CN112779289A

CN112779289A - Human and mammal cell expression vector, expression system, construction method and application thereof

Info

Publication number: CN112779289A
Application number: CN202110113130.6A
Authority: CN
Inventors: 张俊河; 王天云; 张继红; 樊振林; 杨雯雯; 王小引; 肖云喜
Original assignee: Xinxiang Medical University
Current assignee: Xinxiang Medical University
Priority date: 2021-01-27
Filing date: 2021-01-27
Publication date: 2021-05-11

Abstract

The invention relates to a human and mammal cell expression vector, an expression system, a construction method and application thereof. The invention constructs and obtains a MAR 1-68-Spacer-CMV-target gene-PolyA-MAR 1-68 vector by inserting MAR1-68 sequences at the upstream of a promoter and at the downstream of PolyA and inserting a Spacer DNA sequence with the length of 500bp between the MAR sequences and the promoter. The Spacer fragment in the vector is a neutral DNA sequence, and the transgene expression level cannot be improved, so that the influence of the distance effect on the transgene expression can be really discussed. Experiments prove that the expression system of human and mammal cells containing the expression vector can obviously improve the expression level of the carried target gene. Therefore, the mammalian expression vector and the expression system provided by the invention can overcome transgene silencing, promote high-level stable expression of exogenous genes in host cells, and can be used for producing recombinant proteins.

Description

Human and mammal cell expression vector, expression system, construction method and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a human and mammal cell expression vector, an expression system, a construction method and application thereof.

Background

At present, over 70% of the global therapeutic recombinant proteins are produced by human and mammalian cell expression systems, and expression vectors are one of the important factors for recombinant protein production. One problem to be solved in current human and mammalian cell expression systems is the phenomenon of transgene silencing (transgene silencing). The phenomenon of suppression of expression of a foreign gene in a host is called transgene silencing. The occurrence of transgene silencing is related to the location of transgene integration into the host cell gene, i.e., exhibits a "position effect". In addition, the strength of transgene expression is affected by vector DNA sequences, for example, promoter methylation can lead to gradual attenuation of expression during cell culture expansion. The proper selection of the promoter, the optimization of the combination of the promoter and different regulatory elements can improve the expression of recombinant protein, prevent the methylation of the promoter and increase the expression stability. Therefore, the research on the vector for the efficient and stable expression of the mammalian cell expression system can greatly promote the research and development and the production of the therapeutic recombinant protein, and has important theoretical and practical significance.

Currently, with the wide application of transgenic technology, the key problems that transgenic silence, low gene expression level or unstable expression are needed to be solved by transgenic animals and plants. In order to obtain stably expressed transgenic strains, many beneficial attempts have been made to overcome transgene silencing, such as selection of strong promoters for construction of expression vectors, use of enhancers, demethylation during transgenic manipulations, and the like. The utilization of Matrix Attachment Region (MAR) to increase transgene expression is an effective method for overcoming exogenous gene silencing developed in recent years.

MARs are DNA sequences that bind specifically to the nuclear backbone (or nuclear matrix), are non-coding sequences, are 300bp to several kilobases in length, are rich in AT base pairs, and are located in non-coding regions on both sides of the gene. As a new cis-acting element of eukaryote, a plurality of reports about the MAR capable of improving the expression level of foreign genes and reducing transgene silencing exist at present.

The results of previous experiments demonstrated that MARs increased the level of expression of a transgene in stably transfected Chinese Hamster Ovary (CHO) cells (Wang TY, Yang R, Qin C, et al. enhanced expression of a transgene in CHO cells using matrix expression [ J ] Cell Biol Int,2008,32: 1279. sup. 1283.), and that MARs at different insertion sites had different effects on expression of the transgene by inserting EGFP of different lengths between MARs and promoters, which also had different effects on expression of the transgene (Zhang JH, Wang XY, Wang, distance expression of matrix expression on vector expression [ J ] Cell expression [ 2014 ] 12. sup. 12. 9. sup. expression of matrix expression vector). Publication No. CN103642838B discloses a human and mammalian cell expression vector, which is inserted with only human beta-globin MAR sequence (770bp) and EGFP (350 bp and 750bp respectively), and only one MAR is used instead of the full length of MAR sequence although the expression of exogenous gene in mammalian cell can be mediated, and exogenous fragment is derived from EGFP, and the influence of sequence itself on transgene expression may cause some interference. At present, MARs are not ideal in improving the expression level of transgenes, and meanwhile, researches on the position and distance effect adaptability of expression vectors are not reported.

Disclosure of Invention

The invention aims to provide a human and mammal cell expression vector capable of efficiently expressing a target gene.

