414

Claims

1. A eukaryotic promoter CF1, characterized by: comprises a nucleotide sequence shown as SEQ ID No. 1.

2. The eukaryotic promoter CF1 of claim 1, wherein: the promoter CF1 comprises a strong CMV promoter at the 5 '-end and a medium strength EF1 promoter at the 3' -end, and also comprises a connecting region.

3. The eukaryotic promoter CF1 of claim 1, wherein: the CF1 promoter directly drives a plasmid or a vector for expressing a target gene.

4. The eukaryotic promoter CF1 of claim 2, wherein: the use of said plasmid to prepare a cell or cell line expressing a protein product; the vector is used for preparing cells or cell lines expressing protein products.

5. Use of the CF1 promoter-driven expression of a foreign gene according to claim 1, wherein: including the use of growth factors, antibodies, and other protein expression scenarios.

6. A pCF1 recombinant plasmid according to claim 1, wherein: the skeleton of the plasmid is pCAGGS plasmid, the CAG promoter of the pCAGGS plasmid is replaced by the CF1 promoter, and NheI and BsrGI are introduced behind the promoter to be used as multiple cloning sites.