JP6906669B1 - Recombinant fibronectin mutant, its preparation method and its use - Google Patents
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Abstract
【課題】精製過程が簡単であり、細胞増殖活性、接着活性を効果的に促進できる組換えフィブロネクチン変異体及びその使用を提供する。【解決手段】ヌクレオチド配列はある特定の塩基配列、または別のある特定の塩基配列に示す通りである、ことを特徴とする組換えフィブロネクチン変異体。前記ヌクレオチド配列がコードするアミノ酸配列は、他のある特定のアミノ酸配列に示す通りであってよい。該組換えフィブロネクチン変異体は、可溶性の形態で発現されてよい。【選択図】図４PROBLEM TO BE SOLVED: To provide a recombinant fibronectin mutant which has a simple purification process and can effectively promote cell proliferation activity and adhesive activity, and its use. A recombinant fibronectin variant, characterized in that the nucleotide sequence is as shown in one particular base sequence or another particular base sequence. The amino acid sequence encoded by the nucleotide sequence may be as shown in certain other specific amino acid sequences. The recombinant fibronectin variant may be expressed in soluble form. [Selection diagram] Fig. 4
Description
本発明は生物学の分野に属し、具体的には、組換えフィブロネクチン変異体及びその使用に関する。 The present invention belongs to the field of biology and specifically relates to recombinant fibronectin variants and their use.
フィブロネクチン（Ｆｉｂｒｏｎｅｃｔｉｎ，ＦＮ）は、細胞外マトリックスにおける高分子糖タンパク質であり、血漿、さまざまな細胞表面及び細胞マトリックスに広く存在しており、細胞外マトリックスの重要な接着分子の１つであり、フィブロネクチンは、細胞膜上のインテグリン受容体に結合し、細胞間、細胞とマトリックス間の相互作用に非常に重要な機能を果たし、細胞の接着、移動、及び増殖などの調節過程に重要な役割を果たし、創傷の修復と癒合に重要な役割を果たす（Ｋｌｅｉｎ，Ｒ．Ｍ．，ｅｔ ａｌ．（２００３）．“Ｓｔｉｍｕｌａｔｉｏｎ ｏｆ ｅｘｔｒａｃｅｌｌｕｌａｒ ｍａｔｒｉｘ ｒｅｍｏｄｅｌｉｎｇ ｂｙ ｔｈｅ ｆｉｒｓｔ ｔｙｐｅ ＩＩＩ ｒｅｐｅａｔ ｉｎ ｆｉｂｒｏｎｅｃｔｉｎ．”Ｊ Ｃｅｌｌ Ｓｃｉ １１６（Ｐｔ ２２）：４６６３−４６７４．）。 Fibronectin (FN) is a high molecular weight glycoprotein in extracellular matrix, widely present in plasma, various cell surfaces and cell matrix, and one of the important adhesion molecules of extracellular matrix, fibronectin. Binds to the integrin receptor on the cell membrane, plays a very important role in cell-cell and cell-matrix interactions, and plays an important role in regulatory processes such as cell adhesion, migration, and proliferation. It plays an important role in wound repair and healing (Klein, RM, et al. (2003). "Stimulation of extracellular matrix remodeling by the first type III repeat in fibronectin." : 4663-4674.).
フィブロネクチンは、創面修復における重要なタンパク質である。研究により、創傷修復は、修復に関与する細胞と細胞外マトリックスの間の相互作用によって共に完了することがわかった。フィブロネクチンは細胞外マトリックスの重要な構成要素であり、分子間又は細胞間の接着を媒介することによって修復に関与でき、細胞分化を固定し、細胞の動きをガイドすることに役立ち、創傷修復の過程にも重要な調節的役割を果たす。したがって、フィブロネクチンは、臨床的に、主に創傷修復、火傷、角膜修復、歯周修復などに適用される（Ｋｕｂｏｗ，Ｋ．Ｅ．，ｅｔ ａｌ．（２０１５）．“Ｍｅｃｈａｎｉｃａｌ ｆｏｒｃｅｓ ｒｅｇｕｌａｔｅ ｔｈｅ ｉｎｔｅｒａｃｔｉｏｎｓ ｏｆ ｆｉｂｒｏｎｅｃｔｉｎ ａｎｄ ｃｏｌｌａｇｅｎ Ｉ ｉｎ ｅｘｔｒａｃｅｌｌｕｌａｒ ｍａｔｒｉｘ．”Ｎａｔｕｒｅ ｃｏｍｍｕｎｉｃａｔｉｏｎｓ ６：８０２６．）。 Fibronectin is an important protein in wound repair. Studies have shown that wound repair is completed together by the interaction between the cells involved in the repair and the extracellular matrix. Fibronectin is an important component of the extracellular matrix and can be involved in repair by mediating intermolecular or cell-cell adhesion, helping to fix cell differentiation and guide cell movement, and the process of wound repair. Also plays an important regulatory role. Therefore, fibronectin is clinically applied mainly to wound repair, burns, corneal repair, periodontal repair, etc. (Kubow, KE, et al. (2015). and collagen I in extracellular matrix. "Nature communications 6: 8026.).