The second purpose of the invention is to provide a human and mammal cell expression system of the expression vector and a construction method thereof.

The third purpose of the invention is to provide the application of the mammalian expression vector and the expression system.

In order to achieve the purpose, the invention adopts the following technical scheme:

a human and mammalian cell expression vector comprising a nuclear matrix attachment region sequence and a spacer DNA sequence; the nuclear matrix attachment region sequence is MAR1-68, the nucleotide sequence of which is shown as Seq ID No.1 and is positioned at the 5 'end of the promoter and/or the 3' end of the PolyA; the nucleotide sequence of the spacer DNA sequence is shown as Seq ID No.2, is located at the upstream of the promoter, and connects the nuclear matrix attachment region sequence with the promoter.

Preferably, the vector is a modified vector taking pIRESneo 3 as a framework.

Preferably, the promoter of the vector is a CMV promoter.

Further, the vector comprises one or more genes encoding one or more recombinant proteins.

The expression vector can be constructed according to a conventional method in the field of genetic engineering.

A human and mammal cell expression system comprises the expression vector.

The construction process of human and mammal cell expression system includes cloning target gene to the expression vector and transfecting host cell.

Preferably, the host cell is a CHO-K1, CHO-S or CHO-DG44 cell.

The invention also provides the application of the human and mammal cell expression vector and the human and mammal cell expression system in improving the expression level of the recombinant protein.

The invention has the following beneficial effects:

the MAR 1-68-Spacer-CMV-target gene-PolyA-MAR 1-68 vector is constructed by inserting a MAR1-68 sequence at the upstream of a promoter and at the downstream of PolyA and inserting a Spacer DNA sequence with the length of 500bp between the MAR sequence and the promoter. The vector expression cassette contains MAR1-68 sequences at two sides simultaneously, and a Spacer DNA sequence is inserted, the Spacer fragment adopted by the invention is a neutral DNA sequence, and the transgene expression level can not be improved (Ley D, Harraghy N, Le Fourn V, et al. MAR elements and transposons for improved transgene integration and expression [ J ]. PLoS One,2013,8(4): e 62784), thereby really discussing the influence of the distance effect on the transgene expression. Experiments prove that the expression system of human and mammal cells containing the expression vector can obviously improve the expression level of the carried target gene. Therefore, the mammalian expression vector and the expression system provided by the invention can overcome transgene silencing, promote high-level stable expression of exogenous genes in host cells, and can be used for producing recombinant proteins.

Drawings

FIG. 1 is a physical map of pIRESneo 3 vector;

FIG. 2 is a physical map of the expression vector pIRES-CMV-EGFP;

FIG. 3 is a physical map of the expression vector pIRES-MAR-CMV-EGFP-PolyA;

FIG. 4 is a physical map of the expression vector pIRES-CMV-EGFP-PolyA-MAR;

FIG. 5 is a physical map of the expression vector pIRES-MAR-CMV-EGFP-PolyA-MAR;

FIG. 6 is a physical map of the expression vector pIRES-MAR-Spacer-CMV-EGFP-PolyA;

FIG. 7 is a physical map of the expression vector pIRES-Spacer-CMV-EGFP-PolyA-MAR;

FIG. 8 is a physical map of the expression vector pIRES-MAR-Spacer-CMV-EGFP-PolyA-MAR;

FIG. 9 is a physical map of the expression vector pIRES-CMV-EPO;

FIG. 10 is a physical map of the expression vector pIRES-MAR-CMV-EPO-PolyA;

FIG. 11 is a physical map of the expression vector pIRES-CMV-EPO-PolyA-MAR;

FIG. 12 is a physical map of the expression vector pIRES-MAR-CMV-EPO-PolyA-MAR;

FIG. 13 is a physical map of the expression vector pIRES-MAR-Spacer-CMV-EPO-PolyA;

FIG. 14 is a physical map of the expression vector pIRES-Spacer-CMV-EPO-PolyA-MAR;

FIG. 15 is a physical map of the expression vector pIRES-MAR-Spacer-CMV-EPO-PolyA-MAR;

FIG. 16 is a graph comparing the expression levels of EGFP after stable transfection of CHO cells with different expression vectors in example 3;

in the figure, A is an EGFP expression value measured by different expression vector transfection CHO cell flow, and B is a multiple of the expression level of the EGFP improved by the expression vector containing MAR;

FIG. 17 shows the results of ELISA assay of EPO protein expression levels after stable transfection of CHO cells with different expression vectors in example 3.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto; escherichia coli (Escherichia coli) JM109 used in the examples of the present invention, the cell line used, the plasmid vector and the reagent, and the tool enzyme were all commercially available products. pIRESneo 3 plasmid vector was purchased from Clontech Biometrics.

