WO2019024150A1 - Ternary transcription factor and application thereof in mammalian protein expression system - Google Patents

Ternary transcription factor and application thereof in mammalian protein expression system Download PDF

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WO2019024150A1
WO2019024150A1 PCT/CN2017/098859 CN2017098859W WO2019024150A1 WO 2019024150 A1 WO2019024150 A1 WO 2019024150A1 CN 2017098859 W CN2017098859 W CN 2017098859W WO 2019024150 A1 WO2019024150 A1 WO 2019024150A1
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transcription factor
gene
expression system
expression vector
base sequence
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何南海
路杨
杨东晖
王骥
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杭州阿诺生物医药科技股份有限公司
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Definitions

  • the invention relates to the field of genetic engineering technology, in particular to triple transcription factors and their use in mammalian protein expression systems.
  • Protein expression is an important part of modern industrial, medical and basic research, and the use of recombinant technology to produce proteins, especially high-purity, high-activity proteins, plays a pivotal role in biomedical research and pharmaceuticals.
  • prokaryotic expression systems include prokaryotic expression systems, yeast expression systems, insect expression systems, and mammalian expression systems.
  • prokaryotic expression systems and yeast expression systems are characterized by high yield and ease of manipulation, when used for expression of mammalian proteins, there are natural defects: (1) such microbial systems are lacking or difficult to complete after protein translation.
  • Various chemical modifications such as glycosylation, acetylation, etc., and post-translational modification of proteins play an important role in the transport of proteins in cells and in vivo, protein stability and biological activity in organisms. Therefore, the lack of such modifications can have a significant impact on the biological activity of recombinant proteins.
  • the insect expression system has certain post-translational modification ability, due to species differences and genome differences, the protein post-translational modification cannot be compared with mammals in complexity and diversity, and the protein yield of the system is also Not high; (2) Many human proteins cannot be normally folded in a bacterial or insect expression system to form inclusion bodies. Therefore, the purified protein requires a complex renaturation process to restore vitality.
  • the advantage is that the protein can achieve correct folding, with a relatively complete protein post-translational modification, and has complete biological activity, but its disadvantage is that the protein production is usually low and the production cost is high. This is also an important reason why the number and quantity of recombinant proteins derived from mammalian expression systems are less than other systems.
  • Mammalian cells have elaborate regulatory mechanisms for protein expression, including transcription, translation, and Modification regulation. Among them, the upstream gene transcription level largely determines the downstream protein production.
  • SEC Supper Elongation Complex
  • HIV Immunodeficiency Virus (HIV) model is used to describe the two proteins ELL (Elongation factor for RNA Polymerase II) 2 and AFF (AF4/). FMR2 family member)4
  • ELL Elongation factor for RNA Polymerase II
  • AFF AF4/
  • FMR2 family member4 The mechanism of action of HIV HIV on gene transcription, studies have shown that the combination of these two proteins in the complex can greatly promote HIV gene expression, using the reporter gene test showed that this The combination of two proteins can multiply the gene expression of the AIDS promoter, showing A protein complex, in particular the two proteins of the AIDS virus HIV has a very important role.
  • CRTC is a cAMP-responsive element binding protein (CREB)-reactive transcriptional coactivator.
  • the CRTC family has three members (CRTC1-3) and contains CRTC (CREB regulated transcription).
  • ELL2, AFF4 and CRTC2 in combination to mammalian protein expression systems to increase mammalian protein expression.
  • the prior art problem solved by the present invention is that the existing mammalian protein expression system generally has the characteristics of low expression and high cost.
  • the present invention first provides a transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor.
  • the ELL transcription factor is selected from one of ELL2 or ELL1
  • the AFF transcription factor is selected from the group consisting of In one of AFF4 or AFF1
  • the CRTC transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
  • the present invention provides a DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor.
  • the present invention provides a recombinant expression vector obtained by cloning a transcription factor composition or a triple transcription factor into a mammalian expression vector.
  • the present invention also provides a method for preparing a recombinant expression vector, which comprises ligating a gene encoding an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor with a vector to obtain a recombinant vector containing a triple transcription factor; and then transforming the recombinant vector into The recipient cell is screened for a vector for expression of a transcription factor; finally, the expression vector is subjected to DNA sequencing to obtain a recombinant expression vector.
  • the present invention provides a method of preparing a mammalian protein expression system by increasing the protein content by enhancing the transcription of the gene. This will not only greatly reduce the production cost of mammalian recombinant protein, increase the income, but also positively promote the development of life science research and bio-pharmaceutical industry.
  • the preparation method of the mammalian protein expression system of the present invention comprises co-transfecting a recombinant expression vector and a gene expression vector of interest into a cell strain to obtain a recombinant protein.
  • the method for preparing a mammalian protein expression system of the present invention can also integrate a gene encoding an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor into a gene of interest, to obtain a gene of interest containing a gene encoding a triple transcription factor, and then to encode a triple gene.
  • the gene of interest of the gene of the transcription factor is ligated to the vector to obtain a gene expression vector containing the gene encoding the triple transcription factor, and finally transfected into the cell line to obtain a mammalian protein expression system.
  • the invention also provides a method for purifying a mammalian protein expression system, which comprises adding a lysate to the collected cell strain and centrifuging to obtain a supernatant; then, after mixing the supernatant with the label, the supernatant is removed by centrifugation. Adding a high-salt washing solution for washing; then, centrifuging the washing liquid, removing the supernatant, washing with a low-salt washing solution; finally eluting with the labeled peptide, and centrifuging to obtain a supernatant, the supernatant It is a mammalian protein expression system.
  • the present invention proposes the following technical solutions.
  • a transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor, preferably, the ELL transcription factor is selected from one of ELL2 or ELL1, and the AFF transcription factor is selected from one of AFF4 or AFF1, CRTC The transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
  • the ratio of ELL2, AFF4 and CRTC2 is 1:1 to 3: 1-4;
  • the ratio of the ELL2, AFF4 and CRTC2 is 1:1:1.
  • the transcription factor composition wherein the transcription factor composition is selected from one of a human source, a mouse source, a dog source or a pig source; preferably, the transcription factor composition is a human source of.
  • a triple transcription factor comprising the transcription factor composition described above.
  • a DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor; preferably, the base sequence encoding the ELL transcription factor is selected From one of the following base sequences:
  • the base sequence encoding the AFF transcription factor is selected from one of the following base sequences:
  • the base sequence encoding the CRTC transcription factor is selected from one of the following base sequences:
  • (d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 6 and which has a transcriptional activity of the regulatory gene.
  • a recombinant expression vector obtained by cloning the transcription factor composition or the triple transcription factor into a mammalian expression vector.
  • a method for preparing a recombinant expression vector comprising the steps of:
  • the vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
  • transcription factor composition or triple transcription factor in a mammalian protein expression system.
  • a mammalian protein expression system wherein a recombinant expression vector or a recombinant expression vector is co-transfected with a gene expression vector of interest into a cell strain, and the total amount of the recombinant expression vector and the total amount of the expression vector of the target gene are obtained.
  • the ratio is 1:1-5.
  • the cell strain is selected from one of human embryonic kidney cell 293 or Chinese hamster ovary cells.
  • a method of preparing a mammalian protein expression system comprising the steps of:
  • the method for preparing the gene carrier of interest comprises the steps of: ligating the gene of interest and the expression vector to obtain a gene expression vector of interest.
  • a mammalian protein expression system obtained by transfecting a target gene expression vector of a triple transcription factor into a cell strain.
  • the cell strain is selected from one of human embryonic kidney cells or Chinese hamster ovary cells.
  • a method of preparing a mammalian protein expression system comprising the steps of:
  • the expression vector obtained in the step (2) is transfected into the cell strain, and the cells are collected 1-3 days later to obtain a mammalian protein expression system.
  • a method of purifying a mammalian protein expression system comprising the steps of:
  • the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene glycol, and protease.
  • the inhibitor mixture is added in an amount of 1-5 ml of the lysate per 1 ⁇ 10 7 cells.
  • the volume ratio of the supernatant to the label beads is 20-50:1.
  • the high salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene A mixture of alcohol and protease inhibitor, the high salt wash is added in an amount of from 1 to 5 ml.
  • the low-salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene.
  • a mixture of alcohol and protease inhibitor, said low salt wash is added in an amount of from 1 to 5 ml.
  • volume ratio of the label peptide to the label beads is 3-5:1.
  • the beneficial effects obtained by the invention are that the mammalian expression system obtained by the invention has the advantages of low cost and high yield compared with the traditional method, can effectively increase the yield of the recombinant protein, and maintain its complete post-translational modification. Improve biological activity.
  • This mammalian expression system greatly enhances the gene's expression at the transcriptional level by introducing transcription factors in the optimized super-transcriptional elongation complex. To achieve the goal of increasing protein production.
  • the mammalian expression system obtained by the invention has the potential of simple operation, easy large-scale production and rapid industrialization.
  • Example 1 is a schematic diagram showing the expression level of a LIF protein expression system obtained in Example 1;
  • FIG. 2 is a schematic diagram showing the purity of the LIF protein expression system obtained in Example 1;
  • Figure 3 is a schematic diagram showing the comparison of the biological activity of the LIF protein expression system obtained in Example 1 with the same product using the bacterial expression system
  • Figure 3-1 is a schematic diagram of the biological activity of the protein expression system without LIF
  • Figure 3-2 is a diagram showing the biological activity of the protein expression system without LIF.
  • Figures 3-3 to 3-6 are schematic diagrams of the biological activities of the protein expression systems of Example 1 at 2.5 ng/ml, 5 ng/ml, 10 ng/l and 20 ng/ml.
  • 1 is a schematic diagram showing the expression level of a LIF protein expression system obtained in Example 1, wherein 1 is a map showing the expression level of a LIF protein expression system obtained by transfecting a LIF expression vector into a Chinese hamster ovary cell, 2 It is a schematic diagram of the expression level of LIF protein expression system obtained by co-transfection of LIF expression vector and expression vector containing transcription factors ELL2 and AFF4 into Chinese hamster ovary cells, 3 is a LIF expression vector and contains triple transcription factors ELL2, AFF4 and CRTC2. Schematic diagram of the expression level of the LIF protein expression system obtained by co-transfection of the expression vector into Chinese hamster ovary cells. As can be seen from the above figure, the triple transcription factors ELL2, AFF4 and CRTC2 can increase the expression level of LIF by 7 times.
  • Example 2 is a schematic diagram showing the purity of the LIF protein expression system obtained in Example 1. As can be seen from the figure, the purified LIF protein expression system has an extremely high purity and a purity higher than 95%.
  • Figure 3 is a schematic diagram showing the comparison of the biological activity of the LIF protein expression system obtained in Example 1 with a similar product using the bacterial expression system (Cat. No. PHC9484, Thermo Fisher Scientific), wherein Figure 3-1 is a schematic diagram of the biological activity of the protein expression system without LIF.
  • Figure 3-2 is a schematic diagram showing the biological activity of a similar product (10 ng/ml) using a bacterial expression system, and Figures 3-3 to 3-6 are the protein expression systems of Example 1 at 2.5 ng/ml, 5 ng/ml, A schematic diagram of the biological activity of 10 ng/l and 20 ng/ml.
  • the protein expression system has high biological activity and the biological activity exceeds 300% of the similar products using the bacterial expression system.
  • the present invention provides a transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor, preferably, the ELL transcription factor is selected from one of ELL2 or ELL1, and the AFF transcription factor is selected from AFF4. Or one of AFF1, the CRTC transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
  • composition has ELL2, AFF4 and CRTC2.
  • the ratio of the ELL2, AFF4 and CRTC2 is 1:1 to 3: 1-4, and more preferably, the ratio of the ELL2, AFF4 and CRTC2 is 1:1:1.
