CN110863008B - Method for constructing high-efficiency expression vector by using MAR sequence to regulate weak promoter - Google Patents

Abstract

The invention discloses a method for constructing a high-efficiency expression vector by regulating a weak promoter through a MAR sequence, which comprises the steps of selecting a promoter which does not contain an enhancer and contains a CpG island on a transcription initiation site as a target promoter, replacing a CMV promoter on a pEGFP-C1 vector with the target promoter, constructing an intermediate vector, inserting the MAR sequence into an Ase I enzyme cutting site of the intermediate vector, and constructing the expression vector; the expression vector can ensure that the expression quantity of the EGFP gene in the expression frame of the target promoter is continuously, stably and efficiently expressed; the method can be applied to the field of industrial production of antibodies and protein drugs or the construction of expression vectors for gene therapy, improves the production efficiency, reduces the production cost and has good social and economic benefits.

Description

Method for constructing high-efficiency expression vector by using MAR sequence to regulate weak promoter

Technical Field

The invention belongs to the technical field of biology, relates to an expression vector for recombinant antibody or protein drug production and the like and a construction method thereof, and particularly relates to a method for constructing a high-efficiency expression vector by using a MAR sequence to regulate a weak promoter.

Background

Recombinant therapeutic antibodies, vaccines or protein drugs (combined weight group drugs) are effective drugs for major diseases such as cancer, rheumatoid arthritis, hepatitis B, AIDS and the like, and the application of the recombinant drugs in the market mainly has the problems of high research and development cost and high production cost, so that the reduction of the research and development cost and the production cost is an urgent need for recombinant drug production.

The complexity of recombinant drug cell strain screening and the phenomenon of unstable production cell strain due to the reduction of expression amount in the production process are main obstacles in research and development and production, and the key link is to construct a stable and efficient expression vector. Transgene silencing by integration position and epigenetic factors of expression vectors in different cell lines is a major cause of the influence on stable high yield of recombinant drugs. Production instability due to epigenetic mechanisms is particularly complex and elusive, among others, and these epigenetic factors include histone modification (hypoacetylation), DNA methylation, and chromatin remodeling.

In general, a strong promoter with high promoter efficiency is often selected from promoters in an expression frame of a cell expression vector, but the strong promoter is easily resisted by a host cell self-protection mechanism, and chromatin modification and chromatin three-dimensional structural change which obstruct gene expression are formed near a promoter region and a related gene. Therefore, overcoming and avoiding apparent inhibition caused by the three-dimensional structure change of chromatin in the strong promoter region is the key of high-efficiency and stable expression of the expression vector. In the prior art, chromatin regulatory elements such as MARs (Matrix attachment regions) are mainly used to overcome the silencing effect of strong promoters to obtain higher expression, but the comprehensive influence of the change of the three-dimensional structure of chromatin in the regulatory region on the expression capacity of expression vectors is not concerned. MAR sequences were the first DNA elements thought to topologically separate the genome into distinct domains (Phi-Van L, 1996); MAR sequences can sequester transgenes from the negative effects of the surrounding genome (insulating function) and therefore can overcome transgene position effects (Seibler, et al, 2005; McKnight, et al, 1992), enhancing gene transcription efficiency (Bode, et al, 2000); MAR sequences can also support histone acetylation by blocking DNA methylation to maintain long-term high transcription levels (Dang, et al, 2000; Kurre, et al, 2003). However, no solution for regulation of expression levels has been reported when MARs bind to weak promoters.

By analyzing the characteristics of a eukaryotic strong promoter commonly used in genetic engineering, it was found that a number of transcription regulatory elements, which are DNA sequences actively regulating chromatin remodeling in the associated promoter region, exist near the TSS (transcription initiation site) to participate in the apparent regulation of chromatin. Among these, enhancers and CpG islands are of particular importance. Enhancers affect histone methylation at lysine 4 (K4), lysine 9 (K9), and lysine 36 (K36) on histone H3 in different ways. All three lysine residues in the coding region of the enhancer-linked gene are highly trimethylated, whereas the dimethylation of K9 and K36 is not affected by the enhancer; in the enhancer region, only monomethylation occurs at K4 and K9, unlike histone methylation modifications of the coding region of the gene. CpG islands selectively recruit a large amount of chromatin, which often form cysteine methylation deletions, low levels of histone H1, high levels of histone acetylation, and DNaseI high sensitivity sites in the nucleosomal-free regions (Wang, B., J. Sun, et al. (2016), "Small-Activating RNA CangeNuclear localization in Human Fibrobiles." Journal of biomolecular screening 21(6): 634-642; Wang, Huyunzhang (2017), Promoter-targeted activated RNA nucleosome localization, RNA activation, Spger Nature, Chapter 6).