EXAMPLE 1 construction of expression vectors containing MARs in different positions of the expression cassette

1.1 PCR amplification of EGFP

The following primers were designed with reference to the sequence of the Enhanced Green Fluorescent Protein (EGFP) gene fragment (GenBank accession No: U55763.1, base No. 613-1329): p1: 5' -CCGGATATCATGGTGAGCAAGGGC-3′；P2:5′-CGCGCTAGCTCACTTGTACAGCTC-3 ', the 5' end of the primer is introduced with EcoR V and Nhe I enzyme cutting sites respectively. The extracted pEGFP-C1 plasmid is used as a template and is amplified by a conventional PCR method. PCR system and the following:

the PCR procedure was as follows: 95 ℃ for 3min, 94 ℃ for 40s, 60 ℃ for 30s, 72 ℃ for 40s, 30 cycles, 72 ℃ for 3 min. And recovering the amplification product by agarose gel electrophoresis, and sending the amplification product to a biological company for sequencing verification, wherein the result shows that the amplified DNA fragment is completely consistent with the EGFP sequence disclosed by GenBank.

1.2 construction of pIRES-CMV-EGFP expression vector

PCR amplification products of EGFP were digested simultaneously with EcoR V/Nhe I (the correct sequence was verified by sequencing), and pIRESneo 3 plasmid was digested simultaneously with EcoR V/Nhe I (FIG. 1), using a conventional digestion method, at 37 ℃ for 3 h. Identifying the digestion result by agarose gel electrophoresis, and recovering the EGFP fragment and the pIRESneo 3 linear plasmid from the gel; and (3) carrying out enzyme digestion on the recovered EGFP fragment and pIRESneo 3 linear plasmid according to the ratio of 1:5 (molar ratio) and connecting at 16 ℃ overnight; and then adding the ligation product into E.coli JM109 strain competent cell suspension for transformation, inoculating 200 mu l of bacterial liquid on an LB plate containing ampicillin, culturing overnight at 37 ℃, selecting a single colony for subculture, carrying out double enzyme digestion (EcoR V/Nhe I) verification on recombinant plasmids, selecting plasmids with correct enzyme digestion verification, carrying out sequencing verification, and naming the vectors with completely correct target gene sequences as pIRES-CMV-EGFP (the plasmid map is shown in figure 2).

1.3 construction of pIRES-MAR-CMV-EGFP-PolyA expression vector

1.3.1 PCR amplification of fragments of interest

Based on the sequence of MAR1-68 (GenBank accession No: EF694965.1), the following primers were designed: p3: 5' -GTCACGCGTTCGACTCTAGATTAT-3′；P4:5′-AGCTTCGAACTCTAGATGTAGTAC-3 ', the 5' end of the primer is respectively introduced with MluI/BstBI enzyme cutting sites. The extracted human peripheral blood genome DNA is used as a template, and human MAR1-68 is obtained by PCR amplification. PCR system As before, the PCR conditions were as follows: 95 ℃ for 3min, 94 ℃ for 40s, 60-56 ℃ for 30s, 72 ℃ for 40s, 4 cycles per annealing temperature, finally 55 ℃, 30 cycles, 72 ℃ for 3 min. And recovering the amplification product by agarose gel electrophoresis, and sending the amplification product to a biological company for sequencing verification, wherein the result shows that the amplified DNA fragment is completely consistent with the sequence of MAR1-68 disclosed by GenBank.

1.3.2 construction of pIRES-MAR-CMV-EGFP-PolyA vector

The PCR amplification product of MAR1-68 sequence was double digested with MluI/BstBI (correct sequence verified by sequencing), while the pIRES-EGFP plasmid was double digested with MluI/BstBI using conventional digestion method at 37 ℃ for 3 h. Identifying the digestion result by agarose gel electrophoresis, and recovering the MAR sequence segment and the pIRES-EGFP linear plasmid from the gel; and carrying out enzyme digestion on the recovered MAR fragment and pIRES-EGFP linear plasmid DNA according to the ratio of 1:5 (molar ratio) and connecting at 16 ℃ overnight; and then adding the ligation product into E.coli JM109 strain competent cell suspension for transformation, inoculating 200 mu l of bacterial liquid on an LB plate containing ampicillin, culturing overnight at 37 ℃, selecting a single colony for subculture, performing double enzyme digestion (MluI/BstBI) verification on recombinant plasmids, selecting plasmids with correct enzyme digestion verification, performing sequencing verification, and naming the vectors with completely correct target gene sequences as pIRES-MAR-CMV-EGFP-PolyA (the plasmid map is shown in figure 3).