  • the transcription factor composition is selected from one of a human source, a mouse source, a dog source or a pig source, and preferably, the transcription factor composition is of a human origin.
  • the present invention also provides a triple transcription factor expression vector obtained by cloning a triple transcription factor into a mammalian expression vector.
  • the present invention provides a DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor, wherein the ELL transcription factor is encoded
  • a base sequence encoding an ELL transcription factor a base sequence encoding an AFF transcription factor
  • a base sequence encoding a CRTC transcription factor a base sequence encoding a CRTC transcription factor
  • (d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 2 and which has a transcriptional activity of the regulatory gene.
  • the base sequence encoding the AFF transcription factor is selected from one of the following base sequences:
  • (d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 4 and which has a transcriptional activity of the regulatory gene.
  • the base sequence encoding the CRTC transcription factor is selected from one of the following base sequences:
  • (d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 6 and which has a transcriptional activity of the regulatory gene.
  • the present invention provides a recombinant expression vector obtained by cloning a transcription factor composition or a triple transcription factor into a mammalian expression vector containing a CMV promoter.
  • the invention provides a preparation method of a recombinant expression vector, which comprises the following steps:
  • the vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
  • the invention provides a method for preparing a recombinant expression vector comprising the steps of:
  • the gene encoding the ELL transcription factor, the AFF transcription factor, the CRTC transcription factor, and the expression vector are subjected to restriction endonuclease digestion, followed by isolation and purification.
  • the ELL transcription factor, the AFF transcription factor, the CRTC transcription factor gene and the vector after digestion are respectively ligated with the T4 ligase to obtain a recombinant vector containing a triple transcription factor;
  • step (2) mixing the recombinant vector obtained in the step (1) with E. coli competent bacteria, incubating on ice for 30 minutes, and then transforming the recombinant vector by heat shock method (in a 42-degree water bath for 90 seconds) Go into the bacteria. Thereafter, the transformed bacteria were plated on an LB plate containing ampicillin and cultured overnight in a 37-degree incubator.
  • the present invention provides a mammalian protein expression system which is obtained by co-transfecting a recombinant expression vector and a gene expression vector of interest into a cell strain, the total amount of the triple transcription factor expression vector and the expression vector of the target gene.
  • the total ratio is 1:1-5.
  • the cell strain is one selected from the group consisting of human embryonic kidney cell 293 or Chinese hamster ovary cells.
  • the invention provides a preparation method of a mammalian protein expression system, which comprises the following steps:
  • the invention also provides a preparation method of another mammalian protein expression system, comprising the following steps:
  • the expression vector obtained in the step (2) is transfected into the cell strain, and the cells are collected 1-3 days later to obtain a mammalian protein expression system.
  • the present invention provides a method for preparing a mammalian protein expression system comprising the steps of:
  • Integrating an expression vector encoding a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor into a genome of a cell strain to construct a stable cell strain and the specific steps include: (a) transfecting the triple transcription factor expression vector with a transfection reagent Dyeing into the expression cell line; (b) diluting the cells the next day and incubating for 2 weeks at a concentration of 1000 cells/150 mm cell culture plates; (c) randomly selecting 50 Cell clones were used to detect the expression of ELL transcription factor, AFF transcription factor and CRTC by Western blot. Highly expressed cell lines were selected, which were stable cell lines.
  • the expression vector obtained in the step (2) is transfected into the stable cell strain obtained in (1), and the cells are collected 1-3 days later to obtain a desired mammalian protein.
  • the invention provides a method for purifying a mammalian protein expression system, comprising the steps of:
  • the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid (molar concentration is preferably 20 mM, pH is preferably 7.9), glycerin (mass concentration is preferably 10%), NaCl (molar concentration is preferably 0.3 M), EDTA (molar concentration is preferably 0.2 mM, pH is preferably 8.0), ethyl phenyl polyethylene glycol (mass concentration is preferably 0.5%), and Protease inhibitor cocktail, the lysate is added in an amount of 1-5 ml of the lysate was added per 1 ⁇ 10 7 cells.
  • the high salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid (molar concentration is preferably 20 mM, pH is preferably 7.9), glycerin (mass concentration is preferably 10%), NaCl (molar concentration is preferably 0.8 M) ), EDTA (molar concentration is preferably 0.2 mM, pH is preferably 8.0), ethyl phenyl polyethylene glycol (mass concentration is preferably 0.5%), and protease inhibitor cocktail (Protease inhibitor cocktail), the high salt washing solution The amount added is 1-5 ml.
  • the low salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid (molar concentration is preferably 20 mM, pH is preferably 7.9), glycerin (mass concentration is preferably 10%), NaCl (molar concentration is preferably 0.15 M) ), EDTA (molar concentration is preferably 0.2 mM, pH is preferably 8.0), ethyl phenyl polyethylene glycol (mass concentration is preferably 0.5%), and protease inhibitor cocktail (Protease inhibitor cocktail), the low salt washing solution The amount added is 1-5 ml.
  • the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.3 M), EDTA. (0.2mM, pH 8.0), ethyl phenyl polyethylene glycol (NP-40, 0.5%) and Protease inhibitor cocktail;
  • HPES 4-hydroxyethylpiperazineethanesulfonic acid
  • glycerol glycerol
  • NaCl 0.3 M
  • EDTA 0.2mM, pH 8.0
  • NP-40 ethyl phenyl polyethylene glycol
  • Protease inhibitor cocktail Protease inhibitor cocktail
  • the high salt wash solution comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.8 M), EDTA (0.2 mM, pH 8.0), B. Phenyl phenyl polyethylene glycol (NP-40, 0.5%) and protease inhibitor cocktail (Protease inhibitor cocktail);
  • the low salt wash solution comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.15 M), EDTA (0.2 mM, pH 8.0), B. Phenyl phenyl polyethylene glycol (NP-40, 0.5%) and Protease inhibitor cocktail.
  • recombinant human leukemia inhibitory factor is expressed and purified using a lactating protein expression system.
  • ThermoFisher EDTA >98% Sigma NP-40 / Sigma NaCl >99% Sigma Protease inhibitor cocktail >98% Roche Flag beads >98% Sigma Flag peptide >98% Sigma Human embryonic kidney cell / ATCC Chinese hamster ovary cells / ATCC Human leukemia inhibitory factor / ThermoFisher BamHI / ThermoFisher NotI / ThermoFisher HindIII / ThermoFisher T4 ligase / Roche Silver staining kit / ThermoFisher
  • the genes encoding the ELL2 transcription factor, the AFF4 transcription factor and the CRTC2 transcription factor were subjected to polymerase chain reaction (94 ° C, 30 sec / 56 ° C, 30 sec / 68 ° C, 2 min, 30 cycles), electrophoresis
  • the genes encoding ELL2 transcription factor, AFF4 transcription factor and CRTC2 transcription factor were isolated and purified, respectively.
  • the obtained ELL2 transcription factor and AFF4 transcription factor were digested with BamHI and NotI, respectively, and the CRTC2 transcription factor gene was used.
  • the HindIII and NotI enzymes were digested and ligated with the vector by T4 ligase to obtain a recombinant vector containing a gene encoding an ELL2 transcription factor, an AFF4 transcription factor and a CRTC2 transcription factor.
  • the protein molar ratio of the transcription factor was 1:1:1;
  • AFF4 The base sequence of AFF4 is shown in SEQ ID NO.
  • the base sequence of CRTC2 is shown in SEQ ID NO.
  • the vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
  • LIF and expression vector containing cytomegalovirus promoter were digested with HindIII and BamHI, respectively, to obtain LIF and cytomegalovirus vectors with sticky ends, and then LIF was ligated with expression vector using T4 ligase to obtain human leukemia inhibitory factor. (LIF) expression vector.
  • the human leukemia inhibitory factor (LIF) expression vector was transfected into Chinese hamster ovary cells together with different transcription factor expression vector expression vectors. Two days later, the cells were collected and the expression levels were compared. The results are shown in Fig. 1. The first is the amount of LIF in the general expression system, the second is the amount of LIF in the expression system containing ELL2/AFF4, and the third is the amount of LIF in the expression system containing ELL2/AFF4/CRTC2. We clearly see that LIF has the highest expression level in the case of the ELL2/AFF4/CRTC2 triple transcription factor.
  • the recombinant expression vector obtained above and the LIF expression vector are co-transfected into Chinese hamster ovary cells, the ratio of the total amount of the recombinant expression vector to the total amount of the LIF expression vector is 1:2;
  • LIF human leukemia inhibitory factor
  • the purified protein expression system of Example 1 was subjected to protein electrophoresis, and then stained with ThermoFisher's silver staining kit. The results of the assay are shown in Figure 2, and the protein concentration was estimated to be greater than 95%.
  • the protein expression system prepared by the present invention exhibits high activity and can promote the growth of embryonic stem cell cells at a very low concentration in an experiment comparing with a similar product produced by a bacterial expression system. Moreover, compared with the protein expression system to which ELL2 and AFF4 are added, the expression level of the protein expression system prepared by the present invention is increased by 7 times, indicating that this combination (ELL2/AFF4/CRTC2) can greatly enhance the CMV promoter, thereby improving the purpose. Gene expression.

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Abstract

Provided in the present invention are a transcription factor composition comprising transcription factors ELL, AFF and CRTC. A mammalian protein expression system may be obtained by co-transfecting an expression vector comprising a gene encoding the three transcription factors and a target gene expression vector into a cell line. The presence of transcription factors ELL, AFF and CRTC in the expression system promotes the transcription and expression of the target gene and may be applied to the production of mammalian protein.

Description

三重转录因子及其在哺乳动物蛋白表达系统的应用Triple transcription factor and its application in mammalian protein expression system 技术领域Technical field
本发明涉及基因工程技术领域,具体涉及三重转录因子及其在哺乳动物蛋白表达系统的应用。The invention relates to the field of genetic engineering technology, in particular to triple transcription factors and their use in mammalian protein expression systems.
背景技术Background technique
蛋白表达是现代工业、医疗和基础研究领域的重要组成部分,而利用重组技术生产蛋白,特别是高纯度、高活性的蛋白,在生物医学研究和制药学领域当中起着举足轻重的作用。Protein expression is an important part of modern industrial, medical and basic research, and the use of recombinant technology to produce proteins, especially high-purity, high-activity proteins, plays a pivotal role in biomedical research and pharmaceuticals.
目前,常见的重组蛋白表达系统包括原核表达系统、酵母表达系统、昆虫表达系统和哺乳动物表达系统。原核表达系统和酵母表达系统虽然具有高产量和易操作的特点,但是当用于哺乳动物蛋白的表达时,则存在着天然的缺陷:(1)这类微生物系统缺乏或难以完成蛋白翻译后的各种化学修饰例如糖基化、乙酰化等,而蛋白的翻译后修饰对蛋白在细胞以及体内的转运、蛋白稳定性和在有机体内的生物活性都有着非常重要的作用。因此,缺乏此类修饰会对重组蛋白的生物活性有着极大的影响。虽然昆虫表达系统具有一定的蛋白翻译后的修饰能力,但由于种属差异和基因组的不同,其蛋白翻译后修饰在复杂性和多样性上无法与哺乳动物相比,且该系统的蛋白产量亦不高;(2)很多人源蛋白在细菌或昆虫表达系统中无法正常折叠,从而形成包含体,因此,纯化出的蛋白需要进行复杂的复性过程以恢复活力。对于传统哺乳动物表达系统而言,其优点在于蛋白能够实现正确折叠,带有比较完善的蛋白翻译后修饰,具有完整的生物活性,但其缺点是蛋白产量通常较低,生产成本居高不下。这也是市场上来源于哺乳动物表达系统的重组蛋白的种类和数量均较其他系统为少的重要原因。Currently, common recombinant protein expression systems include prokaryotic expression systems, yeast expression systems, insect expression systems, and mammalian expression systems. Although prokaryotic expression systems and yeast expression systems are characterized by high yield and ease of manipulation, when used for expression of mammalian proteins, there are natural defects: (1) such microbial systems are lacking or difficult to complete after protein translation. Various chemical modifications such as glycosylation, acetylation, etc., and post-translational modification of proteins play an important role in the transport of proteins in cells and in vivo, protein stability and biological activity in organisms. Therefore, the lack of such modifications can have a significant impact on the biological activity of recombinant proteins. Although the insect expression system has certain post-translational modification ability, due to species differences and genome differences, the protein post-translational modification cannot be compared with mammals in complexity and diversity, and the protein yield of the system is also Not high; (2) Many human proteins cannot be normally folded in a bacterial or insect expression system to form inclusion bodies. Therefore, the purified protein requires a complex renaturation process to restore vitality. For traditional mammalian expression systems, the advantage is that the protein can achieve correct folding, with a relatively complete protein post-translational modification, and has complete biological activity, but its disadvantage is that the protein production is usually low and the production cost is high. This is also an important reason why the number and quantity of recombinant proteins derived from mammalian expression systems are less than other systems.