In contrast, weak promoters usually do not contain enhancers, but CpG islands may be present on weak promoters. In the prior art, no report that the combination of a MAR sequence and a specific type of weak promoter improves the expression capacity is found, and no report that the MAR sequence and the weak promoter are used together to form an open chromatin three-dimensional structure level which is more beneficial to overcoming the stability of a promoter region is reported.

Disclosure of Invention

The invention aims to solve the technical problem that in the prior art, no expression vector constructed by combining a MAR sequence and a weak promoter is applied to the related fields of recombinant drugs and the like, and provides a method for constructing a high-efficiency expression vector by regulating the weak promoter through the MAR sequence, wherein the constructed expression vector can be continuously, stably and efficiently expressed for more than 32 days so as to meet the requirements of industrial production and popularization.

In order to solve the technical problems, the method for constructing a high-efficiency expression vector by regulating a weak promoter through a MAR sequence comprises the following steps:

(1) selecting a promoter which does not contain an enhancer and contains a CpG island at a transcription initiation site, namely a promoter of the human SOX2 gene as a target promoter;

(2) replacing the CMV promoter in the exogenous gene expression frame on a commercial vector pEGFP-C1 by the target promoter to construct an intermediate vector; in the step of constructing the intermediate vector, a target promoter sequence is amplified by using a double enzyme cutting site primer with AgeI and AseI, wherein the primer is as follows:

SOX2 # Forward primer: CCGTGATTAAT(Ase I) AGACAAGGAAGGTTTTGAGGAC

SOX2 # reverse primer: ATATGACCGGT(Age I) ATCCGGGCTGTTTTTCTGGTT, respectively;

(3) inserting a MAR sequence into an Ase I enzyme cutting site at the upstream of a foreign gene expression frame of the intermediate vector to construct an expression vector, wherein the MAR sequence is shown as a sequence 2; the expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

After the mammalian cell expression vector is integrated into the host cell genome, the expression vector is subjected to a transgenic silencing phenomenon in which the expression is suppressed due to the influence of chromatin remodeling proteins or other various apparent modification factors at the integration site. The mechanism of transgene silencing is generally considered to be influenced by apparent modification, but because the apparent modification is complex and various, it is difficult to generalize, so that the mechanism of transgene silencing, namely the three-dimensional structure of chromatin, needs to be understood from a more macroscopic level. It is generally considered that when the three-dimensional structure of chromatin is not covered by compact nucleosome, the gene transcription or expression at the site is activated; gene transcription at this site is silenced if chromatin is tightly covered by nucleosomes in a three-dimensional structure. In nature, some positions on the genome are always in sensitive regions which can be easily cut by DNase I, and some positions cannot be cut by DNase, which shows that the regions are protected by chromatin to different degrees due to different DNA sequences, so that the DNA sequences play an important role in regulating the three-dimensional structure of the chromatin. Previous studies have shown that DNA sequences capable of modulating chromatin structure include chromatin control elements such as MAR sequences and UCOE sequences; in addition, regulatory elements on promoters such as CpG islands and enhancers also affect chromatin remodeling and thus chromatin three-dimensional structure. In the reported art, MAR sequences linked to certain promoters are capable of up-regulating gene expression, overcoming silencing effects. However, the question of which promoter a MAR can establish a more stable and more efficient expression vector remains unsolved.

The target promoter belongs to a weak promoter, and is a promoter which does not contain an enhancer and has a CpG island at a transcription initiation site; the content of CG in all DNA sequence bases is more than or equal to 60 percent; CG is a dinucleotide.

The invention establishes the inference which is more beneficial to MAR to form chromatin isolated region through the coaction of the CpG island participating in chromatin regulation on a target promoter (such as SOX2 gene promoter) and the MAR sequence, obtains a novel method for constructing a high-efficiency expression vector, and proves that the expression efficiency of a strong promoter which is more than that of an enhancer can be obtained when the MAR sequence and the CpG island at the transcription starting site position on the target promoter are coacted.

The main action mechanism of the method is that the CpG island of the MAR and the target promoter jointly act to change the three-dimensional structure of chromatin nearby the promoter so as to form stable open chromatin or other structures suitable for transcription.

The invention has the beneficial effects that: the method constructs a recombinant vector (expression vector) by combining a MAR sequence with the target promoter, and the expression vector can ensure that the expression quantity of the EGFP gene is higher than that of a commercial vector pEGFP-C1 after being cultured for 32 days after the expression vector is transfected into CHO cells, and is also higher than that of the EGFP gene when the MAR sequence is combined with a promoter containing an enhancer (such as OCT4 or CMV promoters) and other chromatin regulating sequences (such as UCOE sequences) are combined with the target promoter; the invention provides a novel method for constructing a high-efficiency expression vector by combining a MAR sequence and a weak promoter, which can be applied to the fields of recombinant protein and drug production, genetic engineering, gene therapy and the like.

