Eukaryotic high-expression promoter and application thereof

Abstract

The invention belongs to the technical field of biology, and particularly relates to a eukaryotic high-expression promoter and application thereof. Research finds that a promoter (HEP) from a human gene TPRN can be used as a promoter of an eukaryotic expression vector, exogenous gene expression in the expression vector can be efficiently driven in HEK293 cells, and the expression efficiency is improved by dozens or even hundreds of times compared with that of a conventional expression vector. The promoter can be further regulated and controlled by a transcription factor ERR and a regulatory factor PGC1 alpha thereof, so that the expression efficiency is improved. The promoter as well as the expression vector and the expression system thereof have good popularization and application values.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. In particular, the present invention relates to a eukaryotic high-expression promoter and its application. Background Art

[0002] Protein expression systems are tools for producing recombinant proteins, and there are two common types: prokaryotic and eukaryotic. Prokaryotic systems such as Escherichia coli (E. coli) are easy to operate, low in cost, and grow rapidly, suitable for large-scale production, but may not be able to correctly fold complex proteins. Eukaryotic systems include yeast (such as Pichia pastoris), insect cells (such as the baculovirus expression system), and mammalian cells (such as 293F and CHO cells). Yeast grows relatively fast and can perform some post-translational modifications; insect cells and mammalian cells can express proteins closer to the natural state, suitable for complex proteins and proteins that require modification. Each system has its advantages and limitations, and the choice needs to be based on the properties of the target protein and the application requirements.

[0003] As a commonly used mammalian cell expression system, 293 cells are widely used in the fields of recombinant protein production, gene function research, etc., and have many significant advantages. It can grow rapidly in serum-free medium and efficiently express recombinant proteins, and at the same time shows high transfection efficiency for a variety of transfection reagents, suitable for large-scale protein production and the preparation of viral vectors. In addition, 293F cells in the 293 cell series are adapted to serum-free suspension culture, can achieve high-density growth, and are convenient for industrial application. As a mammalian cell, 293F cells can perform complex post-translational modifications, making the expressed proteins closer to the natural state, so they are widely used in biopharmaceuticals and basic research. However, in the prior art, the protein expression efficiency of 293 cells still needs to be further improved. Especially in large-scale production, the improvement of expression efficiency is of great significance for reducing costs and increasing yields. In an expression system, the selection and optimization of promoters are one of the key factors affecting protein expression efficiency. Commonly used promoters in mammalian expression systems include the CMV promoter, CAG promoter, etc., and there are still many problems, such as insufficient promoter activity. Generally speaking, the optimization of promoters in existing eukaryotic expression systems needs to comprehensively consider various factors, including promoter sequences, enhancer combinations, cell culture conditions, etc., and the optimization is difficult. Summary of the Invention

[0004] Enhanced promoters suitable for mammalian expression systems are scarce, and promoters from other species still need to be optimized and modified. Therefore, there is an urgent need in the art for new types of efficient promoters to improve the protein expression efficiency of mammalian expression systems, including the 293 expression system. Through extensive screening and research, the present invention unexpectedly found that the promoter from the human gene TPRN (HEP) exhibits a high basal transcriptional activity in 293 cells. Thus, a highly efficient promoter for protein expression in eukaryotic cells such as 293 cells was discovered, completing the present invention.

[0005] In one aspect of the present invention, the present invention provides a promoter for a eukaryotic high-expression system, which is used to drive the expression of an exogenous gene located on an expression vector in mammalian cells.

[0006] In the present invention, the mammalian cells include human embryonic kidney cells HEK293 cells and CHO cells.

[0007] In one embodiment, the human embryonic kidney cells HEK293 cells include 293T cells and 293F cells.

[0008] The promoter in the present invention is HEP1, HEP3 or HEP4. Among them, HEP1 contains the entire sequence of HEP, and HEP3 and HEP4 are truncated HEP containing partial sequences of HEP. The nucleotide sequence of HEP1 is shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is shown in SEQ ID NO.3.

[0009] There is a recognition site for the transcription factor ERR on HEP. ERR is a member of the orphan nuclear receptor superfamily and has structural and functional similarities with estrogen receptors (ERs). This nuclear receptor subfamily contains three members, ERRα, ERRβ and ERRγ, which recognize the same type of DNA response element and recruit the co-regulator PGC1α to initiate transcription. And HEP can be further regulated by ERR to improve the transcription efficiency.