对于医疗领域而言,目前获得食品和药物管理局(FDA)批准的生物药大部分是重组蛋白,这类药品的生产更是需要保持人体本身的蛋白翻译后修饰,以减少或消除药物进入人体后可能诱发的免疫反应。For the medical field, most of the biopharmaceuticals currently approved by the Food and Drug Administration (FDA) are recombinant proteins. The production of such drugs requires the maintenance of the protein's own post-translational modification to reduce or eliminate the entry of drugs into the human body. The immune response may be induced afterwards.
哺乳动物细胞对于蛋白表达有着精细的调控机制,包括转录、翻译以及 修饰调控。其中,上游的基因转录水平在很大程度上决定了下游的蛋白的产量。Mammalian cells have elaborate regulatory mechanisms for protein expression, including transcription, translation, and Modification regulation. Among them, the upstream gene transcription level largely determines the downstream protein production.
Molecular Cell(Nanhai He,Min Liu,Joanne Hsu,Yuhua Xue,Seemay Chou,Alma Burlingame,Nevan J.Krogan,Tom Alber,and Qiang Zhou.HIV-1 Tat and Host AFF4 Recruit Two Transcription Elongation Factors into a Bifunctional Complex for Coordinated Activation of HIV-1 Transcription.(J)Molecular Cell.2010.05.14(38)428-438)报道了一个多蛋白复合物,后命名为超级延伸复合物(Supper Elongation Complex,SEC),其对转录调控的延伸阶段起着非常关键的作用,以艾滋病病毒HIV(Human Immunodeficiency Virus)为研究模型,具体阐述了这个复合物中的两个蛋白ELL(Elongation factor for RNA Polymerase II)2和AFF(AF4/FMR2 family member)4对艾滋病病毒HIV在基因转录上的作用机制,研究表明,该复合物中的这两个蛋白的组合可以极大的促进HIV的基因表达,利用报告基因的方法测试显示,这两个蛋白的组合可以成百倍的促进艾滋病启动子的基因表达,显示这个蛋白复合物,特别是其中的两个蛋白,对艾滋病病毒HIV有着极为重要的作用。Molecular Cell (Nanhai He, Min Liu, Joanne Hsu, Yuhua Xue, Seemay Chou, Alma Burlingame, Nevan J. Krogan, Tom Alber, and Qiang Zhou. HIV-1 Tat and Host AFF4 Recruit Two Transcription Elongation Factors into a Bifunctional Complex for Coordinated Activation of HIV-1 Transcription. (J) Molecular Cell. 2010.05.14 (38) 428-438) reports a multiprotein complex, hereinafter referred to as the Supper Elongation Complex (SEC), which is transcriptionally responsive. The extension phase of regulation plays a very important role. The HIV Immunodeficiency Virus (HIV) model is used to describe the two proteins ELL (Elongation factor for RNA Polymerase II) 2 and AFF (AF4/). FMR2 family member)4 The mechanism of action of HIV HIV on gene transcription, studies have shown that the combination of these two proteins in the complex can greatly promote HIV gene expression, using the reporter gene test showed that this The combination of two proteins can multiply the gene expression of the AIDS promoter, showing A protein complex, in particular the two proteins of the AIDS virus HIV has a very important role.
CRTC是c AMP(c AMP-responsive element binding protein,CREB)反应元件结合蛋白的转录共激活因子(CREB-regulated transcription coactivator),CRTC家族有3个成员(CRTC1-3),包含CRTC(CREB regulated transcription coactivator)1,CRTC2和CRTC3,其家族可以显著增强CREB靶基因的转录。但是,目前还没有关于ELL2、AFF4和CRTC2组合应用于哺乳动物蛋白表达系统以提高哺乳动物蛋白表达量的报道。CRTC is a cAMP-responsive element binding protein (CREB)-reactive transcriptional coactivator. The CRTC family has three members (CRTC1-3) and contains CRTC (CREB regulated transcription). Coactivator) 1, CRTC2 and CRTC3, whose family can significantly enhance the transcription of CREB target genes. However, there have been no reports on the application of ELL2, AFF4 and CRTC2 in combination to mammalian protein expression systems to increase mammalian protein expression.
发明内容Summary of the invention
本发明所解决的现有技术问题是:现有的哺乳动物蛋白表达系统普遍有着表达量低,成本高的特点。The prior art problem solved by the present invention is that the existing mammalian protein expression system generally has the characteristics of low expression and high cost.
为了解决上述问题,本发明先是提供一种转录因子组合物,含有ELL转录因子、AFF转录因子和CRTC转录因子,优选地,ELL转录因子选自ELL2或ELL1中的一种,AFF转录因子选自AFF4或AFF1中的一种,CRTC转录因子选自CRTC2或CRTC1或CRTC3中的一种。 In order to solve the above problems, the present invention first provides a transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor. Preferably, the ELL transcription factor is selected from one of ELL2 or ELL1, and the AFF transcription factor is selected from the group consisting of In one of AFF4 or AFF1, the CRTC transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
本发明提供了一种DNA分子,所述DNA分子含有编码ELL转录因子的碱基序列,编码AFF转录因子的碱基序列和编码CRTC转录因子的碱基序列。The present invention provides a DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor.
本发明提供了一种重组表达载体,其是将转录因子组合物或三重转录因子克隆到哺乳动物表达载体而得到。The present invention provides a recombinant expression vector obtained by cloning a transcription factor composition or a triple transcription factor into a mammalian expression vector.
本发明还提供了重组表达载体的制备方法,其是将将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因分别与载体连接,得到含有三重转录因子的重组载体;然后将重组载体转化到受体细胞,筛选得到转录因子表达的载体;最后将表达载体进行DNA测序,得到重组表达载体。The present invention also provides a method for preparing a recombinant expression vector, which comprises ligating a gene encoding an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor with a vector to obtain a recombinant vector containing a triple transcription factor; and then transforming the recombinant vector into The recipient cell is screened for a vector for expression of a transcription factor; finally, the expression vector is subjected to DNA sequencing to obtain a recombinant expression vector.
本发明提供了一种哺乳动物蛋白表达系统的制备方法,其是通过利用增强基因转录的方式来提高蛋白含量。这不仅会大大降低哺乳动物重组蛋白的生产成本,增加收益,同时也会对生命科学研究和生物制药业的发展产生积极的促进作用。The present invention provides a method of preparing a mammalian protein expression system by increasing the protein content by enhancing the transcription of the gene. This will not only greatly reduce the production cost of mammalian recombinant protein, increase the income, but also positively promote the development of life science research and bio-pharmaceutical industry.
本发明哺乳动物蛋白表达系统的制备方法是将重组表达载体和目的基因表达载体共同转染到细胞株中表达,得到重组蛋白。The preparation method of the mammalian protein expression system of the present invention comprises co-transfecting a recombinant expression vector and a gene expression vector of interest into a cell strain to obtain a recombinant protein.
本发明哺乳动物蛋白表达系统的制备方法还可以将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因整合到目的基因中,得到含有编码三重转录因子的基因的目的基因,然后将含有编码三重转录因子的基因的目的基因与载体连接,得到含有编码三重转录因子的基因的目的基因表达载体,最后转染到细胞株中得到哺乳动物蛋白表达系统。The method for preparing a mammalian protein expression system of the present invention can also integrate a gene encoding an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor into a gene of interest, to obtain a gene of interest containing a gene encoding a triple transcription factor, and then to encode a triple gene. The gene of interest of the gene of the transcription factor is ligated to the vector to obtain a gene expression vector containing the gene encoding the triple transcription factor, and finally transfected into the cell line to obtain a mammalian protein expression system.
本发明还提供了一种哺乳动物蛋白表达系统的纯化方法,其是在收集的细胞株中加入裂解液,离心得上清液;然后将上清液与标签混合后,离心去除上清液,加入高盐洗涤液进行洗涤;接着对洗涤液进行离心,去除上清液,加入低盐洗涤液进行洗涤;最后用带有标记的肽进行洗脱,离心得到上清液,所述上清液即为哺乳动物蛋白表达系统。The invention also provides a method for purifying a mammalian protein expression system, which comprises adding a lysate to the collected cell strain and centrifuging to obtain a supernatant; then, after mixing the supernatant with the label, the supernatant is removed by centrifugation. Adding a high-salt washing solution for washing; then, centrifuging the washing liquid, removing the supernatant, washing with a low-salt washing solution; finally eluting with the labeled peptide, and centrifuging to obtain a supernatant, the supernatant It is a mammalian protein expression system.
具体来说,本发明提出了如下技术方案。Specifically, the present invention proposes the following technical solutions.
一种转录因子组合物,含有ELL转录因子、AFF转录因子和CRTC转录因子,优选地,ELL转录因子选自ELL2或ELL1中的一种,AFF转录因子选自AFF4或AFF1中的一种,CRTC转录因子选自CRTC2或CRTC1或CRTC3中的一种。 A transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor, preferably, the ELL transcription factor is selected from one of ELL2 or ELL1, and the AFF transcription factor is selected from one of AFF4 or AFF1, CRTC The transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
优选的,对于所述的转录因子组合物,其中,所述组合物所述组合物含有ELL2、AFF4和CRTC2,所述ELL2、AFF4和CRTC2的比例为1:1-3:1-4;更优选的,所述ELL2、AFF4和CRTC2的比例为1:1:1。Preferably, for the transcription factor composition, wherein the composition comprises ELL2, AFF4 and CRTC2, the ratio of ELL2, AFF4 and CRTC2 is 1:1 to 3: 1-4; Preferably, the ratio of the ELL2, AFF4 and CRTC2 is 1:1:1.
优选的,对于所述的转录因子组合物,其中,所述转录因子组合物选自于人源、鼠源、狗源或者猪源中的一种;优选的,所述转录因子组合物为人源的。Preferably, for the transcription factor composition, wherein the transcription factor composition is selected from one of a human source, a mouse source, a dog source or a pig source; preferably, the transcription factor composition is a human source of.
一种三重转录因子,其包含上述的转录因子组合物。A triple transcription factor comprising the transcription factor composition described above.
一种DNA分子,所述DNA分子含有编码ELL转录因子的碱基序列、编码AFF转录因子的碱基序列和编码CRTC转录因子的碱基序列;优选,所述编码ELL转录因子的碱基序列选自以下碱基序列中的一种:A DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor; preferably, the base sequence encoding the ELL transcription factor is selected From one of the following base sequences:
(a)碱基序列如SEQ ID NO.1所示;(a) the base sequence is shown in SEQ ID NO.
