WO2022141441A1 - Follicle-stimulating hormone and in-vitro self-assembly method therefor - Google Patents
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- 102000012673 Follicle Stimulating Hormone Human genes 0.000 title claims abstract description 41
- 108010079345 Follicle Stimulating Hormone Proteins 0.000 title claims abstract description 41
- 229940028334 follicle stimulating hormone Drugs 0.000 title claims abstract description 40
- 238000000338 in vitro Methods 0.000 title claims abstract description 29
- 238000001338 self-assembly Methods 0.000 title claims description 20
- 238000000034 method Methods 0.000 title claims description 14
- 108010076504 Protein Sorting Signals Proteins 0.000 claims abstract description 31
- 238000005215 recombination Methods 0.000 claims abstract description 10
- 230000006798 recombination Effects 0.000 claims abstract description 10
- 230000028327 secretion Effects 0.000 claims abstract description 10
- 238000002360 preparation method Methods 0.000 claims abstract 2
- 108090000623 proteins and genes Proteins 0.000 claims description 27
- 102100030552 Synaptosomal-associated protein 25 Human genes 0.000 claims description 11
- 108040000979 soluble NSF attachment protein activity proteins Proteins 0.000 claims description 11
- 108010060374 FSH Receptors Proteins 0.000 claims description 6
- 102000008175 FSH Receptors Human genes 0.000 claims description 6
- 239000008267 milk Substances 0.000 claims description 6
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- 239000012930 cell culture fluid Substances 0.000 claims description 5
- 239000002504 physiological saline solution Substances 0.000 claims description 5
- 102100027627 Follicle-stimulating hormone receptor Human genes 0.000 claims description 4
- 101000862396 Homo sapiens Follicle-stimulating hormone receptor Proteins 0.000 claims description 4
- 102000018697 Membrane Proteins Human genes 0.000 claims description 3
- 108010052285 Membrane Proteins Proteins 0.000 claims description 3
- 102000035824 beta Subunit Follicle Stimulating Hormone Human genes 0.000 claims description 3
- 108010081485 beta Subunit Follicle Stimulating Hormone Proteins 0.000 claims description 3
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 claims description 3
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/02—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length in solution
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/575—Hormones
- C07K14/59—Follicle-stimulating hormone [FSH]; Chorionic gonadotropins, e.g.hCG [human chorionic gonadotropin]; Luteinising hormone [LH]; Thyroid-stimulating hormone [TSH]
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- the invention relates to the technical field of biomedicine and genetic engineering, in particular to a follicle-stimulating hormone and an in vitro self-assembly method thereof.
- FSH follicle-stimulating hormone
- mammalian cells Purified from urine, there is a problem of protein impurity. Expressed by mammalian cells, during the production process, the yield is low and the price is high. Expressed by yeast or bacteria, it cannot be used in the market because it cannot be glycosylated or is incompletely glycosylated, and the expression yield is high, but it has no activity.
- the technical problem to be solved by the present invention is to provide a follicle-stimulating hormone and its in vitro self-assembly method, which can not only mass-produce highly active FSH, but also maintain the health of individual producing animals, thereby providing possibility for long-term production.
- the embodiments of the present invention provide a follicle-stimulating hormone, which is obtained by self-assembly in vitro after the FSH ⁇ and FSH ⁇ subunit genes are introduced into the secretion signal peptide.
- the secretion signal peptide is the signal peptide of the IgG Kappa chain added in the N segment of the FSH ⁇ and FSH ⁇ subunit genes.
- the buffer used in the in vitro self-assembly is one of cell culture fluid, physiological saline and milk.
- the invention also provides an in vitro self-assembly method of follicle-stimulating hormone, wherein the FSH ⁇ and FSH ⁇ subunit genes are introduced into the secretion signal peptide and self-assembled in vitro.
- Getting follicle-stimulating hormone involves the following steps:
- step (1) comprises the steps:
- METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain
- METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain
- the in vitro self-assembly method of described follicle stimulating hormone also comprises the following steps:
- the sequence of the FSH receptor labeled with SNAP is as follows:
- M R L C I P Q V L L A L F L S M L T G P G E G S is the 5-HT3 membrane protein signal peptide
- SNAP protein is a SNAP protein
- the reconstitution buffer used in step (2) is cell culture fluid, physiological saline or milk.
