WO2021258650A1 - 一种重组类人细胞外基质结构蛋白的制备方法 - Google Patents

一种重组类人细胞外基质结构蛋白的制备方法 Download PDF

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WO2021258650A1
WO2021258650A1 PCT/CN2020/133810 CN2020133810W WO2021258650A1 WO 2021258650 A1 WO2021258650 A1 WO 2021258650A1 CN 2020133810 W CN2020133810 W CN 2020133810W WO 2021258650 A1 WO2021258650 A1 WO 2021258650A1
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fusion protein
human
seq
rhcol
rhfn
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PCT/CN2020/133810
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French (fr)
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项琪
黄亚东
程雅婷
薛来武
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广州暨南大学医药生物技术研究开发中心有限公司
广州市暨达生物科技有限公司
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Priority to US18/003,018 priority Critical patent/US20230272046A1/en
Publication of WO2021258650A1 publication Critical patent/WO2021258650A1/zh

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • 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
    • C07K14/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin or cold insoluble globulin [CIG]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence

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  • the invention belongs to the technical field of biomedicine, and specifically relates to a fusion protein.
  • Collagen is widely distributed in the skin, bones, cartilage, teeth, tendons, blood vessels and ligaments of mammals. It is a group of protein families in the connective tissue of the human body and the most abundant protein in the body, accounting for approximately the total amount of protein. Of 25% to 33%. At present, collagen has become one of the most promising biological raw materials, and can be widely used in cosmetics, medical materials and other fields.
  • Fibronectin is a high molecular weight (220-250KD) extracellular matrix glycoprotein with multiple domains that can bind to cell surface receptors, collagen, fibrin, and affinity sites of sulfated proteoglycan . Fibronectin is widely involved in the processes of cell migration, adhesion, proliferation, hemostasis and tissue repair. It mobilizes the mononuclear phagocyte system to remove harmful substances from damaged tissues and has a growth factor-like effect. As a substrate for cell culture, fibronectin can increase the adhesion rate and confluence rate of a variety of cells, shorten the cell confluence time, make the cell morphology and structure good, increase the metabolic rate, and significantly increase the speed of DNA, RNA and protein synthesis.
  • the present invention provides a recombinant human collagen-human fibronectin fusion protein, which has significantly better effects on cell adhesion and migration than fibronectin expressed alone, and is helpful for hemostasis and healing of wounds. Promote the repair and regeneration of blood vessels and nerves.
  • the fusion protein can be compounded with appropriate excipients, it can be further made into different dosage forms such as freeze-dried powders, liposomes, etc., and added to cosmetics.
  • the fusion protein can also be loaded in gel, sponge and other materials to prepare medical materials.
  • the recombinant human collagen-human fibronectin of the present invention is humanized, has low immunogenicity, and has the outstanding advantages of not being prone to allergic reactions.
  • the present invention provides the following:
  • a fusion protein obtained by fusion of human collagen and human fibronectin.
  • fusion protein according to 1 above, wherein the fusion protein includes at least one domain of human type I collagen alpha chain and at least one domain of human type III fibronectin.
  • fusion protein according to any one of the above 1-3, wherein the human type III fibronectin region (rhFN) of the fusion protein is one or more of 8-15 domains, which The amino acid sequence is SEQ ID NO. 4, SEQ ID NO. 5, or SEQ ID NO. 6.
  • fusion protein according to any one of the above 1-4, wherein the N or C terminal of the human collagen is fused to the N or C terminal of the human fibronectin.
  • fusion protein according to any one of the above 1-5, wherein the human collagen is fused with human fibronectin through a connecting peptide, and the connecting peptide preferably has a general formula (GGGS)n, where n is 1 -5.
  • the fusion protein further includes a Kex2 protease sequence, a secretion signal peptide sequence, a His tag for protein purification, or a combination thereof.
  • fusion protein according to any one of the above 1-6, wherein the amino acid sequence of the fusion protein is SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9.
  • An expression vector comprising the nucleic acid according to 8 above, preferably the expression vector is selected from pPICZ ⁇ A, pHIL, pPIC9k, and/or pPICZ ⁇ B, more preferably pPICZ ⁇ A or pHIL, most preferably pPICZ ⁇ A.
  • a host cell comprising the expression vector according to 9 above, preferably the host cell is a yeast cell, more preferably a Pichia cell, still more preferably GS115, X33 and KM71, most preferably GS115.