Drawings

FIG. 1 is a schematic structural view of the intermediate vector pSOX 2;

FIG. 2 is a schematic structural diagram of the expression vector pSOX 2-MAR;

FIG. 3 is a schematic structural diagram of the control vector I pSOX2-UCOE described in the examples;

FIG. 4 is a schematic diagram of the structure of the control vector II pOCT4-MAR according to the example;

FIG. 5 is a graph showing the expression levels of the recombinant vectors pSOX2, pSOX2-UCOE, pSOX2-S278 and pSOX2-U-S278 in ELISA analysis, compared with the expression level of EGFP protein in the commercial vector pEGFP-C1 (note: the vector with X indicates that the EGFP expression level of the vector is remarkably different from that of the other vectors, and the statistical value P is less than 0.05);

FIG. 6 is a graph showing the comparison of the EGFP gene expression levels of the various expression vectors pSOX2, pSOX2-UCOE, pSOX2-S278 and pSOX2-U-S278 detected by qRT-PCR quantification method relative to the commercial vector pEGFP-C1 (note: the vector with X indicates that the EGFP gene expression level of the vector is remarkably different from that of the other vectors, and the statistical value P is less than 0.05);

FIG. 7 is a graph showing the comparative analysis of the fluorescence intensity test results of the recombinant vectors pSOX2, pSOX2-UCOE, pSOX2-S278 and pSOX2-U-S278 and the commercial vector pEGFP-C1 (note: the vector with x indicates that the EGFP expression amount of the vector is remarkably different from that of the other vectors, and the statistical value P is less than 0.05);

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples.

The method for constructing the high-efficiency expression vector by regulating the weak promoter through the MAR sequence comprises the following steps:

(1) selecting a promoter which does not contain an enhancer and contains a CpG island at a transcription initiation site, namely a promoter of the human SOX2 gene as a target promoter;

(2) replacing the CMV promoter in the exogenous gene expression frame on a commercial vector pEGFP-C1 by the target promoter to construct an intermediate vector; in the step of constructing the intermediate vector, a target promoter sequence is amplified by using a double enzyme cutting site primer with AgeI and AseI, wherein the primer is as follows:

SOX2 # Forward primer: CCGTGATTAAT(Ase I) AGACAAGGAAGGTTTTGAGGAC

SOX2 # reverse primer: ATATGACCGGT(Age I) ATCCGGGCTGTTTTTCTGGTT, respectively;

(3) inserting a MAR sequence into an Ase I enzyme cutting site at the upstream of a foreign gene expression frame of the intermediate vector to construct an expression vector, wherein the MAR sequence is shown as a sequence 2; the expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

Compared with the expression quantity of the conventional commercial vector pEGFP-C1, the expression quantity of the EGFP gene expressed by the expression vector constructed by the method is obviously improved; the gene expression quantity is remarkably improved by transferring the expression vector into CHO cells, culturing for 32 days, then carrying out fluorescence intensity analysis, quantitative PCR analysis and ELISA analysis, comparing the analysis result with a commercial vector pEGFP-C1, wherein the expression quantity of the exogenous EGFP gene expressed by the expression vector is remarkably higher than that of the commercial vector pEGFP-C1 (p is less than 0.05), and the gene expression quantity can be continuously and stably expressed for more than 32 days.

Example (b): the target promoter of the invention refers to a promoter which does not contain an enhancer and contains a CpG island at a transcription initiation site, and the human SOX2 gene promoter meets the requirements of the target promoter, and the method for constructing the expression vector of the invention is illustrated by taking the human SOX2 gene promoter as an example.

I, replacing a CMV promoter on a PEGFP-C1 vector by a human-derived SOX2 gene promoter to construct an intermediate vector pSOX 2.

(1) Cloning Using human SOX2 Gene promoter

Amplified by a human SOX2 gene promoter (ENSG 00000181449), and amplification primers are as follows:

SOX2 # Forward primer: CCGTGATTAAT(Ase I) AGACAAGGAAGGTTTTGAGGAC

SOX2 # reverse primer: ATATGACCGGT(Age I) ATCCGGGCTGTTTTTCTGGTT

Carrying out PCR amplification by using a high-fidelity TAQ enzyme and a 50-microliter system, wherein the amplification conditions are as follows:

5 minutes at 95 ℃; repeating 25 cycles at 95 ℃ for 30 seconds, 55 ℃ for 30 seconds, and 72 ℃ for 45 seconds; 5 minutes at 72 ℃ and stored at normal temperature. The amplified sequence is the promoter sequence containing enhancer and CpG island in the transcription initiation site.