(b)与SEQ ID NO.1序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 1, preferably 95% or more; more preferably 99% or more of the base sequence;
(c)编码SEQ ID NO.2所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO. 2;
(d)在SEQ ID NO.2所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质;(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown in SEQ ID NO. 2 and which has a transcriptional activity of the regulatory gene;
所述编码AFF转录因子的碱基序列选自以下碱基序列中的一种:The base sequence encoding the AFF transcription factor is selected from one of the following base sequences:
(a)碱基序列如SEQ ID NO.3所示;(a) the base sequence is shown in SEQ ID NO. 3;
(b)与SEQ ID NO.3序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 3, preferably 95% or more; more preferably 99% or more of the base sequence;
(c)编码SEQ ID NO.4所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO.
(d)在SEQ ID NO.4所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质;(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown in SEQ ID NO. 4 and which has a transcriptional activity of the regulatory gene;
所述编码CRTC转录因子的碱基序列选自以下碱基序列中的一种:The base sequence encoding the CRTC transcription factor is selected from one of the following base sequences:
(a)碱基序列如SEQ ID NO.5所示;(a) the base sequence is shown in SEQ ID NO. 5;
(b)与SEQ ID NO.5序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 5, preferably 95% or more; more preferably 99% or more of the base sequence;
(c)编码SEQ ID NO.6所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO.
(d)在SEQ ID NO.6所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质。 (d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 6 and which has a transcriptional activity of the regulatory gene.
一种重组表达载体,其是将所述转录因子组合物或者所述三重转录因子克隆到哺乳动物表达载体中而得到。A recombinant expression vector obtained by cloning the transcription factor composition or the triple transcription factor into a mammalian expression vector.
一种重组表达载体的制备方法,其包含下述步骤:A method for preparing a recombinant expression vector, comprising the steps of:
(1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因分别与载体连接,得到含有三重转录因子的重组载体;(1) ligating a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor to a vector, respectively, to obtain a recombinant vector containing a triple transcription factor;
(2)将步骤(1)所得到的重组载体转化到受体细胞,筛选得到转录因子表达的载体;(2) transforming the recombinant vector obtained in the step (1) into a recipient cell, and screening for a vector for expressing a transcription factor;
(3)将步骤(2)所得到的转录因子表达的载体进行DNA测序,得到重组表达载体。(3) The vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
所述转录因子组合物或三重转录因子在哺乳动物蛋白表达系统中的应用。Use of the transcription factor composition or triple transcription factor in a mammalian protein expression system.
一种哺乳动物蛋白表达系统,其是将重组表达载体或重组表达载体与目的基因表达载体共同转染到细胞株中表达得到,所述重组表达载体的总量与目的基因表达载体的总量的比为1:1-5。A mammalian protein expression system, wherein a recombinant expression vector or a recombinant expression vector is co-transfected with a gene expression vector of interest into a cell strain, and the total amount of the recombinant expression vector and the total amount of the expression vector of the target gene are obtained. The ratio is 1:1-5.
优选的,对于所述的哺乳动物蛋白表达系统,其中,所述细胞株选自于人胚胎肾细胞293或者中国仓鼠卵巢细胞中的一种。Preferably, for the mammalian protein expression system, wherein the cell strain is selected from one of human embryonic kidney cell 293 or Chinese hamster ovary cells.
一种哺乳动物蛋白表达系统的制备方法,其包含下述步骤:A method of preparing a mammalian protein expression system, comprising the steps of:
(1)将重组表达载体和目的基因表达载体总量比为1:1-5共同转染到细胞株中;(1) co-transfecting the recombinant expression vector and the target gene expression vector into a cell line in a total ratio of 1:1-5;
(2)1-3天后收集细胞,得到哺乳动物蛋白表达系统。(2) Cells were collected after 1-3 days to obtain a mammalian protein expression system.
优选的,对于所述的制备方法,其特征在于,在步骤(1)中,所述目的基因载体的制备方法包含下述步骤:将目的基因及表达载体连接得到目的基因表达载体。Preferably, in the preparation method, in the step (1), the method for preparing the gene carrier of interest comprises the steps of: ligating the gene of interest and the expression vector to obtain a gene expression vector of interest.
一种哺乳动物蛋白表达系统,其是将三重转录因子的目的基因表达载体转染到细胞株中得到。A mammalian protein expression system obtained by transfecting a target gene expression vector of a triple transcription factor into a cell strain.
优选的,对于所述的哺乳动物蛋白表达系统,其中,所述细胞株选自于人胚胎肾细胞或中国仓鼠卵巢细胞中的一种。Preferably, for the mammalian protein expression system, wherein the cell strain is selected from one of human embryonic kidney cells or Chinese hamster ovary cells.
一种哺乳动物蛋白表达系统的制备方法,其包含下述步骤:A method of preparing a mammalian protein expression system, comprising the steps of:
(1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因整合到目的基因中得到含有编码三重转录因子的基因的目的基因; (1) integrating a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor into a gene of interest to obtain a gene of interest comprising a gene encoding a triple transcription factor;
(2)将含有编码三重转录因子的基因的目的基因与载体连接,得到含有编码三重转录因子基因的目的基因表达载体;(2) ligating a gene of interest containing a gene encoding a triple transcription factor with a vector to obtain a gene expression vector containing a gene encoding a triple transcription factor;
(3)将步骤(2)所得到的表达载体转染到细胞株中,1-3天后收集细胞,得到哺乳动物蛋白表达系统。(3) The expression vector obtained in the step (2) is transfected into the cell strain, and the cells are collected 1-3 days later to obtain a mammalian protein expression system.
一种哺乳动物蛋白表达系统的纯化方法,其包含下述步骤:A method of purifying a mammalian protein expression system, comprising the steps of:
(1)收集哺乳动物蛋白表达系统细胞,加入裂解液,离心得到上清液;(1) collecting mammalian protein expression system cells, adding a lysate, and centrifuging to obtain a supernatant;
(2)将步骤(1)所述的上清液与标签beads混合,离心去除上清液,然后加入高盐洗涤液洗涤;(2) mixing the supernatant described in the step (1) with the label beads, centrifuging to remove the supernatant, and then adding the high salt washing solution to wash;
(3)将步骤(2)所得的洗涤液离心,去除上清液,加入低盐洗涤液洗涤;(3) centrifuging the washing liquid obtained in the step (2), removing the supernatant, and adding the low-salt washing liquid to wash;
(4)用带有标记peptide进行洗脱,然后离心得到上清液,所述上清液即为哺乳动物蛋白表达系统。(4) Elution with a labeled peptide followed by centrifugation to obtain a supernatant, which is a mammalian protein expression system.
优选的,对于所述的纯化方法,其中,在步骤(1)中,所述裂解液包含4-羟乙基哌嗪乙磺酸、甘油、NaCl、EDTA、乙基苯基聚乙二醇和蛋白酶抑制剂混合物,所述裂解液的加入量为每1×107个细胞加入1-5ml的裂解液。Preferably, in the purification method, in the step (1), the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene glycol, and protease. The inhibitor mixture is added in an amount of 1-5 ml of the lysate per 1 × 10 7 cells.
优选的,对于所述的纯化方法,其中,在步骤(2)中,所述上清液与标签beads的体积比为20-50:1。Preferably, in the purification method, in the step (2), the volume ratio of the supernatant to the label beads is 20-50:1.
优选的,对于所述的纯化方法,其中,在步骤(2)中,所述高盐洗涤液包含4-羟乙基哌嗪乙磺酸、甘油、NaCl、EDTA、乙基苯基聚乙二醇和蛋白酶抑制剂混合物,所述高盐洗涤液的加入量为1-5ml。Preferably, in the purification method, in the step (2), the high salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene A mixture of alcohol and protease inhibitor, the high salt wash is added in an amount of from 1 to 5 ml.
优选的,对于所述的纯化方法,其中,在步骤(3)中,所述低盐洗涤液包含4-羟乙基哌嗪乙磺酸、甘油、NaCl、EDTA、乙基苯基聚乙二醇和蛋白酶抑制剂混合物,所述低盐洗涤液的加入量为1-5ml。Preferably, in the purification method, in the step (3), the low-salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene. A mixture of alcohol and protease inhibitor, said low salt wash is added in an amount of from 1 to 5 ml.
优选的,对于所述的纯化方法,其中,所述标签peptide与标签beads的体积比为3-5:1。Preferably, for the purification method, wherein the volume ratio of the label peptide to the label beads is 3-5:1.
哺乳动物蛋白表达系统,其在生物医药中的应用。Mammalian protein expression system, its application in biomedicine.
本发明所取得的有益效果为:本发明所得到的哺乳动物表达系统与传统方法相比,具有低成本、高产量的优势,能够有效提高重组蛋白的产量,并保持其完整的翻译后修饰来提高生物活性。此哺乳动物表达系统通过引入优化的超级转录延伸复合物中的转录因子,在转录水平上极大的提高基因的表 达,从根本上达到增加蛋白产量的目的。同时,本发明所得到的哺乳动物表达系统具有操作简单,易大规模生成和快速产业化的潜能。The beneficial effects obtained by the invention are that the mammalian expression system obtained by the invention has the advantages of low cost and high yield compared with the traditional method, can effectively increase the yield of the recombinant protein, and maintain its complete post-translational modification. Improve biological activity. This mammalian expression system greatly enhances the gene's expression at the transcriptional level by introducing transcription factors in the optimized super-transcriptional elongation complex. To achieve the goal of increasing protein production. At the same time, the mammalian expression system obtained by the invention has the potential of simple operation, easy large-scale production and rapid industrialization.
附图说明DRAWINGS
图1是实施例1所得到的LIF蛋白表达系统的表达量的示意图;1 is a schematic diagram showing the expression level of a LIF protein expression system obtained in Example 1;
图2是实施例1所得到的LIF蛋白表达系统纯度的示意图;Figure 2 is a schematic diagram showing the purity of the LIF protein expression system obtained in Example 1;
图3是实施例1所得到的LIF蛋白表达系统与利用细菌表达系统同类产品生物活性比较示意图,其中,图3-1是没有LIF的蛋白表达系统的生物活性示意图,图3-2是利用细菌表达系统的同类产品的生物活性示意图,图3-3至3-6是实施例1中的蛋白表达系统在2.5ng/ml、5ng/ml、10ng/l和20ng/ml的生物活性示意图。Figure 3 is a schematic diagram showing the comparison of the biological activity of the LIF protein expression system obtained in Example 1 with the same product using the bacterial expression system, wherein Figure 3-1 is a schematic diagram of the biological activity of the protein expression system without LIF, and Figure 3-2 is a diagram showing the biological activity of the protein expression system without LIF. Schematic diagram of the biological activity of the similar products of the expression system, Figures 3-3 to 3-6 are schematic diagrams of the biological activities of the protein expression systems of Example 1 at 2.5 ng/ml, 5 ng/ml, 10 ng/l and 20 ng/ml.
具体实施方式Detailed ways
图1是实施例1中所得到的LIF蛋白表达系统的表达量的示意图,其中,1是只有LIF表达载体转染到中国仓鼠卵巢细胞时所得到的LIF蛋白表达系统的表达量的图,2是LIF表达载体和含有转录因子ELL2和AFF4的表达载体共同转染到中国仓鼠卵巢细胞中所得到的LIF蛋白表达系统表达量的示意图,3是LIF表达载体和含有三重转录因子ELL2、AFF4和CRTC2的表达载体共同转染到中国仓鼠卵巢细胞中所得到的LIF蛋白表达系统表达量的示意图,从上图可以看出,三重转录因子ELL2、AFF4和CRTC2可以把LIF的表达量提高7倍。1 is a schematic diagram showing the expression level of a LIF protein expression system obtained in Example 1, wherein 1 is a map showing the expression level of a LIF protein expression system obtained by transfecting a LIF expression vector into a Chinese hamster ovary cell, 2 It is a schematic diagram of the expression level of LIF protein expression system obtained by co-transfection of LIF expression vector and expression vector containing transcription factors ELL2 and AFF4 into Chinese hamster ovary cells, 3 is a LIF expression vector and contains triple transcription factors ELL2, AFF4 and CRTC2. Schematic diagram of the expression level of the LIF protein expression system obtained by co-transfection of the expression vector into Chinese hamster ovary cells. As can be seen from the above figure, the triple transcription factors ELL2, AFF4 and CRTC2 can increase the expression level of LIF by 7 times.