  • the present invention provides a preparation method and application of recombinant human collagen-human fibronectin fusion protein.
  • the method for preparing the human collagen-human fibronectin fusion protein of the present invention includes the following steps: obtaining a DNA sequence encoding the fusion protein, constructing an appropriate recombinant expression vector and expressing it in Pichia pastoris The fusion protein.
  • the present invention also relates to the amino acid sequence constituting the human collagen-human fibronectin fusion protein of the present invention, the nucleotide sequence encoding the human collagen-human fibronectin fusion protein of the present invention, An expression vector for expressing the human collagen-human fibronectin fusion protein of the present invention, a host strain expressing the human collagen-human fibronectin fusion protein of the present invention, and purification of the human collagen-human fibronectin fusion protein of the present invention .
  • the present invention provides a human collagen (rhCol), which includes the following amino acid sequence: SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3, the human collagen of the present invention Protein is the functional domain structure of type I collagen alpha chain. Truncated collagen can be expressed in a large amount in the Pichia pastoris expression system, and has little effect on its activity. It solves the problem of difficult expression and low expression of full-length collagen. .
  • the present invention provides a human fibronectin (rhFN), which includes the following amino acid sequence: SEQ ID No. 4, SEQ ID No. 5, or SEQ ID No. 6, which is described in the present invention.
  • Human fibronectin is the functional domain structure of fibronectin III. Full-length fibronectin is difficult to secretely express in Pichia pastoris cells.
  • the sequence of the present invention can not only be secreted and expressed in large quantities in yeast, but also has its activity. It has little effect and solves the problem of difficulty in expressing full-length fibronectin and low expression.
  • the present invention provides a connecting peptide.
  • the fusion protein sequence of the present invention also includes a Kex2 protease sequence, a secretion signal peptide sequence, a His tag for protein purification, or a combination thereof.
  • amino acid sequence of the fusion protein of the present invention is as follows: SEQ ID NO. 7, SEQ ID NO. 8, or SEQ ID NO. 9.
  • the fusion protein and expression vector of the present invention includes the nucleic acid according to any one of 7 above, preferably the expression vector is pPICZ ⁇ A, and the recombinant expression plasmid pPICZ ⁇ A-rhCol-rhFN is constructed.
  • the expression strain of the fusion protein of the present invention is selected from GS115, X33 and KM71, preferably GS115.
  • Figure 1 Recombinant expression plasmid map: A: pPICZ ⁇ A-rhCol-rhFN; B: pPICZ ⁇ A-rhCol; C: pPICZ ⁇ A-rhFN;
  • Target fragment rhCol-rhFN amplification picture (M is DL10000 DNA marker; Lane 1 represents the target fragment rhCol-rhFN, about 1149bp; Lane 2 is the target fragment rhFN, about 378bp; Lane 3 is the target fragment rhCol, about 774bp) ;
  • Figure 3 Induced expression, purification and Western blot identification of proteins rhCol-rhFN, rhFN and rhCol (A: rhCol-rhFN protein induced expression diagram; B: rhFN protein induced expression diagram; C: rhCol protein induced expression diagram; Lane 1: Uninduced samples; lanes 2-4 are samples after induction for 24h, 48h, and 72h respectively; D and d: SDS-PAGE and Western blot of purified rhCol-rhFN protein; E and e: SDS-PAGE and Western blot of purified rhFN protein Western blot; F and f: SDS-PAGE and Western blot of purified rhCol protein);
  • FIG. 6 Local lymph node test of the fusion protein rhCol-rhFN (A: Lymphocyte ATP content in mouse lymph nodes. ###Compared with control p ⁇ 0.001; *Compared with rhCol-rhFN p ⁇ 0.05; B: Mouse Ear weight.####Compared with control p ⁇ 0.0001; *Compared with rhCol-rhFN p ⁇ 0.05).
  • the present invention provides a method for efficiently expressing human collagen-human fibronectin rhCol-rhFN fusion protein in Pichia pastoris.
  • the method includes the following steps:
  • Protein purification the fermentation broth is centrifuged and the supernatant is collected, and the affinity chromatography column is first equilibrated with the balance solution, and the recombinant protein carrying His-tag is separated and purified by the nickel column affinity chromatography method.