フィブロネクチンは、多機能、高活性、純粋天然の生物学的タンパク質であり、分子量が４５０ＫＤである。フィブロネクチンは、６つの機能性領域と１つのＲＧＤ配列をそれぞれ有する２つの類似なサブユニットを有する。その６つの機能性領域は、それぞれ特異的配位子に結合できる。アミノ末端から第１の領域はヘパリン、フィブリル、アクチン、細菌及び血液凝固因子ＸＩＩＩａに結合可能であり、第２の領域はコラーゲン及びゼラチンに結合可能であり、第３の領域はフィブリノーゲンに結合可能であり、第４の領域は細菌に結合可能であり、第５の領域はヘパリンに結合可能であり、第６の領域はフィブリルに結合可能である。そのＲＧＤ配列は１１種類のインテグリンに結合し、さらに強力な生物学的機能を果たすことができる（Ｘｕ，Ｊ．ａｎｄ Ｄ．Ｍｏｓｈｅｒ（２０１１）．Ｆｉｂｒｏｎｅｃｔｉｎ ａｎｄ ｏｔｈｅｒ ａｄｈｅｓｉｖｅ ｇｌｙｃｏｐｒｏｔｅｉｎｓ．Ｔｈｅ ｅｘｔｒａｃｅｌｌｕｌａｒ ｍａｔｒｉｘ： ａｎ ｏｖｅｒｖｉｅｗ，Ｓｐｒｉｎｇｅｒ：４１−７５．）。フィブロネクチンは複数の機能性領域を有し、各機能性領域は細胞表面の受容体に結合することで、対応する生物学的機能を果たす。研究によると、フィブロネクチンの第３の領域は細胞接着に非常に重要であり、組換え発現されたフィブロネクチンフラグメントは細胞接着を著しく促進できる。ＦＮは細胞表面のα４β７受容体に結合することで、線維芽細胞の分化を促進し、ＦＮにおけるＥＤＡドメインは重要な役割を果たし、また、ＥＤＡドメインを含むＦＮはＴＧＦ−β１の発現を促進できる。フィブロネクチンＦＮ又はＥＤＡドメインを含むフィブロネクチンは、Ｅｒｋ１／２シグナル伝達経路を活性化できる（Ｋｏｈａｎ，Ｍ．，ｅｔ ａｌ．（２０１０）．“ＥＤＡ−ｃｏｎｔａｉｎｉｎｇ ｃｅｌｌｕｌａｒ ｆｉｂｒｏｎｅｃｔｉｎ ｉｎｄｕｃｅｓ ｆｉｂｒｏｂｌａｓｔ ｄｉｆｆｅｒｅｎｔｉａｔｉｏｎ ｔｈｒｏｕｇｈ ｂｉｎｄｉｎｇ ｔｏ α４β７ ｉｎｔｅｇｒｉｎ ｒｅｃｅｐｔｏｒ ａｎｄ ＭＡＰＫ／Ｅｒｋ１／２−ｄｅｐｅｎｄｅｎｔ ｓｉｇｎａｌｉｎｇ．”Ｔｈｅ ＦＡＳＥＢ Ｊｏｕｒｎａｌ ２４（１１）：４５０３−４５１２．）。 Fibronectin is a multifunctional, highly active, purely natural biological protein with a molecular weight of 450 KD. Fibronectin has two similar subunits, each with six functional regions and one RGD sequence. Each of the six functional regions can be attached to a specific ligand. The first region from the amino terminus can bind to heparin, fibril, actin, bacteria and blood coagulation factor XIIIa, the second region can bind to collagen and gelatin, and the third region can bind to fibrinogen. Yes, the fourth region is capable of binding to bacteria, the fifth region is capable of binding to heparin, and the sixth region is capable of binding to fibril. The RGD sequence can bind to 11 types of integrins and perform more potent biological functions (Xu, J. and D. Mosher (2011). Fibronectin and more advanced glycoproteins. The extracellular matrix: Springer: 41-75.). Fibronectin has a plurality of functional regions, and each functional region fulfills a corresponding biological function by binding to a receptor on the cell surface. Studies have shown that a third region of fibronectin is very important for cell adhesion, and recombinantly expressed fibronectin fragments can significantly promote cell adhesion. FN promotes fibroblast differentiation by binding to the α4β7 receptor on the cell surface, the EDA domain in FN plays an important role, and FN containing the EDA domain can promote the expression of TGF-β1. .. Fibronectin FN or fibronectin containing the EDA domain can activate the Erk1 / 2 signaling pathway (Kohan, M., et al. (2010). "EDA-connecting cellular fibronectin induces fibroblast mitogen-activated protein / Erk1 / 2-dependent signaling. "The FASEB Journal 24 (11): 4503-4512.).