图2是实施例1所得到的LIF蛋白表达系统纯度的示意图,从图中可以看出,纯化后的LIF蛋白表达系统具有极高的纯度,纯度高于95%。2 is a schematic diagram showing the purity of the LIF protein expression system obtained in Example 1. As can be seen from the figure, the purified LIF protein expression system has an extremely high purity and a purity higher than 95%.
图3是实施例1所得到的LIF蛋白表达系统与利用细菌表达系统同类产品(货号PHC9484,Thermo Fisher Scientific)生物活性比较示意图,其中,图3-1是没有LIF的蛋白表达系统的生物活性示意图,图3-2是利用细菌表达系统的同类产品(10ng/ml)的生物活性示意图,图3-3至3-6是实施例1中的蛋白表达系统在2.5ng/ml、5ng/ml、10ng/l和20ng/ml的生物活性示意图,从图中可以看出,使用本发明的蛋白表达系统,干细胞较多,说明本发明所得到的LIF 蛋白表达系统生物活性较高,生物活性超出利用细菌表达系统同类产品的300%以上。Figure 3 is a schematic diagram showing the comparison of the biological activity of the LIF protein expression system obtained in Example 1 with a similar product using the bacterial expression system (Cat. No. PHC9484, Thermo Fisher Scientific), wherein Figure 3-1 is a schematic diagram of the biological activity of the protein expression system without LIF. Figure 3-2 is a schematic diagram showing the biological activity of a similar product (10 ng/ml) using a bacterial expression system, and Figures 3-3 to 3-6 are the protein expression systems of Example 1 at 2.5 ng/ml, 5 ng/ml, A schematic diagram of the biological activity of 10 ng/l and 20 ng/ml. As can be seen from the figure, using the protein expression system of the present invention, there are many stem cells, indicating the LIF obtained by the present invention. The protein expression system has high biological activity and the biological activity exceeds 300% of the similar products using the bacterial expression system.
如上所述,本发明提供了一种转录因子组合物,含有ELL转录因子、AFF转录因子和CRTC转录因子,优选地,ELL转录因子选自ELL2或ELL1中的一种,AFF转录因子选自AFF4或AFF1中的一种,CRTC转录因子选自CRTC2或CRTC1或CRTC3中的一种。As described above, the present invention provides a transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor, preferably, the ELL transcription factor is selected from one of ELL2 or ELL1, and the AFF transcription factor is selected from AFF4. Or one of AFF1, the CRTC transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
其中,所述组合物有ELL2、AFF4和CRTC2。优选的,所述ELL2、AFF4和CRTC2的比例为1:1-3:1-4,更优选的,所述ELL2、AFF4和CRTC2的比例为1:1:1。Wherein the composition has ELL2, AFF4 and CRTC2. Preferably, the ratio of the ELL2, AFF4 and CRTC2 is 1:1 to 3: 1-4, and more preferably, the ratio of the ELL2, AFF4 and CRTC2 is 1:1:1.
其中,所述转录因子组合物选自于人源、鼠源、狗源或者猪源中的一种,优选的,所述转录因子组合物为人源的。Wherein the transcription factor composition is selected from one of a human source, a mouse source, a dog source or a pig source, and preferably, the transcription factor composition is of a human origin.
本发明还提供了一种三重转录因子表达载体,其是将三重转录因子克隆到哺乳动物表达载体中而得到。The present invention also provides a triple transcription factor expression vector obtained by cloning a triple transcription factor into a mammalian expression vector.
本发明提供了一种DNA分子,所述DNA分子含有编码ELL转录因子的碱基序列,编码AFF转录因子的碱基序列和编码CRTC转录因子的碱基序列,其中,所述编码ELL转录因子的选自以下碱基序列中的一种:The present invention provides a DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor, wherein the ELL transcription factor is encoded One selected from the following base sequences:
(a)碱基序列如SEQ ID NO.1所示;(a) the base sequence is shown in SEQ ID NO.
(b)与SEQ ID NO.1序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 1, preferably 95% or more; more preferably 99% or more of the base sequence;
(c)编码SEQ ID NO.2所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO. 2;
(d)在SEQ ID NO.2所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质。(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 2 and which has a transcriptional activity of the regulatory gene.
所述编码AFF转录因子的碱基序列选自以下碱基序列中的一种:The base sequence encoding the AFF transcription factor is selected from one of the following base sequences:
(a)碱基序列如SEQ ID NO.3所示;(a) the base sequence is shown in SEQ ID NO. 3;
(b)与SEQ ID NO.3序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 3, preferably 95% or more; more preferably 99% or more of the base sequence;
(c)编码SEQ ID NO.4所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO.
(d)在SEQ ID NO.4所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质。 (d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 4 and which has a transcriptional activity of the regulatory gene.
所述编码CRTC转录因子的碱基序列选自以下碱基序列中的一种:The base sequence encoding the CRTC transcription factor is selected from one of the following base sequences:
(a)碱基序列如SEQ ID NO.5所示;(a) the base sequence is shown in SEQ ID NO. 5;
(b)与SEQ ID NO.5序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 5, preferably 95% or more; more preferably 99% or more of the base sequence;
(c)编码SEQ ID NO.6所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO.
(d)在SEQ ID NO.6所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质。(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 6 and which has a transcriptional activity of the regulatory gene.
本发明提供了一种重组表达载体,其是将转录因子组合物或三重转录因子克隆到含有CMV启动子的哺乳动物表达载体中而得到。The present invention provides a recombinant expression vector obtained by cloning a transcription factor composition or a triple transcription factor into a mammalian expression vector containing a CMV promoter.
本发明提供了一种重组表达载体的制备方法,其包含下述步骤:The invention provides a preparation method of a recombinant expression vector, which comprises the following steps:
(1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因分别与载体连接,得到含有三重转录因子的重组载体;(1) ligating a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor to a vector, respectively, to obtain a recombinant vector containing a triple transcription factor;
(2)将步骤(1)所得到的重组载体转化到受体细胞,筛选得到转录因子表达的载体;(2) transforming the recombinant vector obtained in the step (1) into a recipient cell, and screening for a vector for expressing a transcription factor;
(3)将步骤(2)所得到的转录因子表达的载体进行DNA测序,得到重组表达载体。(3) The vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
在本发明优选的具体实施方式中,本发明提供了一种重组表达载体的制备方法,其包含下述步骤:In a preferred embodiment of the invention, the invention provides a method for preparing a recombinant expression vector comprising the steps of:
(1)将编码ELL转录因子、AFF转录因子、CRTC转录因子的基因和表达载体分别进行限制性内切酶酶切,之后再进行分离纯化。酶切后的ELL转录因子、AFF转录因子、CRTC转录因子的基因和分别与酶切后的载体通过T4连接酶连接,得到含有三重转录因子的重组载体;(1) The gene encoding the ELL transcription factor, the AFF transcription factor, the CRTC transcription factor, and the expression vector are subjected to restriction endonuclease digestion, followed by isolation and purification. The ELL transcription factor, the AFF transcription factor, the CRTC transcription factor gene and the vector after digestion are respectively ligated with the T4 ligase to obtain a recombinant vector containing a triple transcription factor;
(2)将步骤(1)所得到的重组载体与大肠杆菌感受态细菌混合,冰上孵育30分钟,然后通过热激法(在42度水浴锅中90秒中)的方式,把重组载体转化进入到细菌中去。之后,把转化后的细菌铺到含有氨苄的LB培养板上,在37度的培养箱中培养过夜。(2) mixing the recombinant vector obtained in the step (1) with E. coli competent bacteria, incubating on ice for 30 minutes, and then transforming the recombinant vector by heat shock method (in a 42-degree water bath for 90 seconds) Go into the bacteria. Thereafter, the transformed bacteria were plated on an LB plate containing ampicillin and cultured overnight in a 37-degree incubator.
(3)对(2)中培养板上的细菌进行鉴定,筛选出含有转录因子表达载体的细菌克隆。具体步骤如下:挑选10个细菌克隆分别放入5毫升的含有氨苄的液体LB培养液中,于37度摇床上过夜。第二天,用Invitrogen的小抽质粒 试剂盒提取质粒,然后进行酶切(与(1)中的限制性内切酶酶切一致)。如果能够得两条片段(大片段的为载体,小片段为插入的转录因子基因),则为阳性克隆。(3) Identification of bacteria on the culture plate in (2), and screening for bacterial clones containing a transcription factor expression vector. The specific steps are as follows: 10 bacterial clones were selected and placed in 5 ml of liquid LB medium containing ampicillin, and shaken on a 37-degree shaker overnight. The next day, use Invitrogen's small plasmid The plasmid was extracted from the kit and then digested (consistent with restriction endonuclease digestion in (1)). A positive clone is obtained if two fragments are available (the large fragment is the vector and the small fragment is the inserted transcription factor gene).
(4)将步骤(3)所得到的阳性克隆重组质粒送到测序公司进行DNA测序,确认序列无突变。(4) The positive clone recombinant plasmid obtained in the step (3) was sent to a sequencing company for DNA sequencing, and it was confirmed that the sequence had no mutation.
本发明提供了一种哺乳动物蛋白表达系统,其是通过将重组表达载体与目的基因表达载体共同转染到细胞株中表达得到,所述三重转录因子表达载体的总量与目的基因表达载体的总量的比为1:1-5。The present invention provides a mammalian protein expression system which is obtained by co-transfecting a recombinant expression vector and a gene expression vector of interest into a cell strain, the total amount of the triple transcription factor expression vector and the expression vector of the target gene. The total ratio is 1:1-5.
其中,所述细胞株为选自于人胚胎肾细胞293或者中国仓鼠卵巢细胞的一种。Wherein, the cell strain is one selected from the group consisting of human embryonic kidney cell 293 or Chinese hamster ovary cells.
本发明提供了一种哺乳动物蛋白表达系统的制备方法,其包含下述步骤:The invention provides a preparation method of a mammalian protein expression system, which comprises the following steps:
(1)将三重转录因子表达载体和目的基因的表达载体总量比为1:1-5共同转染到细胞株中;(1) co-transfecting the triple transcription factor expression vector and the expression vector of the target gene into a cell line in a ratio of 1:1-5;
(2)1-3天后收集细胞,得到所需的哺乳动物蛋白。(2) Cells are collected after 1-3 days to obtain the desired mammalian protein.
本发明还提供了另一种哺乳动物蛋白表达系统的制备方法,其包含下述步骤:The invention also provides a preparation method of another mammalian protein expression system, comprising the following steps:
(1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因整合到目的基因中得到含有编码三重转录因子的基因的目的基因;(1) integrating a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor into a gene of interest to obtain a gene of interest comprising a gene encoding a triple transcription factor;
(2)将含有编码三重转录因子的基因的目的基因与载体连接,得到含有编码三重转录因子基因的目的基因表达载体;(2) ligating a gene of interest containing a gene encoding a triple transcription factor with a vector to obtain a gene expression vector containing a gene encoding a triple transcription factor;
(3)将步骤(2)所得到的表达载体转染到细胞株中,1-3天后收集细胞,得到哺乳动物蛋白表达系统。(3) The expression vector obtained in the step (2) is transfected into the cell strain, and the cells are collected 1-3 days later to obtain a mammalian protein expression system.