  • SDS-PAGE protein glue Figure 3, D, E and F
  • Western blotting Figure 3, d, e and f
  • the purified protein sample is desalted through a G25 column to obtain a high-purity fusion protein.
  • the present invention clones human collagen-human fibronectin gene, human collagen gene, and human fibronectin gene respectively into Pichia pastoris expression vector pPICZ ⁇ A through gene cloning technology to obtain recombinant expression vector pPICZ ⁇ A-rhCol- rhFN, pPICZ ⁇ A-rhCol, pPICZ ⁇ A-rhFN.
  • Yeast competent cells were transfected by electrotransfection method to obtain recombinant expression strains. Methanol was used to induce expression of the recombinant strain, and the purified fusion protein was obtained by nickel affinity chromatography. The molecular weight and immunological verification of the recombinant protein were verified by SDS-PAGE and Western blot.
  • the present invention provides a high-efficiency expression of human collagen-human fibronectin rhCol-rhFN fusion protein, human collagen rhCol, and human fibronectin rhFN in Pichia pastoris, and detecting the biological activity thereof.
  • the method includes the following step
  • HF-MSC cells ATCC No: CM-1252 were cultured in DMEM/F12 containing 10% FBS, 37°C, and a CO 2 concentration of 5%; firstly, they were washed once with PBS, and then 0.25% Trypsin was added.
  • PBS solution is used as a negative control: the results show that the three proteins of the fusion protein rhCol-rhFN, recombinant rhCol protein, and recombinant rhFN protein provided by the present invention can promote the attachment of HF-MSC (ATCC No: CM-1252) cells.
  • the cell growth state of the fusion protein rhCol-rhFN under the microscope can not only promote cell adhesion, but also promote cell extension.
  • the cell adhesion performance is significantly higher than that of recombinant protein rhCol and recombinant protein rhFN. See Figure 4 for the results.
  • HF-MSC cells are trypsinized and then connected to a 12-well plate, and the number is appropriate to cover the bottom of the plate after adhering to the wall. After the cells have overgrown the bottom of the plate, use a 100 ⁇ l pipette tip perpendicular to the well plate to make cell scratches at the same position along the back of the plate to ensure that the width of each scratch is the same. Aspirate the cell culture medium and rinse the well plate three times with PBS to wash away the cell debris generated by the scratch.
  • a medium (serum concentration of 1%) containing rhCol-rhFN fusion protein, human collagen rhCol, and human fibronectin rhFN (both at a concentration of 1 ⁇ mol/L) was added, and photographed and recorded. Put the culture plate into the incubator for 12h and 24h decibels and take pictures. The result is shown in Figure 5. The healing rate of rhCol-rhFN group was significantly different at 12h and 24h.
  • the present invention expresses human fibronectin rhFN, human collagen rhCol, and human collagen-human fibronectin fusion protein rhCol-rhFN through genetic engineering methods.
  • the in vitro cell experiment results show that the fusion of human Collagen-human fibronectin rhCol-rhFN has a significant effect on cell adhesion promotion and wound repair, and the effect is significantly better than that of human fibronectin rhFN and human collagen rhCol expressed separately.
  • the present invention provides a high-efficiency expression of human collagen-human fibronectin rhCol-rhFN fusion protein in Pichia pastoris to detect skin allergies.
  • the method includes the following steps:
  • mice purchased from Guangdong Medical Experimental Animal Center, animal production license number: SCXK (Guangdong) 2019-0035), 18 ⁇ 22g, half male and half male, on day 1, animals are grouped, labeled, and weighed , Record clinical symptoms.
  • SLS sodium lauryl sulfate
  • the supernatant is centrifuged to obtain the sample to be tested, and the sample to be tested is subjected to chemiluminescence analysis on the computer to obtain the CPS value (the number of fluorescent photons received per second).
  • ATP determination kit purchased from Biyuntian Biological Co., Ltd.
  • RLU relative luminescence units
  • the present invention expresses human fibronectin rhFN, human collagen rhCol, and human collagen-human fibronectin fusion protein rhCol-rhFN through genetic engineering methods.
  • the results of the mouse local lymph node test show that the fusion
  • the human collagen-human fibronectin rhCol-rhFN has a lower sensitization ability, and the sensitization ability is significantly lower than that of animal collagen.