(2) The pEGFP-C1 expression vector was double cut with AgeI and AseI and the glue recovered a large CMV-free fragment.

The pEGFP-C1 vector is a commercially available vector with better expression effect at present, and the method uses a target promoter to be connected to the vector to replace a CMV promoter on the pEGFP-C1 vector so as to construct an intermediate vector pSOX 2.

(3) And (2) synchronously carrying out double enzyme digestion on the PCR product of the SOX2 gene promoter obtained in the step (1) by AgeI and AseI after gel recovery.

(4) Connection of

Ligation system (10 μ L): 5 mu L of a connection mixed solution containing T4 DNA ligase, 0.5 mu L of pEGFP-C1 double-enzyme digestion large fragment and 4.5 mu L of a double-enzyme digestion PCR product; reaction conditions are as follows: ligation was performed at 16 ℃ for 1h (ligation kit was purchased from Dalian Meiren Biotechnology Ltd.).

(5) Transformation of

Preparing ice cakes in advance, adding 100 mu L of competent cells (in an ice-water mixed state) into the connecting system in the step (4), and carrying out ice bath for 30 minutes; turning to 42 ℃ water bath for 90 seconds; transferring into ice bath for 1-2 min; adding LB culture medium, water bath at 37 ℃ for 45 minutes; the centrifuge rotates at the maximum speed of 1 second, most of the supernatant is discarded, mixed evenly and spread on a culture medium containing IPTG and X-gal, and cultured overnight at 37 ℃.

(6) Screening, extracting plasmid, and testing by biological company

Performing liquid culture (containing kanamycin) on colonies screened by colony PCR at 37 ℃ by using a test tube, performing overnight amplification culture by using a triangular flask, and extracting plasmid DNA by using a plasmid small-amount extraction kit; and after enzyme digestion identification by Ase I, observing the enzyme digestion result by electrophoresis. Sequencing the positive clone plasmid by a biological company, and confirming that the SOX2 promoter is correctly replaced to obtain a recombinant vector pSOX2 (an intermediate vector); the structure of the device is schematically shown in figure 1.

II, inserting MAR sequence into Ase I enzyme cutting site at the upstream of the exogenous gene expression frame of the pSOX2 vector to construct an expression vector

(1) Artificially synthesized MAR sequences

MAR sequences used in the present invention are derived from the HUBB-LCR (beta-globin loci control region) sequence (Genebank: NG-052895).

(2) Vector digestion at the Ase I site of the vector pSOX2 for single digestion

Vector restriction (50. mu.L system)

pSOX2 vector: 44 μ L

10×NEB Buffer：3.1 5μL

Ase Ⅰ：1μL

Reaction time: 1h at 37 DEG C

And (3) enzyme digestion vector purification:

a. adding 50 mu L of membrane binding solution into the centrifugal tube of the carrier enzyme digestion system and uniformly mixing;

b. transferring the solution to an adsorption column, and placing the adsorption column in a liquid collecting pipe;

c. the rotating speed of the centrifuge is 13,000 revolutions per minute, and the liquid in the liquid collecting pipe is discarded;

d. adding 700 mul of membrane eluent, centrifuging for 13,000 r/min, and discarding the liquid in the liquid collecting tube;

e. repeating the step d;

f. discarding the liquid in the liquid collecting pipe, and centrifuging for 1 minute;

g. transferring the adsorption column into a 1.5ml centrifuge tube;

h. adding 50 μ l nuclease-free deionized water into the adsorption column, standing at room temperature for 1min, and centrifuging for 1 min; the centrifuged product was collected.

(3) Blunting the digested pSOX2 vector

The digested pSOX2 plasmid was blunt-ended:

mu.l of pSOX2 plasmid, 1. mu.l of 10 Xbuffer, 3. mu.l of sterile water were incubated at 70 ℃ for 5 minutes, 0.5. mu. l T4 DNA polymerase was added, the mixture was gently mixed, reacted at 37 ℃ for 5 minutes, and then ethanol precipitation was performed.

(4) Dephosphorylation of Single enzyme digested product

Dephosphorylation can eliminate the phosphate group protruding from the 5' end, so that the plasmid vector can not form a closed circular structure.