1.4 construction of pIRES-CMV-EGFP-PolyA-MAR expression vector

1.4.1 PCR amplification of fragments of interest

Based on the sequence of MAR1-68 (GenBank accession No: EF694970.1), the following primers were designed: p5: 5-GCGGCCGCTCGACTCTAGATTAT-3′；P6:5′-GGCGCGCCCTCTAGATGTAGTAC-3 ', and the 5' end of the primer is introduced with NotI/AscI restriction enzyme cutting sites respectively. The extracted human peripheral blood genome DNA is used as a template, and human MAR1-68 is obtained by PCR amplification. PCR system As before, the PCR conditions were as follows: 95 ℃ for 3min, 94 ℃ for 40s, 60-56 ℃ for 30s, 72 ℃ for 40s, 4 cycles per annealing temperature, finally 55 ℃, 30 cycles, 72 ℃ for 3 min. And recovering the amplification product by agarose gel electrophoresis, and sending the amplification product to a biological company for sequencing verification, wherein the result shows that the amplified DNA fragment is completely consistent with the sequence of MAR1-68 disclosed by GenBank.

1.4.2 construction of pIRES-CMV-EGFP-PolyA-MAR expression vector

PCR amplification products of MAR1-68 sequences (correct sequences verified by sequencing) were double-digested with NotI/AscI, while pIRES-EGFP plasmid was double-digested with NotI/AscI, using conventional digestion method, at 37 ℃ for 3 h. Identifying the digestion result by agarose gel electrophoresis, and recovering the MAR sequence segment and the pIRES-EGFP linear plasmid from the gel; and carrying out enzyme digestion on the recovered MAR fragment and pIRES-EGFP linear plasmid DNA according to the ratio of 1:5 (molar ratio) and connecting at 16 ℃ overnight; and then adding the ligation product into E.coli JM109 strain competent cell suspension for transformation, inoculating 200 mu l of bacterial liquid on an LB plate containing ampicillin, culturing at 37 ℃ overnight, selecting a single colony for subculture, performing double enzyme digestion (NotI/AscI) verification on recombinant plasmids, selecting plasmids with correct enzyme digestion verification, performing sequencing verification, and naming the vectors with completely correct target gene sequences as pIRES-CMV-EGFP-PolyA-MAR (the plasmid map is shown in figure 4).

1.5 construction of pIRES-MAR-CMV-EGFP-PolyA-MAR expression vector

PCR amplification products of MAR1-68 sequences (correct sequence verified by sequencing) were double digested with NotI/AscI, while pIRES-MAR-CMV-EGFP-PolyA plasmid was double digested with NotI/AscI, using conventional digestion method, at 37 ℃ for 3 h. Identifying the digestion result by agarose gel electrophoresis, and recovering the MAR sequence fragment and the pIRES-MAR-CMV-EGFP-PolyA linear plasmid from the gel; and carrying out enzyme digestion on the recovered MAR fragment and pIRES-MAR-CMV-EGFP-PolyA linear plasmid DNA according to the ratio of 1:5 (molar ratio) and connecting at 16 ℃ overnight; and then adding the ligation product into E.coli JM109 strain competent cell suspension for transformation, inoculating 200 mu l of bacterial liquid on an LB plate containing ampicillin, culturing at 37 ℃ overnight, selecting a single colony for subculture, performing double enzyme digestion (NotI/AscI) verification on recombinant plasmids, selecting plasmids with correct enzyme digestion verification, performing sequencing verification, and naming the vectors with completely correct target gene sequences as pIRES-MAR-CMV-EGFP-PolyA-MAR (the plasmid map is shown in figure 5).

Example 2 expression vector construction containing MAR and spacer DNA sequences

2.1 Synthesis of Spacer DNA fragment

With reference to the sequence of the Spacer DNA fragment (NCBI Ref Seq: NM-011682.4), a 500bp length of Spacer DNA was first synthesized and gene synthesis was performed by the company Santa Clarke, Beijing.

2.2 construction of expression vectors containing distance fragments

Respectively constructing 500bp Spacer DNA fragments to upstream of CMV expression cassettes of pIRES-MAR-CMV-EGFP-PolyA, pIRES-CMV-EGFP-PolyA and pIRES-MAR-CMV-EGFP-PolyA-MAR vectors by adopting a seamless cloning method, selecting plasmids with correct enzyme digestion verification, and respectively constructing vectors containing MAR1-68 and distance fragments at different positions, wherein the vectors are respectively named as pIRES-MAR-Spacer-CMV-EGFP-PolyA, RESpIRES-Spacer-CMV-EGFP-PolyA and pIRES-MAR-Spacer-CMV-EGFP-PolyA (plasmid maps are shown in figures 6-8).