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Abstract

属于基因工程领域，更具体地公开了一种表达人胶原蛋白与人纤连蛋白的融合蛋白的基因，通过基因工程的方法在体外进行表达纯化。利用毕赤酵母作为表达宿主细胞，可以提供一种新型的、具有高活性的重组融合蛋白。

Description

一种重组类人细胞外基质结构蛋白的制备方法 技术领域
本发明属于生物医药技术领域，具体涉及一种融合蛋白。
背景技术
胶原蛋白(collagen)广泛分布于哺乳动物的皮肤、骨骼、软骨、牙齿、肌腱、血管和韧带中，是人体结缔组织的一组蛋白质家族，也是体内含量最多的一种蛋白质，约占蛋白质总量的25％～33％。目前胶原蛋白已成为最具有发展前景的生物原材料之一，可广泛应用于化妆品、医疗材料等领域。
纤连蛋白(Fibronectin)，是一种高分子量(220-250KD)胞外基质的糖蛋白，具有多个结构域，可与细胞表面受体、胶原、纤维蛋白以及硫酸蛋白多糖的亲和部位结合。纤连蛋白广泛参与细胞迁移、粘附、增殖、止血及组织修复等过程，调动单核吞噬细胞系统清除损伤组织处有害物质，具有生长因子样作用。纤连蛋白作为细胞培养的基质，可提高多种细胞的贴壁率、汇合率，缩短细胞汇合时间，使细胞形态结构良好，代谢率增强，DNA、RNA及蛋白质合成速度显著提高。
发明内容
本发明提供一种重组人胶原蛋白-人纤连蛋白的融合蛋白，其对细胞的粘附、迁移等作用明显优于单独表达的纤连蛋白，且有助于创口的止血、愈合，同时可以促进血管和神经的修复再生。可以将融合蛋白与适当的辅料复配后，可进一步制成冻干粉、脂质体等不同的剂型，添加在化妆品中。也可以将融合蛋白负载在凝胶、海绵等材料中制备医疗材料。本发明的重组人胶原蛋白-人纤连蛋白具有人源化，免疫原性低，具有不易发生过敏反应的突出优点。
因此，本发明提供以下各项：
1.一种融合蛋白，其由人胶原蛋白与人纤连蛋白融合获得。
2.根据以上1所述的融合蛋白，其中所述融合蛋白包括人I型胶原蛋白α链的至少一个结构域和人Ⅲ型纤连蛋白的至少一个结构域。
3.根据以上1或2所述的融合蛋白，其中所述融合蛋白的人胶原蛋白区域 (rhCol)为10～50个氨基酸重复形成的短肽，其重复次数为10～20次，其氨基酸序列为SEQ ID NO.1、SEQ ID NO.2、或SEQ ID NO.3)。
4.根据以上1-3中任一项所述的融合蛋白，其中所述融合蛋白的人Ⅲ型纤连蛋白区域(rhFN)为8～15个结构域中的一个或多个结构域，其氨基酸序列为SEQ ID NO.4、SEQ ID NO.5、或SEQ ID NO.6。
5.根据以上1-4中任一项所述的融合蛋白，其中所述人胶原蛋白的N或C端与所述人纤连蛋白的N或C端融合。
6.根据以上1-5中任一项所述的融合蛋白，其中所述人胶原蛋白通过连接肽与人纤连蛋白融合，所述连接肽优选通式是(GGGS)n，其中n是1-5的整数，优选地所述融合蛋白还包括Kex2蛋白酶序列、分泌信号肽序列、用于蛋白纯化的His标签、或它们的组合。
7.根据以上1-6中任一项所述的融合蛋白，其中所述融合蛋白的氨基酸序列为SEQ ID NO.7、SEQ ID NO.8、或SEQ ID NO.9。
8.编码根据以上1-7中任一项所述的融合蛋白的核酸。
9.表达载体，其包括根据以上8所述的核酸，优选所述表达载体选自pPICZαA、pHIL、pPIC9k、和/或pPICZαB，更优选pPICZαA或pHIL，最优选pPICZαA。
10.包含根据以上9所述的表达载体的宿主细胞，优选所述宿主细胞为酵母细胞，更优选毕赤酵母细胞，还更优选GS115、X33和KM71，最优选GS115。
更具体地，本发明提供了一种重组人胶原蛋白-人纤连蛋白融合蛋白的制备方法与应用。