[0010] In one aspect of the present invention, the present invention provides the application of the promoter of the human gene TPRN (HEP) in a eukaryotic expression system. The promoter is used to drive the expression of a eukaryotic expression vector containing an exogenous gene in mammalian cells. The promoter is HEP1, HEP3 or HEP4. Among them, the nucleotide sequence of HEP1 is shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is shown in SEQ ID NO.3.

[0011] In one embodiment, the promoter of the present invention is upstream of the foreign gene on the expression vector, driving the expression of the downstream foreign gene in mammalian cells.

[0012] In one embodiment, the mammalian cells include human embryonic kidney cells HEK293 cells and CHO cells.

[0013] In one aspect of the present invention, there is provided a eukaryotic expression vector, and the promoter in the promoter region of the expression vector is HEP1, HEP3 or HEP4; wherein, the nucleotide sequence of HEP1 is as shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is as shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is as shown in SEQ ID NO.3.

[0014] In one aspect of the present invention, there is provided the use of the eukaryotic expression vector of the present invention in protein expression.

[0015] In one aspect of the present invention, there is provided a eukaryotic expression kit, and the expression kit contains the eukaryotic expression vector of the present invention.

[0016] In one embodiment, the reagent and the kit of the present invention further contain a eukaryotic expression vector expressing the transcription factor ERR and / or its regulator; preferably, the transcription factor ERR is one or more of ERRα, ERRβ, ERRγ; and the regulator is PGC1α.

[0017] In one aspect of the present invention, there is provided the use of the eukaryotic expression kit of the present invention in protein expression.

[0018] Beneficial effects The present invention has invented a highly efficient promoter HEP that can be used in eukaryotic expression systems, greatly improving the expression efficiency of foreign genes, being able to increase the foreign protein expression ability by dozens of times, reducing the expression cost of highly complex proteins, and having good application value. Brief description of the drawings

[0019] Figure 1 : Luciferase test results of HEP1, HEP3, and HEP4. The standard deviation is obtained from the data of 3 replicate wells.

[0020] Figure 2 : Luciferase test results of the HEP promoter regulated by the transcription factor ERR. The standard deviation is obtained from the data of 3 replicate wells.

[0021] Figure 3:Observation results of the fluorescence microscope of cells 293T transfected with three vectors HEP1-GFP, HEP3-GFP, and HEP4-GFP expressing GFP, and the fluorescence was significantly stronger than that of the control pCAG-GFP.

[0022] Figure 4 :Results of the analysis of the proportion of the area of GFP-expressing particles using ImageJ software.

[0023] Figure 5 :SDS-PAGE was used to detect the PLA2G12B protein after preliminary affinity purification using the expression vector of the present invention. Detailed implementation manners

[0024] In order to make the objectives, technical solutions and advantages of the present invention more clear and understandable, the present invention will be further described in detail below in conjunction with embodiments. The equipment and reagents used in each embodiment and test example can be obtained from commercial channels without special instructions. Unless otherwise specified, the reagents used in the present invention are all analytical grade reagents. The experimental operations involved in the present invention can be based on "Molecular Cloning: A Laboratory Manual" edited by M.R. Green, J. Sambrook, etc. published by Science Press; "Cell: A Laboratory Manual" edited by D.L. Spector, etc. published by Science Press. The kits and reagents involved are all operated according to the instructions of the manufacturers. The specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0025] Example 1: Detection of the activities of HEP1, HEP3, and HEP4 using Dual-luciferase assay The dual-luciferase reporter assay is a gene regulation research technology based on the principle of luciferase luminescence. In the experiment, firefly luciferase is used as the reporter gene, and Renilla luciferase is used as the internal reference gene. By cloning the target gene regulatory element upstream of the firefly luciferase gene to construct a reporter gene plasmid, and detecting the luciferase activity after transfection of cells, it can be accurately measured by a luminometer. The specific test steps are as follows: (1)Construction of the reporter gene vector: HEP1, HEP3, and HEP4 were constructed between the KpnI and XhoI restriction enzyme sites of the pGL3-basic vector (Promega), and were named pGL3-HEP1, pGL3-HEP3, and pGL3-HEP4 respectively; among them, the nucleotide sequence of HEP1 is shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is shown in SEQ ID NO.3.

[0026] (2)Culture 293T cells. When passaged to the 4th generation, plate them in a 96-well plate at 10,000 cells per well.