9. mu.g of the blunt pSOX2 vector

Alkaline phosphatase buffer (10X) 15. mu.l

Bacterial alkaline phosphatase (1U/. mu.l) 2. mu.l

Supplementing deionized water without nuclease to 150 mu l, reacting in a water bath kettle at 65 ℃ for 30 minutes, and purifying;

a. adding the membrane binding solution with the same volume as the plasmid or the enzyme digestion product into a centrifuge tube, and uniformly mixing;

b. moving the solution into an adsorption column by using a liquid transfer gun, standing the solution for 3 to 5 minutes at room temperature, and centrifuging the solution for one minute;

c. adding the liquid in the collecting pipe into the adsorption column again, centrifuging for 1 minute again, and removing waste liquid;

d. adding 700ul of membrane eluent, centrifuging for one minute, and removing waste liquid;

e. adding 500ul of membrane eluent, centrifuging for 4 minutes, and removing waste liquid;

f. after the empty column is centrifuged for one minute, the adsorption column is moved into a new centrifugal tube;

g. adding 50ul of nuclease-free deionized water on the central membrane of the column, standing at room temperature for 3-5min, centrifuging for 1min, adding the liquid in the collecting tube into the adsorption column, centrifuging again, and storing the product at 4 ℃.

(5) Ligation the MAR sequence was ligated to the dephosphorylated dicer fragment of step (4) using the same method as described for the ligation of the intermediate vector pSOX 2.

(6) The transformation was performed in the same manner as in the above-described transformation of the intermediate vector pSOX 2.

(7) Screening, plasmid extraction, detection by Biometrics

Screening by colony PCR (polymerase chain reaction) by using the same method as the intermediate vector pSOX2, extracting plasmid DNA, carrying out enzyme digestion identification by Ase I, observing the enzyme digestion result by electrophoresis, sending a positive cloning plasmid to a biological company for sequencing, and confirming that the MAR sequence is correctly replaced, thereby obtaining an expression vector pSOX2-MAR, wherein the expression frame structure regulated and controlled by the expression vector is MAR-SOX2 promoter-EGFP-polyclonal site-SV 40 poly A; the structure of the device is schematically shown in figure 2.

III, in order to compare the expression effect of the expression vector pSOX2-MAR, two additional control vectors were constructed and compared together.

(1) The vector is constructed by combining a chromatin regulatory element UCOE sequence and a human SOX2 gene promoter and is used as a first control:

a UCOE sequence was inserted into the MluI cleavage site of the intermediate vector pSOX2 to construct a control vector I, pSOX2-UCOE vector, the structure of which is schematically shown in FIG. 3.

(2) The MAR sequence was combined with the human OCT4 gene promoter without CpG island but with enhancer to construct a vector as control two:

replacing a CMV promoter in an exogenous gene expression cassette on a commercial vector pEGFP-C1 by using a human OCT4 gene promoter to construct an intermediate vector pOCT 4; a MAR sequence was inserted into the MluI cleavage site of the intermediate vector pOCT4 to construct a control vector II, pOCT4-MAR vector, the structure of which is schematically shown in FIG. 4.

IV, verification of high-efficiency expression of expression vector

The vectors pSOX2, pSOX2-MAR, pSOX2-UCOE and pOCT4-MAR obtained by the construction are respectively subjected to fluorescence intensity analysis, quantitative PCR analysis and ELISA analysis for verification, and the specific method is as follows:

1. large extract plasmid (using Promega corporation kit)

(1) Taking a 50ml centrifugal tube for marking, pouring the culture bacterial liquid containing the plasmids into the centrifugal tube for multiple times of centrifugation (4000 g/min,10 min), removing supernatant and leaving precipitate;

(2) adding 10ml solution I, vibrating/blowing and uniformly mixing;

(3) adding 10ml solution II, and slightly turning over the mixture up and down for 8-10 times within 2-3 min;

(4) then adding 5ml of precooled N3 buffer, and slightly turning for 10 times to obtain lysate;

(5) preparing an injector, pulling out the piston, and pouring the lysate into a gun barrel after a cap is arranged at an outlet;

(6) taking down the cap, placing a new centrifuge tube below the cap, gently inserting the piston, and collecting liquid through injection;

(7) measuring the liquid volume, adding an ETR solution of 1/10 volumes of the liquid volume, and turning 10 times;

(8) inserting the mixture into ice for ice bath for 10min, turning and uniformly mixing for several times in the ice bath process, and clarifying the liquid from turbidity;

(9) putting into 42 deg.C water bath, and water-bathing for 5min to obtain clear turbid liquid;

(10) centrifuging at 4000g for 5min until a layer of blue substance appears at the bottom of the tube, transferring the supernatant into a new centrifuge tube, adding 1/2 volume of anhydrous ethanol of the supernatant volume, slightly turning over for 6-7 times, and standing at room temperature for 1-2min to obtain a clear solution;

(11) column balancing: taking out HiBind DNA Maxi columns (a green centrifuge tube provided with an adsorption column), adding 3ml of GPS Buffer, standing at room temperature for 4min, centrifuging at 4000g for 3min, and pouring the waste liquid;

(12) transferring the clear liquid into a Hibind column, centrifuging for 3min at 4000g, and pouring the waste liquid;

(13) repeating the previous step until all clear liquid is filtered;