1、具体而言，本发明的人胶原蛋白-人纤连蛋白融合蛋白的制备方法包括下述步骤：获得编码所述融合蛋白的DNA序列，构建适当的重组表达载体于毕赤酵母中表达所述融合蛋白。
2、具体而言：本发明还涉及到构成本发明的人胶原蛋白-人纤连蛋白的融合蛋白的氨基酸序列，编码本发明人胶原蛋白-人纤连蛋白的融合蛋白的核苷酸序列，表达本发明人胶原蛋白-人纤连蛋白的融合蛋白的表达载体，表达本发明人胶原蛋白-人纤连蛋白的融合蛋白的宿主菌株，纯化本发明人胶原蛋白-人纤连蛋白的融合蛋白。
3、具体而言，本发明提供了一种人胶原蛋白(rhCol)，其包括如下氨基酸序 列：SEQ ID NO.1、SEQ ID NO.2或SEQ ID NO.3，本发明所述的人胶原蛋白为I型胶原蛋白α链的功能域结构，截断的胶原蛋白可以在毕赤酵母表达体系中大量表达，而且对其活性影响不大，解决了全长胶原蛋白表达困难且表达量低的问题。
4、具体而言，本发明提供了一种人纤连蛋白(rhFN)，其包括如下氨基酸序列：SEQ ID NO.4、SEQ ID NO.5、或SEQ ID NO.6，本发明所述的人纤连蛋白为纤连蛋白Ⅲ的功能域结构，全长的纤连蛋白很难在毕赤酵母细胞中分泌表达，本发明所述的序列不仅可以在酵母中大量分泌表达，而且对其活性影响不大，解决了全长纤连蛋白表达困难且表达量低的问题。
5、具体而言，本发明提供了一种连接肽，连接肽的通式是(GGGS)n，其中n＝1～5的整数，优选n＝3。
6、具体而言，本发明所述的融合蛋白序列，还包括Kex2蛋白酶序列、分泌信号肽序列、用于蛋白纯化的His标签、或它们的组合。
7、具体而言，本发明所述的融合蛋白，其氨基酸序列如下：SEQ ID NO.7、SEQ ID NO.8、或SEQ ID NO.9。
8、具体而言，本发明所述的融合蛋白，表达载体，其包括根据以上7中任一项所述的核酸，优选所述表达载体为pPICZαA，构建重组表达质粒pPICZαA-rhCol-rhFN。
9、具体而已，本发明所述的融合蛋白，表达菌株选自GS115、X33和KM71，优选GS115。
10、表达根据权利要求1-7所述融合蛋白在于组织工程、药学或美容护肤领域的活性添加剂。
附图说明
图1.重组表达质粒图谱：A:pPICZαA-rhCol-rhFN；B:pPICZαA-rhCol；C:pPICZαA-rhFN；
图2.目的片段rhCol-rhFN扩增图片(M为DL10000 DNA marker；泳道1表示目的片段rhCol-rhFN，约1149bp；泳道2为目的片段rhFN，约378bp；泳道3为目的片段rhCol，约774bp)；
图3.蛋白rhCol-rhFN、rhFN与rhCol的诱导表达、纯化和Western blot鉴定(A: rhCol-rhFN蛋白诱导表达图；B：rhFN蛋白诱导表达图；C：rhCol蛋白诱导表达图；泳道1：未诱导样品；泳道2-4分别为诱导24h、48h、72h后样品；D与d：纯化后rhCol-rhFN蛋白的SDS-PAGE与Western blot；E与e：纯化后rhFN蛋白的SDS-PAGE与Western blot；F与f：纯化后rhCol蛋白的SDS-PAGE与Western blot)；
图4.融合蛋白rhCol-rhFN对细胞的促粘附作用(A：结晶紫染色观察细胞形态；B：细胞附着及伸展的数量统计。**与对照相比p＜0.01；ns：没有显著性差异)；
图5.细胞划痕愈合实验示意图；
图6.融合蛋白rhCol-rhFN的局部淋巴结试验(A：小鼠淋巴结内淋巴细胞ATP含量。###与对照相比p<0.001；*与rhCol-rhFN相比p<0.05；B：小鼠耳廓重量。####与对照相比p<0.0001；*与rhCol-rhFN相比p<0.05)。
本发明涉及的序列
Figure PCTCN2020133810-appb-000001
Figure PCTCN2020133810-appb-000002