在本发明另一种具体的实施方式中,本发明提供了一种哺乳动物蛋白表达系统的制备方法,其包含下述步骤:In another specific embodiment of the present invention, the present invention provides a method for preparing a mammalian protein expression system comprising the steps of:
(1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因的表达载体整合到细胞株的基因组,构建稳定细胞株,具体步骤包括(a)用转染试剂将三重转录因子表达载体转染到表达细胞株中;(b)第二天将细胞稀释,以1000个细胞/150毫米的细胞培养板的浓度培养2周;(c)随机挑选50 个细胞克隆,用Western blot的方法检测ELL转录因子、AFF转录因子和CRTC这三个蛋白的表达,挑选高表达的细胞株,此则为稳定细胞株。(1) Integrating an expression vector encoding a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor into a genome of a cell strain to construct a stable cell strain, and the specific steps include: (a) transfecting the triple transcription factor expression vector with a transfection reagent Dyeing into the expression cell line; (b) diluting the cells the next day and incubating for 2 weeks at a concentration of 1000 cells/150 mm cell culture plates; (c) randomly selecting 50 Cell clones were used to detect the expression of ELL transcription factor, AFF transcription factor and CRTC by Western blot. Highly expressed cell lines were selected, which were stable cell lines.
(2)将目的基因与载体连接,得到含有编码目的基因表达载体;(2) ligating the gene of interest to the vector to obtain an expression vector containing the gene of interest;
(3)将步骤(2)所得到的表达载体转染到(1)中所得的稳定细胞株中,1-3天后收集细胞,得到所需哺乳动物蛋白。(3) The expression vector obtained in the step (2) is transfected into the stable cell strain obtained in (1), and the cells are collected 1-3 days later to obtain a desired mammalian protein.
本发明提供了一种哺乳动物蛋白表达系统的纯化方法,其包含下述步骤:The invention provides a method for purifying a mammalian protein expression system, comprising the steps of:
(1)收集哺乳动物蛋白表达系统细胞,加入裂解液,离心得到上清液;(1) collecting mammalian protein expression system cells, adding a lysate, and centrifuging to obtain a supernatant;
(2)将步骤(1)所述的上清液与标签混合,离心去除上清液,然后加入高盐洗涤液洗涤;(2) mixing the supernatant described in the step (1) with a label, removing the supernatant by centrifugation, and then washing with a high-salt washing solution;
(3)将步骤(2)所得的洗涤液离心,去除上清液,加入低盐洗涤液洗涤;(3) centrifuging the washing liquid obtained in the step (2), removing the supernatant, and adding the low-salt washing liquid to wash;
(4)用带有标记的肽进行洗脱,然后离心得到上清液,所述上清液即为哺乳动物蛋白表达系统。(4) Elution is carried out using a labeled peptide, followed by centrifugation to obtain a supernatant, which is a mammalian protein expression system.
其中,所述裂解液包含4-羟乙基哌嗪乙磺酸(摩尔浓度优选为20mM,pH优选为7.9)、甘油(质量浓度优选为10%)、NaCl(摩尔浓度优选为0.3M)、EDTA(摩尔浓度优选为0.2mM,pH优选为8.0)、乙基苯基聚乙二醇(质量浓度优选为0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail),所述裂解液的加入量为每1×107个细胞加入1-5ml的裂解液。Wherein the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid (molar concentration is preferably 20 mM, pH is preferably 7.9), glycerin (mass concentration is preferably 10%), NaCl (molar concentration is preferably 0.3 M), EDTA (molar concentration is preferably 0.2 mM, pH is preferably 8.0), ethyl phenyl polyethylene glycol (mass concentration is preferably 0.5%), and Protease inhibitor cocktail, the lysate is added in an amount of 1-5 ml of the lysate was added per 1 × 10 7 cells.
其中,所述高盐洗涤液包含4-羟乙基哌嗪乙磺酸(摩尔浓度优选为20mM,pH优选为7.9)、甘油(质量浓度优选为10%)、NaCl(摩尔浓度优选为0.8M)、EDTA(摩尔浓度优选为0.2mM,pH优选为8.0)、乙基苯基聚乙二醇(质量浓度优选为0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail),所述高盐洗涤液的加入量为1-5ml。Wherein, the high salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid (molar concentration is preferably 20 mM, pH is preferably 7.9), glycerin (mass concentration is preferably 10%), NaCl (molar concentration is preferably 0.8 M) ), EDTA (molar concentration is preferably 0.2 mM, pH is preferably 8.0), ethyl phenyl polyethylene glycol (mass concentration is preferably 0.5%), and protease inhibitor cocktail (Protease inhibitor cocktail), the high salt washing solution The amount added is 1-5 ml.
其中,所述低盐洗涤液包含4-羟乙基哌嗪乙磺酸(摩尔浓度优选为20mM,pH优选为7.9)、甘油(质量浓度优选为10%)、NaCl(摩尔浓度优选为0.15M)、EDTA(摩尔浓度优选为0.2mM,pH优选为8.0)、乙基苯基聚乙二醇(质量浓度优选为0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail),所述低盐洗涤液的加入量为1-5ml。Wherein, the low salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid (molar concentration is preferably 20 mM, pH is preferably 7.9), glycerin (mass concentration is preferably 10%), NaCl (molar concentration is preferably 0.15 M) ), EDTA (molar concentration is preferably 0.2 mM, pH is preferably 8.0), ethyl phenyl polyethylene glycol (mass concentration is preferably 0.5%), and protease inhibitor cocktail (Protease inhibitor cocktail), the low salt washing solution The amount added is 1-5 ml.
在本发明一种优选的具体实施方式中,所述裂解液包含4-羟乙基哌嗪乙磺酸(HEPES,20mM,pH7.9)、甘油(10%)、NaCl(0.3M)、EDTA(0.2mM, pH8.0)、乙基苯基聚乙二醇(NP-40,0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail);In a preferred embodiment of the present invention, the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.3 M), EDTA. (0.2mM, pH 8.0), ethyl phenyl polyethylene glycol (NP-40, 0.5%) and Protease inhibitor cocktail;
所述高盐洗涤液包含4-羟乙基哌嗪乙磺酸(HEPES,20mM,pH7.9)、甘油(10%)、NaCl(0.8M)、EDTA(0.2mM,pH8.0)、乙基苯基聚乙二醇(NP-40,0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail);The high salt wash solution comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.8 M), EDTA (0.2 mM, pH 8.0), B. Phenyl phenyl polyethylene glycol (NP-40, 0.5%) and protease inhibitor cocktail (Protease inhibitor cocktail);
所述低盐洗涤液包含4-羟乙基哌嗪乙磺酸(HEPES,20mM,pH7.9)、甘油(10%)、NaCl(0.15M)、EDTA(0.2mM,pH8.0)、乙基苯基聚乙二醇(NP-40,0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail)。The low salt wash solution comprises 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.15 M), EDTA (0.2 mM, pH 8.0), B. Phenyl phenyl polyethylene glycol (NP-40, 0.5%) and Protease inhibitor cocktail.
在本发明优选的实施方式中,使用哺乳蛋白表达系统对重组人白血病抑制因子(LIF)进行表达和纯化。In a preferred embodiment of the invention, recombinant human leukemia inhibitory factor (LIF) is expressed and purified using a lactating protein expression system.
下面对本实施例所用的原料及设备的生产厂家,以及产品分析使用的设备和分析方法进行说明如下,其中所述的化学物质没有标明的均为常规试剂的化学纯级别。其中,实施例中所用到的原料的信息和设备分别如表1和表2所示。The following is a description of the manufacturer of the raw materials and equipment used in the present embodiment, as well as the equipment and analytical methods used for product analysis, wherein the chemical substances are not indicated to be chemically pure grades of conventional reagents. The information and equipment of the raw materials used in the examples are shown in Tables 1 and 2, respectively.
表1 所用到的原料的信息Information on the raw materials used in Table 1
原料raw material 纯度purity 厂家factory
HEPESHEPES 1M1M ThermoFisher公司ThermoFisher
EDTAEDTA >98%>98% Sigma公司Sigma
NP-40NP-40 // Sigma公司Sigma
NaClNaCl >99%>99% Sigma公司Sigma
Protease inhibitor cocktailProtease inhibitor cocktail >98%>98% 罗氏公司(Roche)Roche
Flag beadsFlag beads >98%>98% Sigma公司Sigma
Flag peptideFlag peptide >98%>98% Sigma公司Sigma
人胚胎肾细胞Human embryonic kidney cell // ATCC公司ATCC
中国仓鼠卵巢细胞Chinese hamster ovary cells // ATCC公司ATCC
人白血病抑制因子Human leukemia inhibitory factor // ThermoFisher公司ThermoFisher
BamHIBamHI // ThermoFisher公司ThermoFisher
NotINotI // ThermoFisher公司ThermoFisher
HindIIIHindIII // ThermoFisher公司ThermoFisher
T4连接酶T4 ligase // 罗氏公司(Roche)Roche
银染试剂盒Silver staining kit // ThermoFisher公司ThermoFisher
表2所用到的实验设备Experimental equipment used in Table 2
名称name 型号model 生产厂家Manufacturer
离心机Centrifuge 5417C5417C Eppendorf公司Eppendorf
细胞培养箱Cell culture incubator 31313131 ThermoFisher公司ThermoFisher
细菌培养箱Bacterial incubator IMC18IMC18 ThermoFisher公司ThermoFisher
PCR仪PCR instrument 44840734484073 ThermoFisher公司ThermoFisher
不锈钢水浴锅Stainless steel water bath 89032-18989032-189 VWR公司VWR Corporation
漩涡式震荡器Vortex oscillator 444-0203444-0203 VWR公司VWR Corporation
轨道式摇床Orbital shaker Standard 1000Standard 1000 VWR公司VWR Corporation
滚筒式混合器Drum mixer 444-0501444-0501 VWR公司VWR Corporation
凝胶成像系统Gel imaging system Gel Doc XR+Gel Doc XR+ Bio-Rad公司Bio-Rad
电源power supply 164-5050164-5050 Bio-Rad公司Bio-Rad
电泳槽Electrophoresis tank 164-0300164-0300 Bio-Rad公司Bio-Rad
实施例一 Embodiment 1
(一)重组表达载体的制备及蛋白表达量的比较(1) Preparation of recombinant expression vector and comparison of protein expression
(1)将编码ELL2转录因子、AFF4转录因子和CRTC2转录因子的基因分别进行聚合酶链式反应(94℃,30秒/56℃,30秒/68℃,2分钟,循环30次),电泳分离纯化分别得到编码ELL2转录因子、AFF4转录因子和CRTC2转录因子的基因;将所得到的编码ELL2转录因子和AFF4转录因子分别用BamHI和NotI两个酶进行酶切,将CRTC2转录因子的基因用HindIII和NotI两个酶进行酶切,然后分别与载体用T4连接酶进行连接,得到含有编码ELL2转录因子、AFF4转录因子和CRTC2转录因子的基因的重组载体,所述转录因子的蛋白质摩尔比例为1:1:1;(1) The genes encoding the ELL2 transcription factor, the AFF4 transcription factor and the CRTC2 transcription factor were subjected to polymerase chain reaction (94 ° C, 30 sec / 56 ° C, 30 sec / 68 ° C, 2 min, 30 cycles), electrophoresis The genes encoding ELL2 transcription factor, AFF4 transcription factor and CRTC2 transcription factor were isolated and purified, respectively. The obtained ELL2 transcription factor and AFF4 transcription factor were digested with BamHI and NotI, respectively, and the CRTC2 transcription factor gene was used. The HindIII and NotI enzymes were digested and ligated with the vector by T4 ligase to obtain a recombinant vector containing a gene encoding an ELL2 transcription factor, an AFF4 transcription factor and a CRTC2 transcription factor. The protein molar ratio of the transcription factor was 1:1:1;
其中ELL2的碱基序列如SEQ ID NO.1所示;Wherein the base sequence of ELL2 is as shown in SEQ ID NO.