(14) adding 10ml HBC Buffer into the column, centrifuging for 3min at 4000g, and pouring the waste liquid;

(15) adding 15ml of DNA Wash Buffer into the column, centrifuging for 3min at 4000g, and pouring the waste liquid;

(16) adding 10ml of DNA Wash Buffer into the column, centrifuging for 3min at 4000g, and pouring the waste liquid;

(17) centrifuging 4000g of empty column for 10min, putting the adsorption column into a new centrifuge tube, adding 3ml of Endo-freeElution Buffer (endotoxin-free eluent), and standing at room temperature for 5min;

(18) centrifuging at 4000g for 5min, transferring the liquid into the column again, standing for 5min, and centrifuging at 4000g for 5min;

(19) the collected DNA was dispensed into 1.5ml centrifuge tubes and labeled.

2. Transfection

Cell preparation: 1-2 days before the electrotransfer, transferring the cells to a T75 cell culture flask until the confluence degree of the cells before the transformation is about 50-70%, wherein the number of the cells is about 2X 107, and each electrotransfer needs about 106 cells; slowly washing cells with 2.5-10 ml PBS buffer solution, sucking out PBS, digesting cells with 0.4ml 0.25% Trypsin-EDTA pancreatin digestive juice, adding 2.6ml culture medium containing serum, and neutralizing pancreatin; the cells were harvested by centrifugation and resuspended in 1ml HEPES Buffer to a density of 2.5X 106 cells/ml.

3. Electric shock conversion

(1) Setting a electrotransfer program for 280V and 20 ms;

(2) add plasmid to the cuvette (20 ug);

(3) adding 1 × 106 cells, about 400ul, into the cuvette, and mixing by inversion;

(4) placing the electric shock cup into an electric shock groove, performing pulse electric shock once, and placing the electric shock cup on ice for 5min after electric shock;

(5) sucking the shocked cells into a 12-well plate containing 0.8ml of culture medium;

(6) gently shaking the 12-well plate, mixing the cells uniformly, and culturing in a CO2 incubator.

4. G418 screening

(1) And (3) culturing after transfection: culturing for 24 hours or more after transfection until the cell density is increased to 50% -70% confluence;

(2) g418 is added, the culture solution is removed, PBS is washed once, and G418 prepared according to the optimal screening concentration is added for screening and culture;

(3) and (4) changing the screening culture medium every 3-5 days according to the color of the culture medium and the growth condition of cells. When there is massive cell death, the G418 concentration can be halved for maintenance screening. After 10-14 days of screening, resistant clones can be seen;

(4) and (3) monoclonal identification: and extracting total RNA from the monoclonal cell obtained by the limiting dilution method, and detecting whether the target gene exists by RT-PCR.

5. Quantitative detection of EGFP expression level

In a recombinant vector: in the intermediate vector pSOX2, the expression vector pSOX2-MAR, the control vector I pSOX2-UCOE and the control vector II pOCT4-MAR, Enhanced Green Fluorescent Protein (EGFP) on an expression frame is used as a marker protein, and the expression efficiency of the vectors is observed by analyzing the marker protein.

6. Fluorescence intensity analysis

By using a fluorescence microscope, the unified photographing parameters are 10 times of focal length, the green light intensity, the blue light intensity and the white light intensity are all 70%, 4 photos are randomly taken from different angles in each hole, and the fluorescence intensity analysis is carried out by using ImageJ software. The comparative analysis results are shown in FIG. 7.

7. qRT-PCR analysis

(1) Extraction of Total cellular RNA

RNA extraction was performed with Trizol reagent and reverse transcription was performed to cDNA using Promega reverse transcription kit.

(2) Quantitative PCR

The PCR reaction system required by the experiment is 20 ul, the internal reference is GAPDH, and the primers are universal detection primers of EGFP and GAPDH genes respectively. The prepared sample and the internal reference are respectively repeated for 3 times on a Roche quantitative PCR instrument. Use 2^－△△CTThe method analyzes relative change of gene expression.

The comparative analysis results are shown in FIG. 6.

8. ELISA (enzyme-linked immunosorbent assay)

(1) Coating: 0.1ml of antibody solution (blank wells, negative control wells and positive control wells) diluted with 0.05M of carbonate coating buffer pH7.6 to a protein content of 1-10. mu.g/ml was added to reaction wells of a 96-well polystyrene plate and left overnight at 4 ℃. The next day, the well solutions were discarded and washed 3 times with wash buffer for 3 minutes each. (abbreviated as washing, the same applies below);

(2) sample adding: adding 0.1ml of diluted sample to be detected into the coated reaction hole, marking, incubating for 1 hour at 37 ℃, and then washing;

(3) adding an enzyme-labeled antibody: adding 0.1ml of freshly diluted enzyme-labeled antibody (the dilution after titration) into each reaction hole, incubating for 0.5-1 hour at 37 ℃, and washing;

(4) adding a substrate solution for color development: adding 0.1ml of temporarily prepared TMB substrate solution into each reaction hole, and keeping the temperature at 37 ℃ for 10-30 minutes;

(5) and (3) terminating the reaction: adding 0.05ml of 2mol/L sulfuric acid into each reaction hole;

(6) and (4) judging a result: the wavelength was adjusted to 450nm in a microplate reader, and the OD value of each well was measured after being zeroed with a blank control well.