Figure PCTCN2020133810-appb-000003
Figure PCTCN2020133810-appb-000004
Figure PCTCN2020133810-appb-000005
具体实施方式
本发明提供了一种在毕赤酵母中高效表达人胶原蛋白-人纤连蛋白rhCol-rhFN融合蛋白的方法，所述方法包括如下步骤：
1.构建重组表达融合蛋白载体pPICZαA-rhCol-rhFN：人工合成目的片段人胶原蛋白(rhCol)、人纤连蛋白(rhFN)、人胶原蛋白-人纤连蛋白(rhCol-rhFN)(上海捷瑞生物公司)，人胶原蛋白(rhCol)核苷酸序列(SEQ ID NO.10)两端加有EcoR1和Not1酶切位点、人纤连蛋白(rhFN)核苷酸序列(SEQ ID NO.11)两端加有Xho1和Not1酶切位点、人胶原蛋白-人纤连蛋白融合蛋白rhCol-rhFN核苷酸序列(SEQ ID NO.12)的两端加上限制性酶切位点Xho1和Xba1，分别单独利用EcoR1和Not1、Xho1和Not1、Xho1和Xba1切割表达质粒pPICZαA(BioVector质粒载体菌种细胞基因保藏中心)并进行回收，利用T4连接酶对酶切后相对应的目的片段和质粒进行连接，构建重组表达质粒pPICZαA-rhCol、pPICZαA-rhFN、pPICZαA-rhCol-rhFN。质粒图谱参见图1。
2、重组质粒转染毕赤酵母GS115菌株：将经Sal1内切酶线性化的重组质粒pPICZαA-rhCol-rhFN(10μg)、Sac1内切酶线性化的重组质粒pPICZαA-rhCol(10μg)、 Sal1内切酶线性化的重组质粒pPICZαA-rhFN(10μg)分别与80μL毕赤酵母GS115(购于生物风网站)感受态细胞混匀，混合液转入0.2cm预冷的电转杯中，电击4-10毫秒，加入1mL冰预冷的1mol/L的山梨醇溶液并混匀液体，涂布在YPD培养基平板上(含有1％酵母提取物、2％蛋白胨、2％琼脂粉以及Zeocin 100μg/ml)，30℃倒置培养2天，待平板上长出单菌落。
3、重组表达菌的筛选：以YPD平板上的克隆子为模板，加入两端引物rhCol-rhFN-F(SEQ ID NO.13)和rhCol-rhFN-R(SEQ ID NO.14)、rhCol-F(SEQ ID NO.15)和rhCol-R(SEQ ID NO.16)、rhFN-F(SEQ ID NO.17)和rhFN-R(SEQ ID NO.18)(引物由华大基因合成)(引物浓度10μM)各0.5μL，再加入Premix Taq TM(TaKaRa Taq TM Version 2.0plus dye)预混酶，加水至总体积为20μL，按照PCR反应条件(95℃变性3min后进入循环，循环参数第一步为95℃变性60秒，60℃退火30秒，72℃延伸120秒，30个循环，72℃延伸10min)进行目的片段的扩增。核酸电泳检测目的片段大小。结果如图2所示，获得了目标片段。
4、融合蛋白rhCol-rhFN、重组蛋白rhCol、重组蛋白rhFN的表达：将筛选到的阳性转化菌株接种于10mL的YPD培养基中，230rpm、30℃，培养18-20h。按1％接种量吸取培养液于25mL的YPG培养基中，230rpm、30℃，培养18-20h至OD600＝1.0，而后将培养液3000rpm离心5min收集菌体，用25ml的YPM重悬菌体并置于250mL的三角瓶中，230rpm、30℃，培养72h，培养期间每隔24h添加终浓度为1％的甲醇进行诱导表达
5、蛋白的纯化：将发酵液经离心后收集上清，先用平衡液对亲和层析柱进行平衡，通过镍柱亲和层析方法，分离纯化携带His-tag的重组蛋白。通过SDS-PAGE蛋白胶(图3，D、E和F)以及Western blotting(图3，d、e和f)对重组蛋白的分子量大小和免疫学验证(抗体为6X-His tag多克隆抗体，购自赛默飞生物世尔科技)。将纯化后的蛋白样经过G25柱进行脱盐处理，得到高纯度的融合蛋白。