[0027] (3) Co - transfect the reference plasmid to be detected and the reporter gene plasmid into cells using the transfection reagent lipo2000.

[0028] (4) After 6 hours of transfection, aspirate the culture medium, replace it with fresh medium, and culture for another 18 hours.

[0029] (5) Use the Dual - Glo kit (Promega) for testing and read the plate using a multimode microplate reader (PerkinElmer). Finally, calculate the ratio of firefly luciferase to Renilla luciferase activity in each well to correct the experimental results and perform normalization calculation using the control values.

[0030] Test results: See Figure 1 The results showed that compared with pGL3 - basic without any inserted sequence, the fluorescence signal of HEP1 was about 40 - fold, that of HEP3 was about 58 - fold, and that of HEP4 was about 30 - fold.

[0031] Example 2: Dual - reporter luciferase experiment to test the regulation of the transcriptional factor ERR and its regulator on the activity of pGL3 - HEP1 Through sequence analysis, a binding site for the transcriptional factor ERR was identified on the HEP sequence. ERRα is a member of the nuclear receptor family and can initiate gene transcription by binding to the co - activator PGC1α. In this example, ERRα, PGC1α, and pGL3 - HEP1 were co - transfected simultaneously. The Dual - reporter luciferase detection system was used to detect the effects of ERRα and PGC1α on the activity of pGL3 - HEP1.

[0032] The specific detection steps are as follows: (1) Construct protein expression vectors: Clone the full - length sequences of ERRα and PGC1α into the pCAG (Miaoling Bio, SP0182) and pCDNA3.1 (Invitrogen) vectors for full - length expression respectively.

[0033] (2) Culture 293T cells. When passaged to the 4th generation, seed 10000 cells per well in a 96 - well plate.

[0034] (3) Co - transfect the protein expression plasmid to be detected, the reporter gene plasmid (pGL3 - HEP1), and the reference plasmid into cells using the transfection reagent lipo2000.

[0035] (4) After 6 hours of transfection, aspirate the culture medium, replace it with fresh medium, and culture for another 18 hours.

[0036] (5) Use the Dual-Glo kit (Promega) for testing and read the plates using a multimode microplate reader (PerkinElmer). Finally, calculate the ratio of firefly luciferase to Renilla luciferase activity in each well to correct the experimental results and perform normalization calculations using the control values.

[0037] Test results: See Figure 2 As shown, when comparing with only transfecting HEP1, co-transfecting ERRα and HEP1, the fluorescence signal increased by 1.8 times; the fluorescence signal of co-transfecting ERRα, PGC1α and pGL3-HEP1 was 3 times that of only transfecting pGL3-HEP1 and 120 times that of the empty vector control. It shows that the system of the present invention can further activate ERR to up-regulate the expression of downstream proteins and has regulatory value.

[0038] Example 3: GFP fluorescence protein expression test using the promoter of the present invention The GFP fluorescence protein expression test is a commonly used biotechnology method for detecting gene expression, protein localization and functional research. In this example, HEP was ligated into the pCAG (Miaoling Biology, SP0182) vector that can express GFP green fluorescent protein. After transfecting the cells, the expression of GFP was analyzed by observing the green fluorescence of GFP using a fluorescence microscope.

[0039] The specific experimental steps are as follows: (1) Construct the HEP-GFP expression vector: ligate HEP1, HEP3, and HEP4 between EcoRI and KpnI of the pCAG vector respectively, and this vector can express eGFP; that is, obtain three different expression vectors of HEP1-GFP, HEP3-GFP, and HEP4-GFP.

[0040] (2) Culture 293T cells. When passaged to the 4th generation, plate them at 50,000 cells per well in a 24-well plate.

[0041] (3) Use the transfection reagent lipo2000 to transfect the expression plasmid to be detected into the cells.

[0042] (4) After 6 hours of transfection, aspirate the culture medium, replace it with a new culture medium, and culture for another 18 hours.

[0043] (5) Observe and photograph using an inverted fluorescence microscope with an exposure of 400 ms.

[0044] Test results: See Figure 3 , Observe the GFP expression 12 hours after transfection. The green fluorescence of GFP linked to the HEP expression was significantly stronger than that of the control plasmid under the fluorescence microscope. The fluorescence intensity is positively correlated with the GFP expression level. See Figure 4, the proportion of GFP-expressing particle area was analyzed using ImageJ software, showing that the proportion of HEP1-GFP was approximately 100 times that of the control pCAG-GFP. HEP1 and its truncated fragment HEP3 were the strongest, and the short fragment HEP4 was slightly lower.