Example 3 vector construction containing EPO, MAR and spacer DNA sequences

(1) Construction of pIRES-CMV-EPO vector containing Erythropoietin (EPO)

PCR primers P7 and P8 were designed based on the cDNA sequence of human EPO gene (NCBI Ref Seq: NM-000799.4), and EcoR V/Nhe I cleavage sites were introduced into the 5' ends of the primers for directional cloning, the primer sequences were as follows: p7: 5' -CCGGATATCATGGGGGTGCACGAA-3′；P8：5′-CGCGCTAGCTCATCTGTCCCCTGT-3'. The extracted human peripheral blood genome DNA is used as a template, and human EPO is obtained by PCR amplification. PCR system As before, the PCR conditions were as follows: 95 ℃ for 3min, 94 ℃ for 40s, 60-56 ℃ for 30s, 72 ℃ for 40s, 4 cycles per annealing temperature, finally 55 ℃, 30 cycles, 72 ℃ for 3 min. And recovering the amplification product by agarose gel electrophoresis, and sending the amplification product to a biological company for sequencing verification, wherein the result shows that the amplified DNA fragment is completely consistent with the EPO sequence disclosed by GenBank.

PCR amplification products (correct sequence verified by sequencing) of the EPO gene are subjected to double digestion by EcoR V/Nhe I, and pIRES-CMV-EGFP vectors are subjected to double digestion by the EcoR V/Nhe I by a conventional digestion method at 37 ℃ for 3 h. Identifying the restriction enzyme cutting result by agarose gel electrophoresis, and recovering an EPO gene sequence fragment and pIRES-CMV-EGFP linear plasmid by gel; and (3) digesting and recovering the EPO gene cDNA sequence fragment and pIRES-CMV-EGFP linear plasmid DNA according to the ratio of 1:5 (molar ratio) and connecting at 16 ℃ overnight; and then adding the ligation product into E.coli JM109 strain competent cell suspension for transformation, inoculating on an ampicillin-containing LB plate, culturing at 37 ℃ overnight, selecting a single colony for subculture, performing double digestion on the recombinant plasmid vector by using EcoR V/Nhe I, selecting a plasmid with correct digestion verification for sequencing verification, and naming the vector with the completely correct target gene sequence as pIRES-CMV-EPO (the plasmid map is shown in figure 9).

(2) Construction of expression vectors containing EPO and MAR

Respectively replacing EGFP in expression vectors of pIRES-MAR-CMV-EGFP-PolyA, pIRES-CMV-EGFP-PolyA-MAR and pIRES-MAR-CMV-EGFP-PolyA-MAR with EPO by adopting a molecular cloning technology, and further constructing vectors respectively containing EPO and MAR, wherein the vectors are respectively named as pIRES-MAR-CMV-EPO-PolyA, pIRES-CMV-EPO-PolyA and pIRES-MAR-CMV-EPO-PolyA (plasmid maps are shown in figures 10-12).

(3) Construction of expression vectors containing EPO, MAR and spacer DNA sequences

Respectively replacing EGFP in expression vectors of pIRES-MAR-Spacer-CMV-EGFP-PolyA, pIRES-Spacer-CMV-EGFP-PolyA-MAR and pIRES-MAR-Spacer-CMV-EGFP-PolyA-MAR with EPO by adopting a molecular cloning technology, and further constructing vectors respectively containing EPO, MAR and Spacer DNA sequences, wherein the vectors are respectively named as pIRES-MAR-Spacer-CMV-EPO-PolyA, pIRES-Spacer-CMV-EPO-POLY and pIRES-MAR-Spacer-CMV-EPO-PolyA (plasmid maps are shown in figures 13-15).

Test example 1

The expression vector provided by the invention is used for expressing EGFP in Chinese Hamster Ovary (CHO) cells:

(1) establishment of CHO cell expression system transfected by different expression vectors