综上所述：本发明通过基因克隆技术将人胶原蛋白-人纤连蛋白基因、人胶原蛋白基因、人纤连蛋白基因分别克隆到毕赤酵母表达载体pPICZαA，获得重组表达载体pPICZαA-rhCol-rhFN、pPICZαA-rhCol、pPICZαA-rhFN。通过电转方法转染酵母感受态细胞，获得重组表达菌株。利用甲醇对重组菌株进行诱导表达，并通过镍亲和层析得到纯化的融合蛋白，通过SDS-PAGE和Western blot对重组蛋白的分子量 大小和免疫学验证。
本发明提供了一种在毕赤酵母中高效表达人胶原蛋白-人纤连蛋白rhCol-rhFN融合蛋白、人胶原蛋白rhCol、人纤连蛋白rhFN，对其生物活性进行检测，所述方法包括如下步骤
细胞粘附性测定：将HF-MSC细胞(ATCC No:CM-1252)用含10％FBS的DMEM/F12培养，37℃，CO 2浓度5％；首先用PBS清洗一次，然后添加0.25％Trypsin-EDTA胰酶液进行消化，离心收集细胞；用DMEM/F12进行重悬，细胞密度控制在6×10 4个/mL，将细胞悬液(细胞密度控制在1.5×10 4个/mL)分别接种到底层铺有rhCol-rhFN蛋白、rhCol蛋白、rhFN蛋白(各蛋白浓度均为1μmol/L)膜低粘附96孔板中。37℃培养5h，维持CO 2浓度为5％；用PBS冲洗掉没有贴壁的细胞；在相差显微镜下计数以及用MTT法比较各组细胞数。PBS溶液作为阴性对照：结果显示：本发明提供的融合蛋白rhCol-rhFN、重组rhCol蛋白、重组rhFN蛋白三种蛋白均可促进HF-MSC(ATCC No:CM-1252)细胞贴壁，贴壁后的细胞生长状态良好，显微镜下观察融合蛋白rhCol-rhFN不仅可以促进细胞的贴壁，还可以明显促进细胞延伸，细胞粘附性能显著高于重组蛋白rhCol和重组蛋白rhFN。结果参见图4。
细胞划痕愈合能力的检测：HF-MSC细胞用胰蛋白酶消化后接入12孔板，数量以贴壁后铺满板底为宜。细胞长满板底后，用100μl枪头垂直于孔板，沿板背面划线的相同位置制造细胞划痕，尽量保证各个划痕宽度一致。吸去细胞培养液，用PBS冲洗孔板三次，洗去划痕产生的细胞碎片。加入含rhCol-rhFN融合蛋白、人胶原蛋白rhCol、人纤连蛋白rhFN(浓度均为1μmol/L)的培养基(血清浓度为1％)，拍照记录。将培养板放入培养箱培养12h、24h分贝取出拍照，结果如图5。rhCol-rhFN组的愈合率在12h与24h均有显著性差异。
综上所述：本发明通过基因工程的方法分别表达了人纤连蛋白rhFN、人胶原蛋白rhCol以及人胶原蛋白-人纤连蛋白的融合蛋白rhCol-rhFN，体外细胞实验结果显示融合后的人胶原蛋白-人纤连蛋白rhCol-rhFN对细胞的促粘附作用以及创伤修复有很明显作用，且作用效果明显优于单独表达的人纤连蛋白rhFN与人胶原蛋白rhCol。
本发明提供了一种在毕赤酵母中高效表达人胶原蛋白-人纤连蛋白rhCol-rhFN 融合蛋白，对其皮肤变态反应进行检测，所述方法包括如下步骤：
选用32只KM小鼠(购于广东省医学实验动物中心，动物生产许可证号：SCXK(粤)2019-0035)，18～22g，雌雄各半，第1天，动物分组、标记、称重、记录临床症状。将1％十二烷基硫酸钠(SLS)均匀涂抹到小鼠双侧耳朵背部皮肤。刷子或棉签应浸泡在SDS溶液中，并在小鼠每个耳朵背部重复涂抹4～5次。1h后再分别涂抹rhCol-rhFN融合蛋白、rhCol、rhFN、小分子动物胶原(购于山东隆贝生物科技有限公司，目录号SC13137078101884)、大分子动物胶原(购于西亚化学科技(山东)有限公司，目录号A15847)或阳性对照(0.01％SLS溶液)25μL/耳。