- the present invention expresses the two subunits of FSH: FSH ⁇ and FSH ⁇ separately, and then recombines in vitro into active molecules, which can not only produce high-activity FSH in large quantities, but also maintain the health of the individual producing animals, thereby providing long-term production. possible.
- the FSH protein reconstituted by the method of the present invention is a protein with complete structure, rational glycosylation and high biological activity.
- Fig. 1 is a schematic diagram of the in vitro self-assembly of separately expressed FSH ⁇ and FSH ⁇ subunit genes in a recombination buffer in the present invention
- Figure 2 is the glycosylation analysis of FSH ⁇ and FSH ⁇ subunits in glutathione
- Fig. 3 is the activity analysis diagram of FSH protein after recombination
- Figure 4 shows the activity of SNAP to identify the recombined FSH protein.
- the invention provides a follicle-stimulating hormone, which is obtained by self-assembly in vitro after FSH ⁇ and FSH ⁇ subunit genes are introduced into secretion signal peptides.
- the secretion signal peptide is the signal peptide of the IgG Kappa chain added to the N segment of the FSH ⁇ and FSH ⁇ subunit genes.
- the buffer used in the in vitro self-assembly is one of cell culture fluid, physiological saline and milk.
- the invention also provides an in vitro self-assembly method of follicle-stimulating hormone, which is characterized in that the FSH ⁇ and FSH ⁇ subunit genes are introduced into the secretion signal peptide and self-assembled in vitro.
- Getting follicle-stimulating hormone involves the following steps:
- METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain
- METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain
- FIG. 2 shows the glycosylation analysis of FSH ⁇ and FSH ⁇ subunits in glutathione, wherein FIG. 2A is the glycosylation analysis of the FSH ⁇ subunit, and FIG. 2B is the glycosylation analysis of the FSH ⁇ subunit.
- FIG. 3 is the activity analysis diagram of FSH protein after recombination, wherein, Fig. 3A is the experimental result diagram of rat ovary staining, ovarian weight gain assay, injected separately, and combined with hcg; Fig. 3B is 1.5, 3, 6 pmol/l Ovulation images of FSH-treated mouse oocytes.
- M R L C I P Q V L L A L F L S M L T G P G E G S is the 5-HT3 membrane protein signal peptide
- SNAP protein is a SNAP protein
- Figure 4 shows the activity of SNAP to identify the recombined FSH protein, wherein Figure 4A shows that fluorescently labeled FSHR is presented on the cell membrane; Figure 4B shows the internalization of cells under the stimulation of FSH recombined in vitro.
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Abstract
Provided is a follicle-stimulating hormone (FSH). The preparation thereof comprises the following steps: introducing a secretion signal peptide into the two subunits FSHα and FSHβ of the FSH, and then individually expressing same, followed by in-vitro recombination to form an active molecule.
Description
本发明涉及生物医药与基因工程技术领域,具体涉及一种促卵泡激素及其体外自组装方法。The invention relates to the technical field of biomedicine and genetic engineering, in particular to a follicle-stimulating hormone and an in vitro self-assembly method thereof.
FSH(促卵泡激素)在人的临床应用以及畜牧业繁殖生产方面有极大市场。目前商业化生产主要从尿液中提取或者由哺乳细胞表达纯化。从尿液提纯,存在蛋白不纯的问题。由哺乳细胞表达,生产过程中,产量低、价格高。由酵母或者细菌表达,由于不能糖基化或者糖基化不完全,表达产量高,却没有活性,因此也不能应用于市场。FSH (follicle-stimulating hormone) has a huge market in human clinical application and animal husbandry reproduction production. At present, commercial production is mainly extracted from urine or expressed and purified from mammalian cells. Purified from urine, there is a problem of protein impurity. Expressed by mammalian cells, during the production process, the yield is low and the price is high. Expressed by yeast or bacteria, it cannot be used in the market because it cannot be glycosylated or is incompletely glycosylated, and the expression yield is high, but it has no activity.