AFF4的碱基序列如SEQ ID NO.3所示;The base sequence of AFF4 is shown in SEQ ID NO.
CRTC2的碱基序列如SEQ ID NO.5所示;The base sequence of CRTC2 is shown in SEQ ID NO.
(2)将步骤(1)所得到的重组载体转化到受体细胞,筛选得到转录因子表达的载体;(2) transforming the recombinant vector obtained in the step (1) into a recipient cell, and screening for a vector for expressing a transcription factor;
(3)将步骤(2)所得到的转录因子表达的载体进行DNA测序,得到重组表达载体。(3) The vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
(二)人白血病抑制因子(LIF)表达载体的制备 (II) Preparation of human leukemia inhibitory factor (LIF) expression vector
将LIF和含有巨细胞病毒启动子的表达载体分别用HindIII和BamHI进行酶切,得到露出粘性末端的LIF和巨细胞病毒载体,然后用T4连接酶将LIF与表达载体连接,得到人白血病抑制因子(LIF)表达载体。LIF and expression vector containing cytomegalovirus promoter were digested with HindIII and BamHI, respectively, to obtain LIF and cytomegalovirus vectors with sticky ends, and then LIF was ligated with expression vector using T4 ligase to obtain human leukemia inhibitory factor. (LIF) expression vector.
(三)表达量的比较(3) Comparison of expression levels
将人白血病抑制因子(LIF)表达载体与不同的转录因子表达载体组合表达载体共同转染到中国仓鼠卵巢细胞中,两天后收集细胞,对表达量进行比较,结果见图1。第一道是LIF在一般的表达系统中的量,第二道是LIF在含有ELL2/AFF4的表达系统中的量,第三道是LIF在含有ELL2/AFF4/CRTC2的表达系统中的量。我们清楚的看到,LIF在含有ELL2/AFF4/CRTC2三重转录因子的情况下,表达量最高。The human leukemia inhibitory factor (LIF) expression vector was transfected into Chinese hamster ovary cells together with different transcription factor expression vector expression vectors. Two days later, the cells were collected and the expression levels were compared. The results are shown in Fig. 1. The first is the amount of LIF in the general expression system, the second is the amount of LIF in the expression system containing ELL2/AFF4, and the third is the amount of LIF in the expression system containing ELL2/AFF4/CRTC2. We clearly see that LIF has the highest expression level in the case of the ELL2/AFF4/CRTC2 triple transcription factor.
实施例二 Embodiment 2
(一)人白血病抑制因子的蛋白的制备(1) Preparation of human leukemia inhibitory factor protein
(1)将上述所得到的重组表达载体与LIF表达载体共同转染到中国仓鼠卵巢细胞中,所述重组表达载体的总量与LIF表达载体的总量的比例为1:2;(1) The recombinant expression vector obtained above and the LIF expression vector are co-transfected into Chinese hamster ovary cells, the ratio of the total amount of the recombinant expression vector to the total amount of the LIF expression vector is 1:2;
(2)两天后收集细胞,得到人白血病抑制因子(LIF)的蛋白。(2) The cells were collected two days later to obtain a protein of human leukemia inhibitory factor (LIF).
(二)人白血病抑制因子(LIF)蛋白的纯化(II) Purification of human leukemia inhibitory factor (LIF) protein
(1)收集上述所得到的LIF蛋白表达系统,加入裂解液,所述裂解液包含4-羟乙基哌嗪乙磺酸(HEPES,20mM,pH7.9)、甘油(10%)、NaCl(0.3M)、EDTA(0.2mM,pH8.0)、乙基苯基聚乙二醇(NP-40,0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail),所述裂解液的加入量是1×107个细胞中加入1ml裂解液,在冰上放置15分钟,在转速为15000rpm下离心10min,得到上清液;(1) Collecting the LIF protein expression system obtained above, and adding a lysate containing 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl ( 0.3 M), EDTA (0.2 mM, pH 8.0), ethyl phenyl polyethylene glycol (NP-40, 0.5%) and Protease inhibitor cocktail, the lysate was added in an amount of 1 ×10 7 cells were added with 1 ml of lysate, placed on ice for 15 minutes, and centrifuged at 15,000 rpm for 10 min to obtain a supernatant;
(2)将步骤(1)所得到的上清液与flag beads混合,在4°孵育90min,所述上清液与flag beads的体积比为20:1;然后在转速为5000rpm下离心30s,去除上清液,最后加入高盐洗涤液洗涤,所述高盐洗涤液包含4-羟乙基哌嗪乙磺酸(HEPES,20mM,pH7.9)、甘油(10%)、NaCl(0.8M)、EDTA(0.2mM,pH8.0)、乙基苯基聚乙二醇(NP-40,0.5%)和蛋白酶抑制剂混合物(Protease  inhibitor cocktail),然后在转速为5000rpm下离心30s,去除上清液,重复2次,所加入的高盐洗涤液的体积为1ml;(2) mixing the supernatant obtained in the step (1) with the flag beads, incubating at 4 ° for 90 min, the volume ratio of the supernatant to the flag beads is 20:1; then centrifuging at 5000 rpm for 30 s, The supernatant was removed and finally washed with a high salt wash containing 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9), glycerol (10%), NaCl (0.8 M). ), EDTA (0.2 mM, pH 8.0), ethyl phenyl polyethylene glycol (NP-40, 0.5%) and protease inhibitor mixture (Protease) Inhibitor cocktail), then centrifuged at 5000 rpm for 30 s, remove the supernatant, repeat 2 times, the volume of the added high salt wash solution is 1 ml;
(3)采用与步骤(2)相同的方法,用低盐洗涤液进行洗涤,重复3次,所述低盐洗涤液包含4-羟乙基哌嗪乙磺酸(HEPES,20mM,pH7.9)、甘油(10%)、NaCl(0.15M)、EDTA(0.2mM,pH8.0)、乙基苯基聚乙二醇(NP-40,0.5%)和蛋白酶抑制剂混合物(Protease inhibitor cocktail),所使用的低盐洗涤液的体积为1ml;(3) Washing with a low salt washing solution containing 4-hydroxyethylpiperazineethanesulfonic acid (HEPES, 20 mM, pH 7.9) in the same manner as in the step (2), washing with a low salt washing solution ), glycerol (10%), NaCl (0.15M), EDTA (0.2 mM, pH 8.0), ethyl phenyl polyethylene glycol (NP-40, 0.5%) and Protease inhibitor cocktail The volume of the low salt washing liquid used is 1 ml;
(4)用终浓度为0.5mg/ml的flag peptide(溶在低盐洗涤液)在室温下洗脱25min,所述的洗脱体积与flag beads的体积比为4:1,然后进行离心,得到上清液,所述上清液即为纯化的人白血病抑制因子(LIF)的蛋白表达系统。(4) eluting with a flag peptide (dissolved in a low-salt washing solution) having a final concentration of 0.5 mg/ml for 25 min at room temperature, the volume ratio of the elution volume to the flag beads was 4:1, and then centrifuging. The supernatant is obtained, which is the protein expression system of purified human leukemia inhibitory factor (LIF).
将实施例一中纯化好的蛋白表达系统进行蛋白电泳,然后用ThermoFisher公司的银染试剂盒进行染色,测定结果见图2,对蛋白浓度进行估算,其纯度大于95%。The purified protein expression system of Example 1 was subjected to protein electrophoresis, and then stained with ThermoFisher's silver staining kit. The results of the assay are shown in Figure 2, and the protein concentration was estimated to be greater than 95%.
实施例三 Embodiment 3
生物活性的验证Bioactivity verification
采用小鼠胚胎干细胞进行生物活性验证的实验,实验结果见图3。由于人和鼠LIF具有很高的同源性,人源的LIF被验证对于鼠源的胚胎干细胞也起作用,及抑制鼠源胚胎干细胞的分化,让其保持不断分裂增殖的能力。我们用含ELL2/AFF4/CRTC2三重转录因子的表达系统所产生的LIF能够较ThermoFisher公司的LIF在相同浓度下有着更高的活性,体现为产生了更多胚胎干细胞(因为更多的胚胎干细胞能够分裂增殖),从图三中可以看出,使用本发明的蛋白表达系统的胚胎干细胞较多,说明本发明的蛋白表达系统能够促进干细胞生长且抑制干细胞分化的生物活性。Experiments were carried out using mouse embryonic stem cells for bioactivity verification. The experimental results are shown in Figure 3. Due to the high homology between human and murine LIF, human LIF has been validated for murine embryonic stem cells and inhibits the differentiation of murine embryonic stem cells, allowing them to maintain their ability to divide and proliferate. The LIF produced by the expression system containing the ELL2/AFF4/CRTC2 triple transcription factor is more active than the ThermoFisher LIF at the same concentration, which means that more embryonic stem cells are produced (because more embryonic stem cells can As shown in Fig. 3, it can be seen that the embryonic cells using the protein expression system of the present invention are more, indicating that the protein expression system of the present invention can promote stem cell growth and inhibit the biological activity of stem cell differentiation.
综上所述,本发明所制备得到的蛋白表达系统与利用细菌表达系统所产生的同类产品进行对比的实验中,表现出高活性,能够在极低浓度下促进胚胎干细胞生长的活性。并且与加入ELL2和AFF4的蛋白表达系统相比较,本发明所制备的蛋白表达系统的表达量提高7倍,说明这种组合(ELL2/AFF4/CRTC2)能极大地增强CMV启动子,从而提高目的基因的表达。 In summary, the protein expression system prepared by the present invention exhibits high activity and can promote the growth of embryonic stem cell cells at a very low concentration in an experiment comparing with a similar product produced by a bacterial expression system. Moreover, compared with the protein expression system to which ELL2 and AFF4 are added, the expression level of the protein expression system prepared by the present invention is increased by 7 times, indicating that this combination (ELL2/AFF4/CRTC2) can greatly enhance the CMV promoter, thereby improving the purpose. Gene expression.
以上所述,仅是本发明实施的较佳实施例,并非对本发明做任何形式上的限制,凡在本发明的精神和原则之内所做的修改、等同替换和改进等,均需要包含在本发明的保护范围之内。 The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention are required to be included in the present invention. Within the scope of protection of the present invention.

Claims (22)

  1. 一种转录因子组合物,其特征在于,含有ELL转录因子、AFF转录因子和CRTC转录因子,优选地,ELL转录因子选自ELL2或ELL1中的一种,AFF转录因子选自AFF4或AFF1中的一种,CRTC转录因子选自CRTC2或CRTC1或CRTC3中的一种。A transcription factor composition comprising an ELL transcription factor, an AFF transcription factor and a CRTC transcription factor, preferably, the ELL transcription factor is selected from one of ELL2 or ELL1, and the AFF transcription factor is selected from AFF4 or AFF1. In one case, the CRTC transcription factor is selected from one of CRTC2 or CRTC1 or CRTC3.
  2. 根据权利要求1所述的转录因子组合物,其特征在于,所述组合物含有ELL2、AFF4和CRTC2,所述ELL2、AFF4和CRTC2的比例为1:1-3:1-4;优选的,所述ELL2、AFF4和CRTC2的比例为1:1:1。The transcription factor composition according to claim 1, wherein said composition contains ELL2, AFF4 and CRTC2, and the ratio of said ELL2, AFF4 and CRTC2 is from 1:1 to 3: 1-4; preferably, The ratio of ELL2, AFF4 and CRTC2 is 1:1:1.