[0045] Example 4: Expression of Secreted Membrane Surface Protein Using the Expression Vector of the Promoter of the Present Invention In this example, the effect of HEP on protein expression was further tested in a suspension cell protein expression system. PLA2G12B (Phospholipase A2 Group XIIB, G12B) is a secreted phospholipase A2-like protein belonging to the PLA2 enzyme family and is involved in cholesterol homeostasis, low-density lipoprotein particles, lipid droplet transport, etc. G12B is a membrane surface protein containing 7 pairs of disulfide bonds, and large amounts of soluble protein in the supernatant cannot be expressed in prokaryotic and insect cells.

[0046] The specific experimental steps are as follows: (1) Construction of the HEP3-G12B expression vector: The HEP3-G12B sequence was ligated between EcoRI and KpnI of the pCAG (Miaoling Biology, SP0182) vector, and a his tag and a stop codon were added after G12B to make the vector express only G12B. After successful construction, a kit was used to extract the plasmid with endotoxin removal.

[0047] (2) Cell transfection: Expi293F (gibco) cells were diluted to a density of 1.5 × 10 6 cell / mL, 200 mL / 1L shake flask, and cultured in suspension. The HEP3-G12B plasmid was transfected into Expi293F using the transfection reagent PEI.

[0048] (3) Harvesting cell supernatant: After 96 hours of transfection, the supernatant was centrifuged at low speed first and then at high speed, and the supernatant was harvested and filtered.

[0049] (4) Enrichment of hG12B-His protein by nickel column: The hG12B-His protein in the supernatant was enriched using an AKTA system and a pre-packed nickel column (Cytiva). After collecting and concentrating the protein, the concentration was measured by the Bradford method, and SDS-PAGE gel was used for detection.

[0050] Test results: After detection, the expression level of the HEP3-G12B vector using the HEP3 promoter reached 0.4 mg / L. If ERR / PGC1α was co-transfected, its expression level could be further increased to 0.6 mg / L. In contrast, the amount of G12B protein expressed without using the HEP3 promoter was only 0.08 mg / L. That is, using the promoter and system of the present invention, the protein expression efficiency was increased by 5 - 8 times. See Figure 5, to obtain the PLA2G12B protein after preliminary affinity purification using the expression vector of the present invention.

[0051] The above content is a further detailed description of the present invention in combination with specific embodiments. It cannot be determined that the specific implementation of the present invention is only limited to these descriptions. For those of ordinary skill in the technical field to which the present invention pertains, without departing from the concept of the present invention, several simple deductions or substitutions can still be made, and all should be regarded as falling within the protection scope determined by the claims submitted for the present invention.

Claims

1. A promoter of a eukaryotic high-expression system, characterized in that, The promoter is used to drive the expression of an exogenous gene located on an expression vector in mammalian cells. The promoter is HEP1, HEP3 or HEP4. Among them, the nucleotide sequence of HEP1 is shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is shown in SEQ ID NO.

3.

2. Application of the promoter of human gene TPRN in eukaryotic expression system, characterized in that, The promoter is used to drive the expression of an exogenous gene located on an expression vector in mammalian cells. The promoter is HEP1, HEP3 or HEP4. Among them, the nucleotide sequence of HEP1 is shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is shown in SEQ ID NO.

3.

3. The application according to claim 2, wherein The mammalian cells are human embryonic kidney cells HEK293 cells and CHO cells.

4. A eukaryotic expression vector, characterized in that, The promoter of the expression vector is HEP1, HEP3 or HEP4; among them, the nucleotide sequence of HEP1 is shown in SEQ ID NO.1, the nucleotide sequence of HEP3 is shown in SEQ ID NO.2, and the nucleotide sequence of HEP4 is shown in SEQ ID NO.

3.

5. Use of the eukaryotic expression vector according to claim 4 in protein expression.

6. A eukaryotic expression kit, characterized in that, The expression kit contains the expression vector according to claim 4.

7. The kit according to claim 6, wherein The kit further contains a eukaryotic expression vector expressing the transcription factor ERR and / or its regulator.

8. The kit according to claim 7, wherein The transcription factor ERR is one or more of ERRα, ERRβ, and ERRγ; the regulator is PGC1α.

9. Use of the eukaryotic expression kit according to any one of claims 6-8 in protein expression.

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