如果从大型哺乳动物乳腺生物反应器中进行表达,已有实验证明,其表达量高,糖基化也可满足药物活性要求,但是由于体内高表达,将导致FSH泄漏到血液中,因而给生产动物带来诸多健康问题。If it is expressed from a large mammalian mammary gland bioreactor, experiments have shown that its high expression level and glycosylation can also meet the requirements of drug activity. However, due to high expression in vivo, it will cause FSH to leak into the blood. Animals bring many health problems.
发明内容SUMMARY OF THE INVENTION
本发明要解决的技术问题是提供一种促卵泡激素及其体外自组装方法,既可以大量生产高活性的FSH,又能保持生产动物个体的健康,进而为长期生产提供可能。The technical problem to be solved by the present invention is to provide a follicle-stimulating hormone and its in vitro self-assembly method, which can not only mass-produce highly active FSH, but also maintain the health of individual producing animals, thereby providing possibility for long-term production.
为解决上述技术问题,本发明的实施例提供一种促卵泡激素,是在FSHα和FSHβ亚基基因引入分泌信号肽后,在体外自组装获得。In order to solve the above technical problems, the embodiments of the present invention provide a follicle-stimulating hormone, which is obtained by self-assembly in vitro after the FSHα and FSHβ subunit genes are introduced into the secretion signal peptide.
其中,所述分泌信号肽为在FSHα和FSHβ亚基基因的N段加入的IgG Kappa链的信号肽。Wherein, the secretion signal peptide is the signal peptide of the IgG Kappa chain added in the N segment of the FSHα and FSHβ subunit genes.
其中,体外自组装所用缓冲液为细胞培养液、生理盐水和牛奶中的一种。Wherein, the buffer used in the in vitro self-assembly is one of cell culture fluid, physiological saline and milk.
本发明还提供一种促卵泡激素的体外自组装方法,FSHα和FSHβ亚基基因引入分泌信号肽后在体外自组装。得到促卵泡激素,包括以下步骤:The invention also provides an in vitro self-assembly method of follicle-stimulating hormone, wherein the FSHα and FSHβ subunit genes are introduced into the secretion signal peptide and self-assembled in vitro. Getting follicle-stimulating hormone involves the following steps:
(1)制备含有引导信号肽的FSHα亚基氨基酸序列和FSHβ亚基氨基酸序列;(1) preparing the FSHα subunit amino acid sequence and the FSHβ subunit amino acid sequence containing the guide signal peptide;
(2)分开表达的FSHα和FSHβ亚基基因在重组缓冲液内体外自组装成促卵泡激素。(2) The separately expressed FSHα and FSHβ subunit genes self-assemble into follicle-stimulating hormone in vitro and in the recombination buffer.
其中,步骤(1)包括如下步骤:Wherein, step (1) comprises the steps:
(1-1)在FSHα亚基基因的N段加入IgG Kappa链的信号肽,制备得到含有引导信号肽的FSHα亚基氨基酸序列:(1-1) Add the signal peptide of the IgG Kappa chain to the N segment of the FSHα subunit gene to prepare the FSHα subunit amino acid sequence containing the guiding signal peptide:
其中,
METDTLLLWVLLLWVP是IgG kappa分泌肽信号链;
Wherein, METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain;
(1-2)在FSHβ亚基基因的N段加入IgG Kappa链的信号肽,制备含有引导信号肽的FSHβ亚基氨基酸序列:(1-2) Add the signal peptide of the IgG Kappa chain to the N segment of the FSHβ subunit gene to prepare the FSHβ subunit amino acid sequence containing the guiding signal peptide:
其中,
METDTLLLWVLLLWVP是IgG kappa分泌肽信号链;
Wherein, METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain;
其中,所述的促卵泡激素的体外自组装方法还包括如下步骤:Wherein, the in vitro self-assembly method of described follicle stimulating hormone also comprises the following steps:
(3)促卵泡激素活性的验证:用SNAP标记FSH受体,显微镜下读取表达株的产量。(3) Verification of follicle-stimulating hormone activity: FSH receptors were labeled with SNAP, and the yield of the expression strain was read under a microscope.