  3. 根据权利要求1或2所述的转录因子组合物,其特征在于,所述转录因子组合物选自于人源、鼠源、狗源或者猪源中的一种;优选的,所述转录因子组合物为人源的。The transcription factor composition according to claim 1 or 2, wherein the transcription factor composition is selected from one of human, murine, dog or porcine sources; preferably, the transcription factor The composition is of human origin.
  4. 一种三重转录因子,其含有权利要求1-3中任一项所述的转录因子组合物。A triple transcription factor comprising the transcription factor composition of any one of claims 1-3.
  5. 一种DNA分子,其特征在于,所述DNA分子含有编码ELL转录因子的碱基序列、编码AFF转录因子的碱基序列和编码CRTC转录因子的碱基序列;优选,所述编码ELL转录因子的碱基序列选自以下碱基序列中的一种:A DNA molecule comprising a base sequence encoding an ELL transcription factor, a base sequence encoding an AFF transcription factor, and a base sequence encoding a CRTC transcription factor; preferably, the ELL transcription factor is encoded The base sequence is selected from one of the following base sequences:
    (a)碱基序列如SEQ ID NO.1所示;(a) the base sequence is shown in SEQ ID NO.
    (b)与SEQ ID NO.1序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 1, preferably 95% or more; more preferably 99% or more of the base sequence;
    (c)编码SEQ ID NO.2所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO. 2;
    (d)在SEQ ID NO.2所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质;(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown in SEQ ID NO. 2 and which has a transcriptional activity of the regulatory gene;
    所述编码AFF转录因子的碱基序列选自以下碱基序列中的一种:The base sequence encoding the AFF transcription factor is selected from one of the following base sequences:
    (a)碱基序列如SEQ ID NO.3所示;(a) the base sequence is shown in SEQ ID NO. 3;
    (b)与SEQ ID NO.3序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 3, preferably 95% or more; more preferably 99% or more of the base sequence;
    (c)编码SEQ ID NO.4所示的氨基酸序列组成的蛋白质的基因; (c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO.
    (d)在SEQ ID NO.4所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质;(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown in SEQ ID NO. 4 and which has a transcriptional activity of the regulatory gene;
    所述编码CRTC转录因子的碱基序列选自以下碱基序列中的一种:The base sequence encoding the CRTC transcription factor is selected from one of the following base sequences:
    (a)碱基序列如SEQ ID NO.5所示;(a) the base sequence is shown in SEQ ID NO. 5;
    (b)与SEQ ID NO.5序列的同源性在90%以上的碱基序列,优选95%以上的碱基序列;更优选在99%以上的碱基序列;(b) a base sequence having 90% or more homology with the sequence of SEQ ID NO. 5, preferably 95% or more; more preferably 99% or more of the base sequence;
    (c)编码SEQ ID NO.6所示的氨基酸序列组成的蛋白质的基因;(c) a gene encoding a protein consisting of the amino acid sequence shown in SEQ ID NO.
    (d)在SEQ ID NO.6所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸且具有调控基因转录活性的由(c)衍生的蛋白质。(d) a protein derived from (c) which has been substituted, deleted or added with one or several amino acids in the amino acid sequence shown by SEQ ID NO. 6 and which has a transcriptional activity of the regulatory gene.
  6. 一种重组表达载体,其特征在于,其是将权利要求1-3中任一项所述的转录因子组合物或者权利要求4所述的三重转录因子克隆到哺乳动物表达载体中而得到。A recombinant expression vector obtained by cloning the transcription factor composition according to any one of claims 1 to 3 or the triple transcription factor according to claim 4 into a mammalian expression vector.
  7. 一种权利要求6所述的重组表达载体的制备方法,其特征在于,其包含下述步骤:A method for preparing a recombinant expression vector according to claim 6, comprising the steps of:
    (1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因分别与载体连接,得到含有三重转录因子的重组载体;(1) ligating a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor to a vector, respectively, to obtain a recombinant vector containing a triple transcription factor;
    (2)将步骤(1)所得到的重组载体转化到受体细胞,筛选得到转录因子表达的载体;(2) transforming the recombinant vector obtained in the step (1) into a recipient cell, and screening for a vector for expressing a transcription factor;
    (3)将步骤(2)所得到的转录因子表达的载体进行DNA测序,得到重组表达载体。(3) The vector expressing the transcription factor obtained in the step (2) is subjected to DNA sequencing to obtain a recombinant expression vector.
  8. 权利要求1-3中任一项所述的转录因子组合物或权利要求4所述的三重转录因子在哺乳动物蛋白表达系统中的应用。Use of the transcription factor composition of any one of claims 1 to 3 or the triple transcription factor of claim 4 in a mammalian protein expression system.
  9. 一种哺乳动物蛋白表达系统,其特征在于,其通过将权利要求6所述的重组表达载体或用权利要求7制备得到的重组表达载体与目的基因表达载体共同转染到细胞株中表达得到,所述重组表达载体的总量与目的基因表达载体的总量的比为1:1-5。A mammalian protein expression system, which is obtained by co-transfecting the recombinant expression vector of claim 6 or the recombinant expression vector prepared by the method of claim 7 with a gene expression vector of interest into a cell strain. The ratio of the total amount of the recombinant expression vector to the total amount of the gene expression vector of interest is 1:1-5.
  10. 根据权利要求9所述的哺乳动物蛋白表达系统,其特征在于,所述细胞株选自于人胚胎肾细胞293或者中国仓鼠卵巢细胞中的一种。 The mammalian protein expression system according to claim 9, wherein the cell strain is selected from the group consisting of human embryonic kidney cell 293 or Chinese hamster ovary cells.
  11. 一种权利要求9或10所述的哺乳动物蛋白表达系统的制备方法,其包含下述步骤:A method of preparing a mammalian protein expression system according to claim 9 or 10, comprising the steps of:
    (1)将重组表达载体和目的基因表达载体总量比为1:1-5共同转染到细胞株中;(1) co-transfecting the recombinant expression vector and the target gene expression vector into a cell line in a total ratio of 1:1-5;
    (2)1-3天后收集细胞,得到哺乳动物蛋白表达系统。(2) Cells were collected after 1-3 days to obtain a mammalian protein expression system.
  12. 根据权利要求11所述的制备方法,其特征在于,在步骤(1)中,所述目的基因载体的制备方法包含下述步骤:将目的基因及表达载体连接得到目的基因表达载体。The preparation method according to claim 11, wherein in the step (1), the method for preparing the gene carrier of interest comprises the step of ligating the gene of interest and the expression vector to obtain a gene expression vector of interest.
  13. 一种哺乳动物蛋白表达系统,其通过含有权利要求1-3任一项所述的三重转录因子的目的基因表达载体转染到细胞株中得到。A mammalian protein expression system obtained by transfecting a target gene expression vector containing the triple transcription factor of any one of claims 1 to 3 into a cell strain.
  14. 根据权利要求11所述的哺乳动物蛋白表达系统,其中,所述细胞株选自于人胚胎肾细胞或中国仓鼠卵巢细胞中的一种。The mammalian protein expression system according to claim 11, wherein the cell strain is selected from one of human embryonic kidney cells or Chinese hamster ovary cells.
  15. 一种权利要求13或14所述的哺乳动物蛋白表达系统的制备方法,其包含下述步骤:A method of preparing a mammalian protein expression system according to claim 13 or 14, comprising the steps of:
    (1)将编码ELL转录因子、AFF转录因子和CRTC转录因子的基因整合到目的基因中得到含有编码三重转录因子的基因的目的基因;(1) integrating a gene encoding an ELL transcription factor, an AFF transcription factor, and a CRTC transcription factor into a gene of interest to obtain a gene of interest comprising a gene encoding a triple transcription factor;
    (2)将含有编码三重转录因子的基因的目的基因与载体连接,得到含有编码三重转录因子基因的目的基因表达载体;(2) ligating a gene of interest containing a gene encoding a triple transcription factor with a vector to obtain a gene expression vector containing a gene encoding a triple transcription factor;
    (3)将步骤(2)所得到的表达载体转染到细胞株中,1-3天后收集细胞,得到哺乳动物蛋白表达系统。(3) The expression vector obtained in the step (2) is transfected into the cell strain, and the cells are collected 1-3 days later to obtain a mammalian protein expression system.
  16. 一种权利要求11-15任一项哺乳动物蛋白表达系统的纯化方法,其包含下述步骤:A method of purifying a mammalian protein expression system according to any of claims 11-15, comprising the steps of:
    (1)收集哺乳动物蛋白表达系统细胞,加入裂解液,离心得到上清液;(1) collecting mammalian protein expression system cells, adding a lysate, and centrifuging to obtain a supernatant;
    (2)将步骤(1)所述的上清液与标签beads混合,离心去除上清液,然后加入高盐洗涤液洗涤;(2) mixing the supernatant described in the step (1) with the label beads, centrifuging to remove the supernatant, and then adding the high salt washing solution to wash;
    (3)将步骤(2)所得的洗涤液离心,去除上清液,加入低盐洗涤液洗涤;(3) centrifuging the washing liquid obtained in the step (2), removing the supernatant, and adding the low-salt washing liquid to wash;
    (4)用带有标记peptide进行洗脱,然后离心得到上清液,所述上清液即为哺乳动物蛋白表达系统。 (4) Elution with a labeled peptide followed by centrifugation to obtain a supernatant, which is a mammalian protein expression system.
  17. 根据权利要求16所述的纯化方法,其特征在于,在步骤(1)中,所述裂解液包含4-羟乙基哌嗪乙磺酸、甘油、NaCl、EDTA、乙基苯基聚乙二醇和蛋白酶抑制剂混合物,所述裂解液的加入量为每1×107个细胞加入1-5ml的裂解液。The purification method according to claim 16, wherein in the step (1), the lysate comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, ethylphenyl polyethylene. A mixture of an alcohol and a protease inhibitor is added in an amount of 1 to 5 ml of a lysate per 1 x 10 7 cells.
  18. 根据权利要求16或17所述的纯化方法,其特征在于,在步骤(2)中,所述上清液与标签beads的体积比为20-50:1。The purification method according to claim 16 or 17, wherein in the step (2), the volume ratio of the supernatant to the label beads is 20-50:1.
  19. 根据权利要求16-18任一项所述的纯化方法,其特征在于,在步骤(2)中,所述高盐洗涤液包含4-羟乙基哌嗪乙磺酸、甘油、NaCl、EDTA、乙基苯基聚乙二醇和蛋白酶抑制剂混合物,所述高盐洗涤液的加入量为1-5ml。The purification method according to any one of claims 16 to 18, wherein in the step (2), the high salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, A mixture of ethyl phenyl polyethylene glycol and a protease inhibitor is added in an amount of from 1 to 5 ml.
  20. 根据权利要求11-19任一项所述的纯化方法,其特征在于,在步骤(3)中,所述低盐洗涤液包含4-羟乙基哌嗪乙磺酸、甘油、NaCl、EDTA、乙基苯基聚乙二醇和蛋白酶抑制剂混合物,所述低盐洗涤液的加入量为1-5ml。The purification method according to any one of claims 11 to 19, wherein in the step (3), the low-salt washing liquid comprises 4-hydroxyethylpiperazineethanesulfonic acid, glycerin, NaCl, EDTA, A mixture of ethyl phenyl polyethylene glycol and a protease inhibitor is added in an amount of from 1 to 5 ml.
  21. 根据权利要求11-20任一项所述的纯化方法,其特征在于,所述标签peptide与标签beads的体积比为3-5:1。The purification method according to any one of claims 11 to 20, wherein the volume ratio of the label peptide to the label beads is 3-5:1.
  22. 权利要求9或10或13或14所述的哺乳动物蛋白表达系统或11或12或15所述方法得到的哺乳动物蛋白表达系统,其在生物医药中的应用。 The mammalian protein expression system of claim 9 or 10 or 13 or 14, or the mammalian protein expression system obtained by the method of 11 or 12 or 15, for use in biomedicine.
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