The comparative analysis results are shown in FIG. 5.

9. Evaluation of expression efficiency of expression vector

(1) Description of the SOX2 Gene promoter

The SOX2 gene promoter was truncated 1200bp from the 5' UTR region of the SOX2 gene, and contained a TATA box and a transcription start site located in the CpG island region. The intermediate vector pSOX2 plasmid was constructed by replacing the CMV promoter expressing the foreign gene in the commercial vector pEGFP-C1 with the SOX2 gene promoter. Combined analysis of qRT-PCR, ELISA expression and fluorescence intensity 32 days after transfection of CHO cells with the pSOX2 plasmid revealed that the pSOX2 plasmid promoted expression of downstream EGFP protein in CHO cells in an amount lower than the commercial vector pEGFP-C1 (FIGS. 5-7).

(2) The plasmid vector pSOX2-MAR (expression vector) constructed was higher than the control plasmid pSOX2 (intermediate vector)

The results of analysis by qRT-PCR, ELISA and fluorescence intensity analysis showed that the expression level and expression stability of EGFP protein were better than those of control plasmid pSOX2 without MAR regulation 32 days after the pSOX2-MAR plasmid was transformed into CHO cells (FIGS. 5-7).

(3) The constructed plasmid vector pSOX2-MAR was higher than the control plasmid pSOX2-UCOE (control vector I)

Results of qRT-PCR, ELISA and fluorescence intensity analysis show that the expression amount of EGFP protein is remarkably increased after the pSOX2-MAR plasmid is transformed into CHO cells for 32 days compared with the pSOX2-UCOE plasmid, and the expression efficiency and stability of MAR are higher than the combined action of a chromatin regulatory sequence UCOE and a SOX2 promoter when the MAR acts on the SOX2 promoter (FIGS. 5-7).

(4) The constructed plasmid vector pSOX2-MAR was higher than the control plasmid pOCT4-MAR (control vector II)

The results of analysis by qRT-PCR, ELISA and fluorescence intensity analysis showed that the expression level and expression stability of EGFP protein were better than those of MAR and the control plasmid pOCT4-MAR containing enhancer but no CpG islands after the pSOX2-MAR plasmid was transformed into CHO cells for 32 days (FIGS. 5-7).

The method of the invention obtains the following conclusion by comparing the constructed expression vector with the comparison vector:

(1) binding of the MAR sequence to the target promoter enables a greater efficiency of activation of the target promoter than if the MAR sequence had acted together with the enhancer-containing promoter.

(2) The effect of MAR sequence on the expression efficiency of the target promoter is significantly higher than the effect of the co-action of another chromatin-regulating sequence UCOE sequence (Homo sapiens chromosome 7, GRCh38.p12: 26200227 to 26201780) with the target promoter.

The above are only some embodiments of the present invention, and the examples are not intended to limit the present invention, and those skilled in the art will appreciate that there are many embodiments that can implement the technical solution of the present invention according to the content of the present specification, and that simple replacement or homogeneous change made according to the technical solution of the present invention should fall into the protection scope of the present invention.

SEQUENCE LISTING

<110> Kunming academy of academic

Method for constructing high-efficiency expression vector by regulating weak promoter through <120> MAR sequence