CHO cells with good growth status were selected and inoculated on 6-well culture plates, and transfection was performed when the plating density reached about 80%. The specific operation steps are as follows: adding 10 μ L lipofectamine2000 to 240 μ L serum-free DMEM/F12 medium, standing in an incubator at 37 ℃ for 5min, mixing the serum-free DMEM/F12 medium with 250 μ L (5 μ g) of the expression vector, and standing in the incubator at 37 ℃ for 20 min; cells in 6-well plates were washed three times with PBS and 2mL of serum-free DMEM/F12 cell culture medium was added. Transfection was divided into 7 groups in total according to experimental design: pIRES-CMV-EGFP (MAR-free control vector), pIRES-MAR-CMV-EGFP-PolyA, pIRES-CMV-EGFP-PolyA-MAR, pIRES-MAR-CMV-EGFP-PolyA-MAR, pIRES-MAR-Spacer-CMV-EGFP-PolyA, pIRES-Spacer-CMV-EGFP-PolyA-MAR, pIRES-MAR-Spacer-CMV-EGFP-PolyA-MAR. Then, the mixed solution of the liposome and each group of plasmid DNA is gently dripped into the hole, and the culture plate is gently shaken as soon as possible to be uniformly mixed; adding 5% CO₂After culturing at 37 ℃ for 6 hours in the cell culture box of (1), the serum-free DMEM/F12 medium was replacedDMEM/F12 complete medium is obtained and placed into a cell culture box for continuous culture.

(2) Different expression vectors stably transfect the EGFP gene expression condition of CHO cells

After 48h of transfection, 600. mu.g/mL of G418 drug was added to the transfection wells and replaced with fresh DMEM/F12 complete medium every 48h, beginning with massive cell death from the fifth day. Two weeks after selection, the G418 concentration was adjusted to a maintenance concentration of 300. mu.g/mL and the culture was continued. After drug screening was complete, when the cells formed a stable cell pool (about 14 days), the cells were harvested for flow cytometry to analyze the effect of MAR and distance fragments on reporter EGFP expression.

The results are shown in FIG. 16, where A is the EGFP expression value measured by different expression vector transfection CHO cell flow, and B is the fold of the MAR-containing expression vector for increasing the EGFP expression level. As a result, the EGFP expression level of the vector pEAM (F/F) containing MAR and Spacer DNA sequences on both sides of the expression cassette plus 500bp Spacer (pIRES-MAR-Spacer-CMV-EGFP-PolyA-MAR) is the highest and is 2.95 times that of a control vector without MAR; the expression level of EGFP of a vector pEAM3(F) containing MAR and Spacer DNA sequences at the downstream of the PolyA is 1.65 times that of a control vector without the MAR and the expression level of EGFP of a vector pEAM5(F) containing MAR and Spacer DNA sequences at the upstream of the promoter is 1.7 times that of a control vector without the MAR; other MAR-containing vector pEAM (F/F) (pIRES-MAR-CMV-EGFP-PolyA-MAR) on both sides or MAR-containing vector pEAM5(F) (pIRES-MAR-CMV-EGFP-PolyA) on one side and pEAM3(F) (pIRES-CMV-EGFP-PolyA-MAR) can improve the expression level of EGFP, and the fold of the vector containing MAR and the spacer DNA sequence is not correspondingly improved obviously (P is less than 0.05, the difference is obvious), which shows that the spacer DNA sequence inserted in the upstream of the promoter has obvious promotion effect on improving the transgene expression by the MAR1-68 sequence at the 5 'end of the upstream promoter and the 3' end of the downstream PolyA, particularly when the expression cassette contains MAR1-68 sequences on both sides and the spacer DNA sequence is inserted (pEAM (F/F) +500bp spacer), the distance effect generated by the spacer DNA sequence and the synergistic effect of the MAR1-68 sequences on both sides, can obviously improve the expression of target genes.

Test example 2

The expression vector of the invention is used for expressing EPO gene in CHO cell:

CHO cells with good growth status were selected and inoculated on 6-well culture plates, and transfection was performed when the plating density reached about 80%. The specific operation steps are as follows: adding 10 μ L lipofectamine2000 to 240 μ L serum-free Opti-MEM medium, standing in 37 deg.C incubator for 5min, mixing the serum-free Opti-MEM medium with 250 μ L (5 μ g) expression vector, and standing in 37 deg.C incubator for 20 min; cells in 6-well plates were washed three times with PBS at the same time, and 2mL of Opti-MEM cell culture medium without serum was added. Transfection was divided into 7 groups in total according to experimental design: pIRES-CMV-EPO (MAR-free control vector), pIRES-MAR-CMV-EPO-PolyA, pIRES-CMV-EPO-PolyA-MAR, pIRES-MAR-CMV-EPO-PolyA-MAR, pIRES-MAR-Spacer-CMV-EPO-PolyA, pIRES-MAR-Spacer-CMV-EPO-PolyA-MAR. Then, the mixed solution of the liposome and each group of plasmid DNA is gently dripped into the hole, and the culture plate is gently shaken as soon as possible to be uniformly mixed; adding 5% CO₂After culturing at 37 ℃ for 6 hours, the cell culture chamber of (1) was replaced with a serum-free Opti-MEM complete medium, and the medium was placed in the cell culture chamber for further culture. After 48h 600. mu.g/mL of G418 drug was added to the transfection wells and replaced with fresh Opti-MEM complete medium every 48h, beginning with massive cell death from the fifth day. Two weeks after selection, the G418 concentration was adjusted to a maintenance concentration of 300. mu.g/mL and the culture was continued. After drug selection was complete, when cells formed stable cell pools (about 14 days), they were changed to serum-free medium (CD OptiCHO)^TMMedium, available from Gibco).