其中,用SNAP标记FSH受体序列如下:Among them, the sequence of the FSH receptor labeled with SNAP is as follows:
其中,
M R L C I P Q V L L A L F L S M L
T G P G E G S是5-HT3膜蛋白信号肽;
Wherein, M R L C I P Q V L L A L F L S M L T G P G E G S is the 5-HT3 membrane protein signal peptide;
优选的,步骤(2)所用重组缓冲液为细胞培养液、生理盐水或者牛奶。Preferably, the reconstitution buffer used in step (2) is cell culture fluid, physiological saline or milk.
本发明的上述技术方案的有益效果如下:The beneficial effects of the above-mentioned technical solutions of the present invention are as follows:
1、本发明把FSH的两个亚基:FSHα和FSHβ分开表达,然后体外重组成为有活性的分子,既可以大量生产高活性的FSH,又能保持生产动物个体的健康,进而为长期生产提供可能。1. The present invention expresses the two subunits of FSH: FSHα and FSHβ separately, and then recombines in vitro into active molecules, which can not only produce high-activity FSH in large quantities, but also maintain the health of the individual producing animals, thereby providing long-term production. possible.
2、利用本发明的方法重组的FSH蛋白是结构完整,合理糖基化的,具有很高的生物学活性的蛋白。2. The FSH protein reconstituted by the method of the present invention is a protein with complete structure, rational glycosylation and high biological activity.
图1为本发明中分开表达的FSHα和FSHβ亚基基因在重组缓冲液内体外自组装的示意图;Fig. 1 is a schematic diagram of the in vitro self-assembly of separately expressed FSHα and FSHβ subunit genes in a recombination buffer in the present invention;
图2为谷胱甘肽中FSHα和FSHβ亚基的糖基化分析;Figure 2 is the glycosylation analysis of FSHα and FSHβ subunits in glutathione;
图3为重组后FSH蛋白的活性分析图;Fig. 3 is the activity analysis diagram of FSH protein after recombination;
图4为SNAP鉴定重组后FSH蛋白的活性。Figure 4 shows the activity of SNAP to identify the recombined FSH protein.
为使本发明要解决的技术问题、技术方案和优点更加清楚,下面将结合附图及具体实施例进行详细描述。In order to make the technical problems, technical solutions and advantages to be solved by the present invention more clear, the following will be described in detail with reference to the accompanying drawings and specific embodiments.
本发明提供一种促卵泡激素,是在FSHα和FSHβ亚基基因引入分泌信号肽后,在体外自组装获得。The invention provides a follicle-stimulating hormone, which is obtained by self-assembly in vitro after FSHα and FSHβ subunit genes are introduced into secretion signal peptides.
所述分泌信号肽为在FSHα和FSHβ亚基基因的N段加入的IgG Kappa链的信号肽。体外自组装所用缓冲液为细胞培养液、生理盐水和牛奶中的一种。The secretion signal peptide is the signal peptide of the IgG Kappa chain added to the N segment of the FSHα and FSHβ subunit genes. The buffer used in the in vitro self-assembly is one of cell culture fluid, physiological saline and milk.
本发明还提供一种促卵泡激素的体外自组装方法,其特征在于,FSHα和 FSHβ亚基基因引入分泌信号肽后在体外自组装。得到促卵泡激素,包括以下步骤:The invention also provides an in vitro self-assembly method of follicle-stimulating hormone, which is characterized in that the FSHα and FSHβ subunit genes are introduced into the secretion signal peptide and self-assembled in vitro. Getting follicle-stimulating hormone involves the following steps:
(1)制备含有引导信号肽的FSHα亚基氨基酸序列和FSHβ亚基氨基酸序列;包括如下具体步骤:(1) preparing the FSHα subunit amino acid sequence and the FSHβ subunit amino acid sequence containing the guiding signal peptide; including the following specific steps:
(1-1)在FSHα亚基基因的N段加入IgG Kappa链的信号肽,制备得到含有引导信号肽的FSHα亚基氨基酸序列:(1-1) Add the signal peptide of the IgG Kappa chain to the N segment of the FSHα subunit gene to prepare the FSHα subunit amino acid sequence containing the guiding signal peptide:
其中,
METDTLLLWVLLLWVP是IgG kappa分泌肽信号链;
Wherein, METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain;
(1-2)在FSHβ亚基基因的N段加入IgG Kappa链的信号肽,制备含有引导信号肽的FSHβ亚基氨基酸序列:(1-2) Add the signal peptide of the IgG Kappa chain to the N segment of the FSHβ subunit gene to prepare the FSHβ subunit amino acid sequence containing the guiding signal peptide:
其中,
METDTLLLWVLLLWVP是IgG kappa分泌肽信号链;
Wherein, METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain;
(2)分开表达的FSHα和FSHβ亚基基因在重组缓冲液内体外自组装成促卵泡激素。分开表达的FSHα和FSHβ可以奇妙的重组,重组的缓冲液可以是细胞培养液、生理盐水或者牛奶。图1所示为分开表达的FSHα和FSHβ亚基基因 在重组缓冲液内体外自组装的示意图。图2所示为谷胱甘肽中FSHα和FSHβ亚基的糖基化分析,其中图2A为FSHα亚基的糖基化分析,图2B为FSHβ亚基的糖基化分析。图3为重组后FSH蛋白的活性分析图,其中,图3A为大鼠卵巢染色,卵巢体重增加化验,分别注射,并与hcg联合应用的实验结果图;图3B为1.5、3、6pmol/l的FSH分别处理小鼠卵母细胞的排卵图像。(2) The separately expressed FSHα and FSHβ subunit genes self-assemble into follicle-stimulating hormone in vitro and in the recombination buffer. Separately expressed FSHα and FSHβ can be wonderfully recombined, and the recombination buffer can be cell culture medium, saline or milk. Figure 1 shows a schematic representation of the in vitro self-assembly of separately expressed FSHα and FSHβ subunit genes in recombination buffer. FIG. 2 shows the glycosylation analysis of FSHα and FSHβ subunits in glutathione, wherein FIG. 2A is the glycosylation analysis of the FSHα subunit, and FIG. 2B is the glycosylation analysis of the FSHβ subunit. Fig. 3 is the activity analysis diagram of FSH protein after recombination, wherein, Fig. 3A is the experimental result diagram of rat ovary staining, ovarian weight gain assay, injected separately, and combined with hcg; Fig. 3B is 1.5, 3, 6 pmol/l Ovulation images of FSH-treated mouse oocytes.
(3)促卵泡激素活性的验证:用SNAP标记FSH受体,显微镜下读取表达株的产量;用SNAP标记FSH受体序列如下:(3) Verification of follicle-stimulating hormone activity: label the FSH receptor with SNAP, and read the yield of the expression strain under a microscope; label the FSH receptor sequence with SNAP as follows:
其中,
M R L C I P Q V L L A L F L S M L
T G P G E G S是5-HT3膜蛋白信号肽;
Wherein, M R L C I P Q V L L A L F L S M L T G P G E G S is the 5-HT3 membrane protein signal peptide;
图4为SNAP鉴定重组后FSH蛋白的活性,其中,图4A为荧光标记的FSHR呈现在细胞膜上;图4B为在有体外重组的FSH的刺激下,细胞内在化。Figure 4 shows the activity of SNAP to identify the recombined FSH protein, wherein Figure 4A shows that fluorescently labeled FSHR is presented on the cell membrane; Figure 4B shows the internalization of cells under the stimulation of FSH recombined in vitro.
以上所述是本发明的优选实施方式,应当指出,对于本技术领域的普通技术人员来说,在不脱离本发明所述原理的前提下,还可以作出若干改进和润饰,这些改进和润饰也应视为本发明的保护范围。The above are the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.
Claims (9)
- 一种促卵泡激素,其特征在于,是在FSHα和FSHβ亚基基因引入分泌信号肽后,在体外自组装获得。A follicle-stimulating hormone is characterized in that it is obtained by self-assembly in vitro after FSHα and FSHβ subunit genes are introduced into secretion signal peptides.
- 根据权利要求1所述的促卵泡激素,其特征在于,所述分泌信号肽为在FSHα和FSHβ亚基基因的N段加入的IgG Kappa链的信号肽。The follicle-stimulating hormone according to claim 1, wherein the secretion signal peptide is the signal peptide of the IgG Kappa chain added in the N segment of the FSHα and FSHβ subunit genes.