組換えフィブロネクチンフラグメントは、その複数の種類の生物学的機能により、現在フィブロネクチン研究に注目されるポイントの一つであり、また、組換えフィブロネクチンフラグメントは安定し、調製しやすいため、より効果的に宣伝及び使用できる。フィブロネクチンは、創傷の癒合に非常に重要な役割を果たし、フィブロネクチンは、損傷した部位に沈着し、凝血塊を形成し、出血を止め、皮下組織を保護する。フィブロネクチンは、細胞の移動及び局在化のためのマトリックスを形成し、線維芽細胞、マクロファージを媒介して損傷の修復に関与する。フィブロネクチンは、肉芽組織の発生及び組織化過程における細胞の移動及び成長、並びに結合組織マトリックスの再構築及び再合成を含む、創面の治癒に深い影響を与える。フィブロネクチンは、血漿フィブリンへの線維芽細胞の接着を促進し、創傷でのコラーゲンの沈着を促進する。 Recombinant fibronectin fragments are one of the points of interest in fibronectin research at present due to their multiple types of biological functions, and recombinant fibronectin fragments are more effective because they are stable and easy to prepare. Can be advertised and used. Fibronectin plays a very important role in wound healing, and fibronectin deposits on the injured site, forms clots, stops bleeding, and protects the subcutaneous tissue. Fibronectin forms a matrix for cell migration and localization and mediates fibroblasts, macrophages and is involved in damage repair. Fibronectin has profound effects on wound healing, including cell migration and growth during granulation tissue development and organization, as well as remodeling and resynthesis of connective tissue matrix. Fibronectin promotes fibroblast adhesion to plasma fibrin and promotes collagen deposition in wounds.
フィブロネクチンは、医学、美容、化粧品の分野において、幅広い使用見通しがあるが、人体又は動物の血液、組織から抽出される天然フィブロネクチンは、産出量が極めて限定的であり、コストが高く、また製品の純度が低い。ＤＮＡ組換え技術は、フィブロネクチンの調製の困難さを解決できるが、フィブロネクチンの単量体は分子量が大きいため、ＤＮＡ組換え技術を用いてフィブロネクチンの全長を発現させることは困難であり、タンパク質は安定性が悪く、活性が低い傾向がある。ＤＮＡ組換え技術を用いてフィブロネクチンの機能ドメインを発現させ、安定性が高く、活性が高いフィブロネクチンを得ることは、フィブロネクチンの使用のボトルネックを解決する重要な経路である。 Fibronectin has a wide range of potential uses in the fields of medicine, cosmetology, and cosmetics, but natural fibronectin extracted from human or animal blood and tissues has extremely limited production, high cost, and product products. The purity is low. DNA recombination technology can solve the difficulty of preparing fibronectin, but since the monomer of fibronectin has a large molecular weight, it is difficult to express the full length of fibronectin using DNA recombination technology, and the protein is stable. Poor sex and tends to be less active. Expressing the functional domain of fibronectin using DNA recombination technology to obtain highly stable and highly active fibronectin is an important pathway to solve the bottleneck of fibronectin use.
従来の生化学理論「アンフィンセンのドグマ」によると、タンパク質の折り畳み情報はそのアミノ酸配列によって一定の環境で一意に決定される。しかし、最近の研究によると、異なるＤＮＡが同じアミノ酸に翻訳できるが、タンパク質生成の速度（翻訳速度）が一定ではなく、あるセグメントでは比較的遅くなり、この現象は翻訳一時停止（ｔｒａｎｓｌａｔｉｏｎａｌ ｐａｕｓｉｎｇ又はｔｒａｎｓｌａｔｉｏｎａｌ ａｔｔｅｎｕａｔｉｏｎ）と呼ばれる。翻訳一時停止サイトはタンパク質の折り畳みと高い相関があり、翻訳一時停止サイトが正しくないと遅い場所が早くなり、あるいは早い場所が遅くなることは、いずれもタンパク質の誤った折り畳みと凝集につながり、機能的な可溶性タンパク質が得られない。つまり、タンパク質の立体構造は、アミノ酸配列のみならず、ヌクレオチド配列によっても決定される。 According to the conventional biochemical theory "Anfinsen's dogma", protein folding information is uniquely determined in a certain environment by its amino acid sequence. However, recent studies have shown that different DNAs can be translated into the same amino acid, but the rate of protein production (translation rate) is not constant and is relatively slow in some segments, a phenomenon that is translational pausing or transnational. It is called "attentionation". Translation pause sites are highly correlated with protein folding, and improper translation pause sites can result in faster or slower slow locations, both of which lead to incorrect protein folding and aggregation, and function. Soluble protein cannot be obtained. That is, the three-dimensional structure of a protein is determined not only by the amino acid sequence but also by the nucleotide sequence.
従来技術の一般的な欠点について、本発明は、組換えフィブロネクチン変異体及びその使用を提供する。本発明に係る組換えフィブロネクチン変異体は、精製過程が簡単であり、細胞増殖活性、接着活性を効果的に促進できる。 For the general drawbacks of prior art, the present invention provides recombinant fibronectin variants and their use. The recombinant fibronectin mutant according to the present invention has a simple purification process and can effectively promote cell proliferation activity and adhesive activity.