(2) Different expression vectors stably transfect EPO gene expression of CHO cells

After continuing suspension culture for 6 days, taking cell supernatant for ELISA detection.

ELISA analysis results are shown in FIG. 17, all expression vectors containing Spacer can improve the expression level of EPO to different degrees, wherein the EPO expression level of the vector pEAM (F/F) +500bp Spacer (pIRES-MAR-Spacer-CMV-EPO-PolyA-MAR) containing MAR and Spacer DNA sequences at two sides of the expression cassette is the highest and is 3 times that of a control vector without MAR; the expression level of EPO of a vector pEAM5(F) containing MAR and Spacer DNA sequences at the upstream of the promoter and 500bp Spacer (pIRES-MAR-Spacer-CMV-EPO-PolyA) is 2 times that of a control vector without MAR, the expression level of EPO of a vector pEAM3(F) containing MAR and Spacer DNA sequences at the downstream of PolyA is 1.5 times that of a control vector without MAR, and the difference of EPO content of cells transfected by different expression vectors has statistical significance; while other MAR-only vectors increased EPO expression, no expression vector containing both MAR and spacer was evident.

<110> New countryside medical college

<120> human and mammal cell expression vector, expression system, construction method and application thereof

<160> 10

<170> PatentIn version 3.5

<210> 1

<211> 3614

<212> DNA

<213> Homo sapiens

<221> MAR 1-68

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tatacacaca tatatataaa atatatatat acacacatat atataaaata tatatataca 1560