<130> SOX2 Gene promoter sequence

CAAGGAAGGTTTTGAGGACAGAGGTTTGGGTCTCCTAACTTCTAGTCGGGACTGTGAGAAGGGCGTGAGAGAGTGTTGGCACCTGTAAGGTAAGAGAGGAGAGCGGAAGAGCGCAGTACGGGAGCGGCACCAGAGGGGCTGGAGTTGGGGGGGAGTGCTGTGGATGAGCGGGAGAACAATGACACACCAACTCCTGCACTGGCTGTTTCCAGAAATACGAGTTGGACAGCCGCCCTGAGCCACCCACTGTGCCCTGCCCCACCCCCGCACCTTAGCTGCTTCCCGCGTCCCATCCTCATTTAAGTACCCTGCACCAAAAAGTAAATCAATATTAAGTTTAAAGAAAAAAAAACCCACGTAGTCTTAGTGCTGTTTACCCACTTCCTTCGAAAAGGCGTGTGGTGTGACCTGTTGCTGCGAGAGGGGATACAAAGGTTTCTCAGTGGCTGGCAGGCTGGCTCTGGGAGCCTCCTCCCCCTCCTCGCCTGCCCCCTCCTCCCCCGGCCTCCCCCGCGCGGCCGGCGGCGCGGGAGGCCCCGCCCCCTTTCATGCAAAACCCGGCAGCGAGGCTGGGCTCGAGTGGAGGAGCCGCCGCGCGCTGATTGGTCGCTAGAAACCCATTTATTCCCTGACAGCCCCCGTCACATGGATGGTTGTCTATTAACTTGTTCAAAAAAGTATCAGGAGTTGTCAAGGCAGAGAAGAGAGTGTTTGCAAAAGGGGGAAAGTAGTTTGCTGCCTCTTTAAGACTAGGACTGAGAGAAAGAAGAGGAGAGAGAAAGAAAGGGAGAGAAGTTTGAGCCCCAGGCTTAAGCCTTTCCAAAAAATAATAATAACAATCATCGGCGGCGGCAGGATCGGCCAGAGGAGGAGGGAAGCGCTTTTTTTGATCCTGATTCCAGTTTGCCTCTCTCTTTTTTTCCCCCAAATTATTCTTCGCCTGATTTTCCTCGCGGAGCCCTGCGCTCCCGACACCCCCGCCCGCCTCCCCTCCTCCTCTCCCCCCGCCCGCGGGCCCCCCAAAGTCCCGGCCGGGCCGAGGGTCGGCGGCCGCCGGCGGGCCGGGCCCGCGCACAGCGCCCGCATGTACAACATGATGGAGACGGAGCTGAAGCCGCCGGGCCCGCAGCAAACTTCGGGGGGCGGCGGCGGCAACTCCACCGCGGCGGCGGCCGGCGGCAACCAGAAAAACAGCCCGG

<140> MAR sequence i.e. (HUBB-LCR sequence)

TTAGTAAGACATCACCTTGCATTTTGAAAATGCCATAGACTTTCAAAATTATTTCATACATCGGTCTTTCTTTATTTCAAGAGTCCAGAAATGGCAACATTACCTTTGATTCAATGTAATGGAAAGAGCTCTTTCAAGAGACAGAGAAAAGAATAATTTAATTTCTTTCCCCACACCTCCTTCCCTGTCTCTTACCCTATCTTCCTTCCTTCTACCCTCCCCATTTCTCTCTCTCATTTCTCAGAAGTATATTTTGAAAGGATTCATAGCAGACAGCTAAGGCTGGTTTTTTCTAAGTGAAGAAGTGATATTGAGAAGGTAGGGTTGCATGAGCCCTTTCAGTTTTTTAGTTTATATACATCTGTATTGTTAGAATGTTTTATAATATAAATAAAATTATTTCTCAGTTATATACTAGCTATGTAACCTGTGGATATTTCCTTAAGTATTACAAGCTATACTTAACTCACTTGGAAAACTCAAATAAATACCTGCTTCATAGTTATTAATAAGGATTAAGTGAGATAATGCCCTATAAGATTCCTATTAATAACAGATAAATACATACACACACACACACATTGAAAGGATTCTTACTTTGTGCTAGGAACTATAATAAGTTCATTGATGCATTATATCATTAAGTTCTAATTTCAACACTAGAAGGCAGGTATTATCTAAATTTCATACTGGATACCTCCAAACTCATAAAGATAATTAAATTGCCTTTTGTCATATATTTATTCAAAAGGGTAACTCAAACTATGGCT

Claims (1)

1. A method for constructing a high-efficiency expression vector by using a MAR sequence to regulate a weak promoter, which is characterized by comprising the following steps:

(1) selecting a promoter which does not contain an enhancer and contains a CpG island at a transcription initiation site, namely a promoter of the human SOX2 gene as a target promoter;

(2) replacing the CMV promoter in the exogenous gene expression frame on a commercial vector pEGFP-C1 by the target promoter to construct an intermediate vector; in the step of constructing the intermediate vector, a target promoter sequence is amplified by using a double enzyme cutting site primer with AgeI and AseI, wherein the primer is as follows:

SOX2 # Forward primer: CCGTGATTAAT(Ase I) AGACAAGGAAGGTTTTGAGGAC

SOX2 # reverse primer: ATATGACCGGT(Age I) ATCCGGGCTGTTTTTCTGGTT, respectively;

(3) inserting a MAR sequence into an Ase I enzyme cutting site at the upstream of a foreign gene expression frame of the intermediate vector to construct an expression vector, wherein the MAR sequence is shown as a sequence 2; the expression vector can ensure that the expression quantity of the EGFP gene is continuously, stably and efficiently expressed by verification.

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