上記の目的を達成するために、本発明は下記の技術案を講じた。
組換えフィブロネクチン変異体は、そのヌクレオチド配列がＳＥＱ ＩＤ ＮＯ．１に示す通りである。
ＡＣＧＧＧＣＡＴＣＧＡＣＴＴＣＡＧＣＧＡＴＡＴＣＡＣＣＧＣＧＡＡＣＡＧＣＴＴＣＡＣＣＧＴＴＣＡＣＴＧＧＡＴＣＧＣＧＣＣＡＣＧＴＧＣＧＡＣＧＡＴＣＡＣＣＧＧＣＴＡＴＣＧＣＡＴＣＣＧＣＣＡＴＣＡＣＣＣＧＧＡＡＣＡＣＴＴＴＡＧＣＧＧＴＣＧＴＣＣＡＣＧＣＧＡＡＧＡＴＣＧＣＧＴＴＣＣＧＣＡＴＡＧＣＣＧＣＡＡＴＡＧＣＡＴＣＡＣＧＣＴＧＡＣＣＡＡＴＣＴＧＡＣＣＣＣＧＧＧＣＡＣＣＧＡＡＴＡＴＧＴＴＧＴＧＡＧＣＡＴＣＧＴＧＧＣＧＣＴＧＡＡＣＧＧＣＣＧＣＧＡＡＧＡＡＡＧＣＣＣＡＣＴＧＣＴＧＡＴＴＧＧＣＣＡＧＣＡＧＡＧＣＡＣＣＧＴＧＡＧＴＧＡＴＧＧＴＧＧＣＧＧＴＧＧＣＡＧＣＡＡＴＡＴＴＧＡＴＣＧＣＣＣＧＡＡＡＧＧＴＣＴＧＧＣＣＴＴＣＡＣＧＧＡＴＧＴＧＧＡＣＧＴＧＧＡＣＡＧＣＡＴＣＡＡＡＡＴＣＧＣＧＴＧＧＧＡＡＡＧＣＣＣＡＣＡＡＧＧＣＣＡＡＧＴＴＡＧＣＣＧＣＴＡＣＣＧＣＧＴＧＡＣＣＴＡＴＡＧＣＡＧＣＣＣＧＧＡＡＧＡＴＧＧＣＡＴＣＣＡＣＧＡＡＣＴＧＴＴＴＣＣＧＧＣＧＣＣＧＧＡＴＧＧＴＧＡＡＧＡＡＧＡＴＡＣＣＧＣＧＧＡＡＣＴＧＣＡＡＧＧＴＣＴＧＣＧＴＣＣＧＧＧＣＡＧＣＧＡＡＴＡＣＡＣＧＧＴＴＡＧＣＧＴＧＧＴＧＧＣＧＣＴＡＣＡＴＧＡＴＧＡＴＡＴＧＧＡＡＡＧＣＣＡＧＣＣＧＣＴＡＡＴＡＧＧＣＡＣＡＣＡＡＡＧＣＡＣＡＧＣＧ（ＳＥＱ ＩＤ ＮＯ．１）。
In order to achieve the above object, the present invention has adopted the following technical proposals.
The nucleotide sequence of the recombinant fibronectin mutant is SEQ ID NO. It is as shown in 1.
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTACATGATGATATGGAAAGCCAGCCGCTAATAGGCACACAAAGCACAGCG (SEQ ID NO.1).
好ましくは、前記組換えフィブロネクチン変異体は可溶性の形態で発現される。 Preferably, the recombinant fibronectin variant is expressed in a soluble form.
好ましくは、前記組換えフィブロネクチン変異体はフィブロネクチン変異体を翻訳一時停止して最適化することで得られる。 Preferably, the recombinant fibronectin variant is obtained by suspending translation and optimizing the fibronectin variant.