cacatatata taaaatatat atatacacac atatataaaa tatatatata cacacatata 1620

taaaatatat atatacacat atatataaaa tatatatata cacatatata taaaatatat 1680

atacacacat atatataaaa tatatatata cacacatata tataaaatat atatatacac 1740

atatatataa aatatatata tacacatata tataaaatat atatatatac acatatatat 1800

aaaatatata tacacacata tatataaagt atatatatac acacatatat ataaaatata 1860

tatatacaca tatatataaa atatatatat acacatatat ataaaatata tatatacaca 1920

tatatataaa aatatatata tatatttttt aaaatattcc aattgtctca ctttgtggat 1980

gagaaaaaga agtagttaga ggtcaagtaa cttggcctac atcttttctc aagattgtaa 2040

actcctagtg agcaataacc acatcttcat tttctttgta taaaacaaga aagtttagca 2100

tgaaaaaggt actcaattac aaatgtgttg gattgaattg aagacccttg gaaggggatt 2160

ttgtacctga ggatctcttt cttttggcca tattgttcaa tggacaaaat ttagccttcg 2220

aaggcaggcc gatttgaggt taatactacc tttaccactt gatagctatg tgaccttggc 2280

catgtggttt caacagtctg aacctcattt tctctgtgta tgtgtggtcc tccttacaag 2340

tttgtgaaaa atgtgaagtc cttagccatg atagcccaat ataacaggct aaatgataat 2400

aggtttatgt tcttttcctt tatattctca gataagcact gtccaagttt gaggtgtttt 2460

gaggtctcgc ctgatttgga ttgtttgagt ttatgctatt ctttgaattc tttgagctgt 2520

tctgaagcag tgtatcatga acaaaaacat ccccagttca gtccaaaccc ctggttacat 2580

atcattctta tgccatgtta taaccagttt gagagtgttc cctctgttat tgcatttaag 2640

tttcagcctc acacagaaat tcagcagcca atttctaagc cctaagcata aaatctgggg 2700

tggggggggg ggatggcctg aagagcagca ttatgaatag caccattata attaatgatc 2760

tctcaggaag atttacaatc acaggtagca gataaaacaa atagtactgc ttctgcactt 2820

cccctccttt tattcgctat gaaattttat gggaaatcag tccagtgaaa aatgtaagct 2880

cttaatcttt cccagaaatc ctacctcatt tgatgaatac tttgagggaa tgaattagag 2940

catttttttc ttttatagtc tacttcgcat ttacgaagtg aggacggtag cttaggctgc 3000

ctggccaact gatgagaagg tcagaggcat ttttagagac ctctgttgtc tttcattcat 3060

gttcattttc cacaaggcaa gtaatttcca acaaatcagt gtcttcatta gtaataagat 3120

tattaacaac aataatagtc atagtaacta ttcagtgaga gtccattata tatcaggcat 3180

tctacaaggt actttatata catctgagta aacctcacac aattctacag ggaggtattt 3240

ctatccccat ttaacaaata aggaaacgaa gtccaagtaa attaacttgc ccaaggtcac 3300

acagatagta cctggcagaa caggaattta aacctaaatt tgtccaactc caaaagcagc 3360

cttctatttg ttataaatgc tgcctctcat tatcacatat tttattatta acaacaacaa 3420

acataccaat tagcttaaga tacaatacaa ccagataatc atgatgacaa cagtaattgt 3480

tatactatta taataaaata gatgttttgt atgttactat aatcttgaat ttgaatagaa 3540

atttgcattt ctgaaagcat gttcctgtca tctaatatga ttctgtatct attaaaatag 3600

tactacatct agag 3614

<210> 2

<211> 500

<212> DNA

<213> Artificial sequence

<221> spacer DNA sequence

<400> 2

ccaagtatgg ggaccttgaa gccaggcctg atgatgggca gaacgaattc agtgacatca 60

ttaagtccag atctgatgaa cacaatgatg tacagaagaa aacctttacc aaatggataa 120

acgctcgatt ttccaagagt gggaaaccac ccatcagtga tatgttctca gacctcaaag 180

atgggagaaa gctcttggat cttctcgaag gcctcacagg aacatcattg ccaaaggaac 240

gtggttccac aagggtgcat gccttaaaca atgtcaaccg agtgctacag gttttacatc 300

agaacaatgt ggacttggtg aatattggag gcacggacat tgtggatgga aatcccaagc 360

tgactttagg gttactctgg agcatcattc tgcactggca ggtgaaggat gtcatgaaag 420

atatcatgtc agacctgcag cagacaaaca gcgagaagat cctgctgagc tgggtgcggc 480

agaccaccag gccctacagt 500

<210> 3

<211> 24

<212> DNA

<213> Artificial sequence

<221> P1

<400> 3

ccggatatca tggtgagcaa gggc 24

<210> 4

<211> 24

<212> DNA

<213> Artificial sequence

<221> P2

<400> 4

cgcgctagct cacttgtaca gctc 24

<210> 5

<211> 24

<212> DNA

<213> Artificial sequence

<221> P3

<400> 5

gtcacgcgtt cgactctaga ttat 24

<210> 6

<211> 24

<212> DNA

<213> Artificial sequence

<221> P4

<400> 6

agcttcgaac tctagatgta gtac 24

<210> 7

<211> 23

<212> DNA

<213> Artificial sequence

<221> P5

<400> 7

gcggccgctc gactctagat tat 23

<210> 8

<211> 23

<212> DNA

<213> Artificial sequence

<221> P6

<400> 8

ggcgcgccct ctagatgtag tac 23

<210> 9

<211> 24

<212> DNA

<213> Artificial sequence

<221> P7

<400> 9

ccggatatca tgggggtgca cgaa 24

<210> 10

<211> 24

<212> DNA

<213> Artificial sequence

<221> P8

<400> 10

cgcgctagct catctgtccc ctgt 24

Claims

1. A human and mammalian cell expression vector comprising a nuclear matrix attachment region sequence and a spacer DNA sequence; the nuclear matrix attachment region sequence is MAR1-68, the nucleotide sequence of which is shown as Seq ID No.1 and is positioned at the 5 'end of the promoter and the 3' end of the PolyA; the nucleotide sequence of the spacer DNA sequence is shown as Seq ID No.2, is located at the upstream of the promoter, and connects the nuclear matrix attachment region sequence with the promoter.

2. The expression vector of claim 1, wherein the vector is a modified vector having pIRESneo 3 as a backbone.

3. The expression vector of claim 1, wherein the promoter of the vector is a CMV promoter.

4. The expression vector of claim 1, wherein the vector comprises one or more genes encoding one or more recombinant proteins.

5. A human and mammalian cell expression system comprising the expression vector of any one of claims 1 to 4.

6. The method of claim 5, wherein the expression system is constructed by cloning a gene of interest into the expression vector of any one of claims 1 to 4 and transfecting host cells.

7. The method of claim 6, wherein the host cell is a CHO-K1, CHO-S or CHO-DG44 cell.

8. Use of the expression vector of any one of claims 1 to 4, the human and mammalian cell expression system of claim 5 for increasing the expression level of a recombinant protein.