第2、3、7天操作同第1天，重复0.01％SLS溶液预处理和rhCol-rhFN融合蛋白、rhCol、rhFN、小分子动物胶原、大分子动物胶原涂抹。第4～6天不做任何处理。第8天记录小鼠体重和出现的任何临床症状，第7天0.01％SLS溶液和rhCol-rhFN融合蛋白、rhCol、rhFN、小分子动物胶原、大分子动物胶原涂抹后约24～30h人道处死动物，摘取小鼠双侧颌下淋巴结，同时分离耳廓打孔(直径：6mm)称重。将取出的淋巴结至于裂解液内剪碎，冰上裂解。裂解完成后离心取上清液得到待测样品，待测样品上机进行化学发光分析，得出CPS值(每秒接收的荧光光子数量)。利用ATP测定试剂盒(购于碧云天生物有限公司)测定淋巴结中ATP含量，以相对发光单位(RLU)表示生物发光。每只动物从处死到ATP含量测定时间保持一致，约30min内。每只动物从分离颌下淋巴结到ATP测定系列操作务必在20min内完成，且每只动物处理时间保持一致。
结果如图6所示，在观察期内受试小鼠未出现死亡或异常，与动物胶原相比，重组人胶原蛋白-人纤连蛋白rhCol-rhFN融合蛋白具有更低的致敏能力。
综上所述：本发明通过基因工程的方法分别表达了人纤连蛋白rhFN、人胶原蛋白rhCol以及人胶原蛋白-人纤连蛋白的融合蛋白rhCol-rhFN，小鼠局部淋巴结试验结果显示融合后的人胶原蛋白-人纤连蛋白rhCol-rhFN具有较低的致敏能力，且致敏能力明显低于动物胶原。
本领域技术人员应该理解，尽管参照上述实施例对本发明进行了具体的描述，但是本发明并不限于这些具体的实施例。基于本发明所教导的方法和技术方案，在不背离本发明的精神的前提下，本领域技术人员能够进行适当的修改或改进，由此 所得的等价实施方案都在本发明的范围内。

Claims (10)

  1. 一种融合蛋白，其由人胶原蛋白与人纤连蛋白融合获得。
  2. 根据权利要求1所述的融合蛋白，其中所述融合蛋白包括人I型胶原蛋白α链的至少一个结构域和人Ⅲ型纤连蛋白的至少一个结构域。
  3. 根据权利要求1或2所述的融合蛋白，其中所述融合蛋白的人胶原蛋白区域(rhCol)为10～50个氨基酸重复形成的短肽，其重复次数为10～20次，其氨基酸序列为SEQ ID NO.1、SEQ ID NO.2、或SEQ ID NO.3。
  4. 根据权利要求1-3中任一项所述的融合蛋白，其中所述融合蛋白的人Ⅲ型纤连蛋白区域(rhFN)为8～15个结构域中的一个或多个结构域，其氨基酸序列为SEQ ID NO.4、SEQ ID NO.5、或SEQ ID NO.6。
  5. 根据权利要求1-4中任一项所述的融合蛋白，其中所述人胶原蛋白的N或C端与所述人纤连蛋白的N或C端融合。
  6. 根据权利要求1-5中任一项所述的融合蛋白，其中所述人胶原蛋白通过连接肽与人纤连蛋白融合，所述连接肽优选通式是(GGGS)n，其中n是1-5的整数，优选地所述融合蛋白还包括Kex2蛋白酶序列、分泌信号肽序列、用于蛋白纯化的His标签、或它们的组合。
  7. 根据权利要求1-6中任一项所述的融合蛋白，其中所述融合蛋白的氨基酸序列为SEQ ID NO.7、SEQ ID NO.8、或SEQ ID NO.9。
  8. 编码根据权利要求1-7中任一项所述的融合蛋白的核酸。
  9. 表达载体，其包括根据权利要求8所述的核酸，优选所述表达载体选自pPICZαA、pHIL、pPIC9k、和/或pPICZαB，更优选pPICZαA或pHIL，最优选pPICZαA。
  10. 包含根据权利要求9所述的表达载体的宿主细胞，优选所述宿主细胞为酵母细胞，更优选毕赤酵母细胞，还更优选GS115、X33和KM71，最优选GS115。
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