好ましくは、前記フィブロネクチン変異体のヌクレオチド配列はＳＥＱ ＩＤ ＮＯ．２に示す通りである。
ＡＣＧＧＧＣＡＴＣＧＡＣＴＴＣＡＧＣＧＡＴＡＴＣＡＣＣＧＣＧＡＡＣＡＧＣＴＴＣＡＣＣＧＴＴＣＡＣＴＧＧＡＴＣＧＣＧＣＣＡＣＧＴＧＣＧＡＣＧＡＴＣＡＣＣＧＧＣＴＡＴＣＧＣＡＴＣＣＧＣＣＡＴＣＡＣＣＣＧＧＡＡＣＡＣＴＴＴＡＧＣＧＧＴＣＧＴＣＣＡＣＧＣＧＡＡＧＡＴＣＧＣＧＴＴＣＣＧＣＡＴＡＧＣＣＧＣＡＡＴＡＧＣＡＴＣＡＣＧＣＴＧＡＣＣＡＡＴＣＴＧＡＣＣＣＣＧＧＧＣＡＣＣＧＡＡＴＡＴＧＴＴＧＴＧＡＧＣＡＴＣＧＴＧＧＣＧＣＴＧＡＡＣＧＧＣＣＧＣＧＡＡＧＡＡＡＧＣＣＣＡＣＴＧＣＴＧＡＴＴＧＧＣＣＡＧＣＡＧＡＧＣＡＣＣＧＴＧＡＧＴＧＡＴＧＧＴＧＧＣＧＧＴＧＧＣＡＧＣＡＡＴＡＴＴＧＡＴＣＧＣＣＣＧＡＡＡＧＧＴＣＴＧＧＣＣＴＴＣＡＣＧＧＡＴＧＴＧＧＡＣＧＴＧＧＡＣＡＧＣＡＴＣＡＡＡＡＴＣＧＣＧＴＧＧＧＡＡＡＧＣＣＣＡＣＡＡＧＧＣＣＡＡＧＴＴＡＧＣＣＧＣＴＡＣＣＧＣＧＴＧＡＣＣＴＡＴＡＧＣＡＧＣＣＣＧＧＡＡＧＡＴＧＧＣＡＴＣＣＡＣＧＡＡＣＴＧＴＴＴＣＣＧＧＣＧＣＣＧＧＡＴＧＧＴＧＡＡＧＡＡＧＡＴＡＣＣＧＣＧＧＡＡＣＴＧＣＡＡＧＧＴＣＴＧＣＧＴＣＣＧＧＧＣＡＧＣＧＡＡＴＡＣＡＣＧＧＴＴＡＧＣＧＴＧＧＴＧＧＣＧＣＴＧＣＡＴＧＡＴＧＡＴＡＴＧＧＡＡＡＧＣＣＡＧＣＣＧＣＴＧＡＴＣＧＧＣＡＣＣＣＡＡＡＧＣＡＣＣＧＣＧ（ＳＥＱ ＩＤ ＮＯ．２）；
Preferably, the nucleotide sequence of the fibronectin variant is SEQ ID NO. It is as shown in 2.
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTGCATGATGATATGGAAAGCCAGCCGCTGATCGGCACCCAAAGCACCGCG (SEQ ID NO.2);
好ましくは、前記翻訳一時停止して最適化する過程は、フィブロネクチン変異体の最後の２０コドンのうちの翻訳速度の速いコドンを翻訳速度の遅いコドンに置き換えることである。 Preferably, the process of pausing and optimizing the translation is to replace the faster translation codon of the last 20 codons of the fibronectin variant with a slower translation codon.
好ましくは、前記翻訳速度の速いコドンはＡＴＣ、ＡＣＣ、ＣＴＧを含み、前記翻訳速度の遅いコドンはＡＴＡ、ＡＣＡ、ＣＴＡを含み、翻訳一時停止サイトを作成するソフトウェアにより計算して決定される。 Preferably, the fast translation rate codon comprises ATC, ACC, CTG and the slow translation rate codon comprises ATA, ACA, CTA and is calculated and determined by software creating a translation pause site.
好ましくは、前記ヌクレオチド配列がコードするアミノ酸配列はＳＥＱ ＩＤ ＮＯ．３に示す通りである。
ＴＧＩＤＦＳＤＩＴＡＮＳＦＴＶＨＷＩＡＰＲＡＴＩＴＧＹＲＩＲＨＨＰＥＨＦＳＧＲＰＲＥＤＲＶＰＨＳＲＮＳＩＴＬＴＮＬＴＰＧＴＥＹＶＶＳＩＶＡＬＮＧＲＥＥＳＰＬＬＩＧＱＱＳＴＶＳＤＧＧＧＧＳＮＩＤＲＰＫＧＬＡＦＴＤＶＤＶＤＳＩＫＩＡＷＥＳＰＱＧＱＶＳＲＹＲＶＴＹＳＳＰＥＤＧＩＨＥＬＦＰＡＰＤＧＥＥＤＴＡＥＬＱＧＬＲＰＧＳＥＹＴＶＳＶＶＡＬＨＤＤＭＥＳＱＰＬＩＧＴＱＳＴＡ（ＳＥＱ ＩＤ ＮＯ．３）。
Preferably, the amino acid sequence encoded by the nucleotide sequence is SEQ ID NO. It is as shown in 3.
TGIDFSDITANSFTVHWIAPRATIONGYRIRHPEHFSSGRPREDRVPSHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDGGGGSNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVSET
本発明は、前記組換えフィブロネクチン変異体の細胞増殖活性、接着活性の促進における使用を更に提供する。 The present invention further provides the use of the recombinant fibronectin mutant in promoting cell proliferation activity and adhesive activity.
従来の技術と比較して、本発明に係る組換えフィブロネクチン変異体は、以下の利点を有する。
（１）本発明に係る組換えフィブロネクチン変異体は、細胞増殖活性を効果的に促進し、細胞接着活性を向上させることができる。
（２）本発明に係る組換えフィブロネクチン変異体は、調製過程が簡単であり、精製が容易である。
（３）本発明に係る組換えフィブロネクチンは、新型組換えタンパク質変異体であり、変異体を得る新しい方法を提供する。
Compared with the prior art, the recombinant fibronectin mutant according to the present invention has the following advantages.