- 根据权利要求1所述的促卵泡激素,其特征在于,体外自组装所用缓冲液为细胞培养液、生理盐水和牛奶中的一种。The follicle-stimulating hormone according to claim 1, wherein the buffer used in the in vitro self-assembly is one of cell culture fluid, physiological saline and milk.
- 一种促卵泡激素的体外自组装方法,其特征在于,FSHα和FSHβ亚基基因引入分泌信号肽后在体外自组装。得到促卵泡激素,包括以下步骤:An in vitro self-assembly method of follicle-stimulating hormone is characterized in that, after the FSHα and FSHβ subunit genes are introduced into the secretion signal peptide, they are self-assembled in vitro. Getting follicle-stimulating hormone involves the following steps:(1)制备含有引导信号肽的FSHα亚基氨基酸序列和FSHβ亚基氨基酸序列;(1) preparing the FSHα subunit amino acid sequence and the FSHβ subunit amino acid sequence containing the guide signal peptide;(2)分开表达的FSHα和FSHβ亚基基因在重组缓冲液内体外自组装成促卵泡激素。(2) The separately expressed FSHα and FSHβ subunit genes self-assemble into follicle-stimulating hormone in vitro and in the recombination buffer.
- 根据权利要求4所述的促卵泡激素的体外自组装方法,其特征在于,步骤(1)包括如下步骤:The in vitro self-assembly method of follicle stimulating hormone according to claim 4, is characterized in that, step (1) comprises the steps:(1-1)在FSHα亚基基因的N段加入IgG Kappa链的信号肽,制备得到含有引导信号肽的FSHα亚基氨基酸序列:(1-1) Add the signal peptide of the IgG Kappa chain to the N segment of the FSHα subunit gene to prepare the FSHα subunit amino acid sequence containing the guiding signal peptide:其中, METDTLLLWVLLLWVP是IgG kappa分泌肽信号链; Wherein, METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain;(1-2)在FSHβ亚基基因的N段加入IgG Kappa链的信号肽,制备含有引导信号肽的FSHβ亚基氨基酸序列:(1-2) Add the signal peptide of the IgG Kappa chain to the N segment of the FSHβ subunit gene to prepare the FSHβ subunit amino acid sequence containing the guiding signal peptide:其中, METDTLLLWVLLLWVP是IgG kappa分泌肽信号链; Wherein, METDTLLLWVLLLWVP is the IgG kappa secretory peptide signal chain;
- 根据权利要求4所述的促卵泡激素的体外自组装方法,其特征在于,还包括如下步骤:The in vitro self-assembly method of follicle stimulating hormone according to claim 4, is characterized in that, also comprises the steps:(3)促卵泡激素活性的验证:用SNAP标记FSH受体,显微镜下读取表达株的产量。(3) Verification of follicle-stimulating hormone activity: FSH receptors were labeled with SNAP, and the yield of the expression strain was read under a microscope.
- 根据权利要求6所述的促卵泡激素的体外自组装方法,其特征在于,用SNAP标记FSH受体序列如下:The in vitro self-assembly method of follicle-stimulating hormone according to claim 6, is characterized in that, mark FSH receptor sequence with SNAP as follows:M R L C I P Q V L L A L F L S M L T G P G E G S A S D I G A P A F K S V Q T G E F T M R L C I P Q V L L A L F L S M L T G P G E G S A S D I G A P A F K S V Q T G E F T其中, M R L C I P Q V L L A L F L S M L T G P G E G S是5-HT3膜蛋白信号肽; Wherein, M R L C I P Q V L L A L F L S M L T G P G E G S is the 5-HT3 membrane protein signal peptide;是FSHR的N端部分序列。is the N-terminal partial sequence of FSHR.
- 根据权利要求4所述的促卵泡激素的体外自组装方法,其特征在于,步骤(2)所用重组缓冲液为细胞培养液、生理盐水或者牛奶。The in vitro self-assembly method of follicle stimulating hormone according to claim 4, wherein the recombination buffer used in step (2) is cell culture fluid, physiological saline or milk.
- 一种利用如权利要求4-8中任一项所述的体外自组装方法制备的促卵泡激素在药物制备中的应用。A kind of application of follicle stimulating hormone prepared by the in vitro self-assembly method according to any one of claims 4-8 in the preparation of medicine.
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