(1) The recombinant fibronectin mutant according to the present invention can effectively promote cell proliferation activity and improve cell adhesion activity.
(2) The recombinant fibronectin mutant according to the present invention has a simple preparation process and is easy to purify.
(3) The recombinant fibronectin according to the present invention is a novel recombinant protein mutant, and provides a new method for obtaining the mutant.
以下、具体的な実施例を参照しながら本発明をさらに説明するが、以下の実施例は本発明を説明するためのものに過ぎず、本発明を限定するためのものではなく、本発明と同一又は類似な技術案はすべて本発明の保護の範囲内に含まれることを注意すべきである。特に明記しない限り、実施例で使用される技術的手段は当業者に周知の一般的な手段であり、使用される原材料は市販の製品である。 Hereinafter, the present invention will be further described with reference to specific examples, but the following examples are merely for explaining the present invention, not for limiting the present invention, but with the present invention. It should be noted that all identical or similar technical proposals are within the scope of protection of the present invention. Unless otherwise specified, the technical means used in the examples are common means well known to those skilled in the art and the raw materials used are commercially available products.
本発明の実施例に係る主な材料は以下の通りである。Ｆｅｒｍｅｎｔａｓ社から購入した宿主菌である大腸菌ＢＬ２１（ＤＥ３）（Ｍｅｒｃｋ）、プラスミドｐＥＴ−２８ａ（Ｍｅｒｃｋ）、染色済みタンパク質Ｍａｒｋｅｒ；ＧＥ社から購入したＮｉ ＳｅｐｈａｒｏｓｅＴＭ ６ Ｆａｓｔ Ｆｌｏｗ；米国ＳＩＧＭＡ社から購入したＣＣＫ−８試薬カセット；他の試薬はいずれも分析純試薬である。ＮＴＡ−０緩衝液（２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０＋０．１５ｍｏｌ／Ｌ ＮａＣｌ）、ＮＴＡ−４０緩衝液（２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０＋０．１５ｍｏｌ／Ｌ ＮａＣｌ＋４０ｍｍｏｌ／Ｌイミダゾール）、ＮＴＡ−８０緩衝液（２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０＋０．１５ｍｏｌ／Ｌ ＮａＣｌ＋８０ｍｍｏｌ／Ｌイミダゾール）、ＮＴＡ−２５０緩衝液（２０ｍｍｏｌ／Ｌ Ｔｒｉｓ−ＨＣｌ、ｐＨ８．０＋０．１５ｍｏｌ／Ｌ ＮａＣｌ＋２５０ｍｍｏｌ／Ｌイミダゾール）。 The main materials according to the examples of the present invention are as follows. Escherichia coli BL21 (DE3) (Merck), plasmid pET-28a (Merck), stained protein Marker purchased from Fermentas; Ni SepharoseTM 6 Fast Flow purchased from GE; CCK- purchased from SIGMA, USA 8 Reagent cassette; All other reagents are pure analytical reagents. NTA-0 buffer (20 mmol / L Tris-HCl, pH 8.0 + 0.15 mol / L NaCl), NTA-40 buffer (20 mmol / L Tris-HCl, pH 8.0 + 0.15 mol / L NaCl + 40 mmol / L imidazole), NTA -80 buffer (20 mmol / L Tris-HCl, pH 8.0 + 0.15 mol / L NaCl + 80 mmol / L imidazole), NTA-250 buffer (20 mmol / L Tris-HCl, pH 8.0 + 0.15 mol / L NaCl + 250 mmol / L imidazole) ..
実施例１ 組換えフィブロネクチン担体の構築
生物学的高分子シミュレーションを通じて細胞増殖及び接着を促進するフィブロネクチンの機能ドメインを選択し、新しいフィブロネクチン変異体を設計した。大腸菌のコドンの好み及び翻訳一時停止理論に従って、フィブロネクチン変異体のヌクレオチド配列を最適化した。そして、蘇州泓迅生物科技股フン有限公司に全遺伝子からフィブロネクチン変異体ＤＮＡを合成するよう依頼した。
Example 1 Construction of recombinant fibronectin carrier A new fibronectin mutant was designed by selecting the functional domain of fibronectin that promotes cell proliferation and adhesion through biological polymer simulation. The nucleotide sequences of the fibronectin variants were optimized according to E. coli codon preferences and translational arrest theory. Then, we asked Suzhou Jinjin Biotechnology Co., Ltd. to synthesize fibronectin mutant DNA from all genes.
最適化されていない組換えフィブロネクチンのヌクレオチド配列は以下の通りである。
ＡＣＧＧＧＣＡＴＣＧＡＣＴＴＣＡＧＣＧＡＴＡＴＣＡＣＣＧＣＧＡＡＣＡＧＣＴＴＣＡＣＣＧＴＴＣＡＣＴＧＧＡＴＣＧＣＧＣＣＡＣＧＴＧＣＧＡＣＧＡＴＣＡＣＣＧＧＣＴＡＴＣＧＣＡＴＣＣＧＣＣＡＴＣＡＣＣＣＧＧＡＡＣＡＣＴＴＴＡＧＣＧＧＴＣＧＴＣＣＡＣＧＣＧＡＡＧＡＴＣＧＣＧＴＴＣＣＧＣＡＴＡＧＣＣＧＣＡＡＴＡＧＣＡＴＣＡＣＧＣＴＧＡＣＣＡＡＴＣＴＧＡＣＣＣＣＧＧＧＣＡＣＣＧＡＡＴＡＴＧＴＴＧＴＧＡＧＣＡＴＣＧＴＧＧＣＧＣＴＧＡＡＣＧＧＣＣＧＣＧＡＡＧＡＡＡＧＣＣＣＡＣＴＧＣＴＧＡＴＴＧＧＣＣＡＧＣＡＧＡＧＣＡＣＣＧＴＧＡＧＴＧＡＴＧＧＴＧＧＣＧＧＴＧＧＣＡＧＣＡＡＴＡＴＴＧＡＴＣＧＣＣＣＧＡＡＡＧＧＴＣＴＧＧＣＣＴＴＣＡＣＧＧＡＴＧＴＧＧＡＣＧＴＧＧＡＣＡＧＣＡＴＣＡＡＡＡＴＣＧＣＧＴＧＧＧＡＡＡＧＣＣＣＡＣＡＡＧＧＣＣＡＡＧＴＴＡＧＣＣＧＣＴＡＣＣＧＣＧＴＧＡＣＣＴＡＴＡＧＣＡＧＣＣＣＧＧＡＡＧＡＴＧＧＣＡＴＣＣＡＣＧＡＡＣＴＧＴＴＴＣＣＧＧＣＧＣＣＧＧＡＴＧＧＴＧＡＡＧＡＡＧＡＴＡＣＣＧＣＧＧＡＡＣＴＧＣＡＡＧＧＴＣＴＧＣＧＴＣＣＧＧＧＣＡＧＣＧＡＡＴＡＣＡＣＧＧＴＴＡＧＣＧＴＧＧＴＧＧＣＧＣＴＧＣＡＴＧＡＴＧＡＴＡＴＧＧＡＡＡＧＣＣＡＧＣＣＧＣＴＧＡＴＣＧＧＣＡＣＣＣＡＡＡＧＣＡＣＣＧＣＧ。
The nucleotide sequence of the unoptimized recombinant fibronectin is as follows.
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTGCATGATGATATGGAAAGCCAGCCGCTGATCGGCACCCAAAGCACCGCG.
フィブロネクチン変異体のヌクレオチド配列の最適化されていない発現菌株を、カナマイシンの含有量が５０μｇ／ｍＬのＬＢ培地１Ｌに接種し、上記の発現条件で振とうフラスコで発酵を行って誘導した。４℃、６０００×ｇで１０分間遠心分離して菌体を回収してから、菌体の沈殿物をＮＴＡ−１０緩衝液に１：１０の体積比で再懸濁し、高圧（１０００ｂａｒ）で細胞を均一に破砕した。４℃、２５０００×ｇで３０分間遠心分離して上清を回収し、遠心沈殿物を上清と等体積の８Ｍ尿素含有ＮＴＡ−０緩衝液に溶解し、１２％のＳＤＳ−ＰＡＧＥゲル電気泳動で組換えフィブロネクチン変異体の発現状況を分析した。結果は図１に示すように、ヌクレオチド配列が最適化されていない組換えフィブロネクチン変異体は、主に大腸菌で封入体の形態で発現されることがわかった。実際の使用では、細胞増殖活性の促進剤としてフィブロネクチン変異体をよく使用するが、実際のニーズを満たすために、フィブロネクチン変異体のヌクレオチド配列を最適化する必要がある。 A strain expressing the fibronectin mutant in which the nucleotide sequence was not optimized was inoculated into 1 L of LB medium having a kanamycin content of 50 μg / mL, and fermented in a shaking flask under the above expression conditions to induce the strain. After centrifuging at 4 ° C. and 6000 xg for 10 minutes to collect the cells, the cell precipitate was resuspended in NTA-10 buffer at a volume ratio of 1:10, and the cells were subjected to high pressure (1000 bar). Was uniformly crushed. Centrifuge at 4 ° C., 25,000 xg for 30 minutes to collect the supernatant, dissolve the centrifuge precipitate in the same volume of 8M urea-containing NTA-0 buffer as the supernatant, and perform 12% SDS-PAGE gel electrophoresis. The expression status of the recombinant fibronectin mutant was analyzed in. As a result, as shown in FIG. 1, it was found that the recombinant fibronectin mutant in which the nucleotide sequence was not optimized is expressed mainly in Escherichia coli in the form of inclusion bodies. In practical use, fibronectin variants are often used as promoters of cell proliferation activity, but the nucleotide sequences of the fibronectin variants need to be optimized to meet actual needs.
翻訳一時停止理論に従って、組換えフィブロネクチン変異体をコードするヌクレオチド配列を設計し、組換えフィブロネクチンのアミノ酸配列を変更しない場合、翻訳一時停止サイトを作成するように（ＲｉｂｏＴｅｍｐｏソフトウェアにより計算して決定され、ｈｔｔｐ：／／ｂｉｏｉｎｆｏｒｍａｔｉｃｓ．ｊｎｕ．ｅｄｕ．ｃｎ ／ｓｏｆｔｗａｒｅ ／ｒｉｂｏｔｅｍｐｏ）、組換えフィブロネクチンの最後の２０コドンのうちの翻訳速度の速いコドン（ＡＴＣ、ＡＣＣ、ＣＴＧ）を翻訳速度の遅いコドン（ＡＴＡ、ＡＣＡ、ＣＴＡ）に置き換え、変異体のＲｉｂｏＴｅｍｐｏソフトウェアにより計算された翻訳一時停止曲線をそれぞれ図２に示し、図２において、Ａは最適化前の翻訳曲線を表し、Ｂは翻訳一時停止理論に従って最適化された翻訳曲線を示した。翻訳一時停止曲線は、所定の領域に翻訳一時停止サイトを形成し、要求を満たした。設計された組換えフィブロネクチン変異体をコードするヌクレオチド配列は次の通りである。 Design a nucleotide sequence encoding a recombinant fibronectin variant according to translational suspension theory and, if the amino acid sequence of recombinant fibronectin is not altered, to create a translational suspension site (calculated and determined by RiboTempo software). http: // bioinformatics.nucleotide.edu.cn / software / ribotempo), the codon with the fastest translation rate (ATC, ACC, CTG) among the last 20 codons of recombinant fibronectin, and the codon with the slow translation rate (ATA, ACA , CTA), and the translation pause curves calculated by the RivoTempo software of the variant are shown in FIG. 2, where A represents the translation curve before optimization and B is optimized according to the translation pause theory. The translation curve was shown. The translation pause curve formed a translation pause site in a predetermined area and met the requirements. The nucleotide sequence encoding the designed recombinant fibronectin variant is as follows.
翻訳一時停止して最適化された配列は以下の通りである。
ＡＣＧＧＧＣＡＴＣＧＡＣＴＴＣＡＧＣＧＡＴＡＴＣＡＣＣＧＣＧＡＡＣＡＧＣＴＴＣＡＣＣＧＴＴＣＡＣＴＧＧＡＴＣＧＣＧＣＣＡＣＧＴＧＣＧＡＣＧＡＴＣＡＣＣＧＧＣＴＡＴＣＧＣＡＴＣＣＧＣＣＡＴＣＡＣＣＣＧＧＡＡＣＡＣＴＴＴＡＧＣＧＧＴＣＧＴＣＣＡＣＧＣＧＡＡＧＡＴＣＧＣＧＴＴＣＣＧＣＡＴＡＧＣＣＧＣＡＡＴＡＧＣＡＴＣＡＣＧＣＴＧＡＣＣＡＡＴＣＴＧＡＣＣＣＣＧＧＧＣＡＣＣＧＡＡＴＡＴＧＴＴＧＴＧＡＧＣＡＴＣＧＴＧＧＣＧＣＴＧＡＡＣＧＧＣＣＧＣＧＡＡＧＡＡＡＧＣＣＣＡＣＴＧＣＴＧＡＴＴＧＧＣＣＡＧＣＡＧＡＧＣＡＣＣＧＴＧＡＧＴＧＡＴＧＧＴＧＧＣＧＧＴＧＧＣＡＧＣＡＡＴＡＴＴＧＡＴＣＧＣＣＣＧＡＡＡＧＧＴＣＴＧＧＣＣＴＴＣＡＣＧＧＡＴＧＴＧＧＡＣＧＴＧＧＡＣＡＧＣＡＴＣＡＡＡＡＴＣＧＣＧＴＧＧＧＡＡＡＧＣＣＣＡＣＡＡＧＧＣＣＡＡＧＴＴＡＧＣＣＧＣＴＡＣＣＧＣＧＴＧＡＣＣＴＡＴＡＧＣＡＧＣＣＣＧＧＡＡＧＡＴＧＧＣＡＴＣＣＡＣＧＡＡＣＴＧＴＴＴＣＣＧＧＣＧＣＣＧＧＡＴＧＧＴＧＡＡＧＡＡＧＡＴＡＣＣＧＣＧＧＡＡＣＴＧＣＡＡＧＧＴＣＴＧＣＧＴＣＣＧＧＧＣＡＧＣＧＡＡＴＡＣＡＣＧＧＴＴＡＧＣＧＴＧＧＴＧＧＣＧＣＴＡＣＡＴＧＡＴＧＡＴＡＴＧＧＡＡＡＧＣＣＡＧＣＣＧＣＴＡＡＴＡＧＧＣＡＣＡＣＡＡＡＧＣＡＣＡＧＣＧ。
The sequence optimized by pausing translation is as follows.
ACGGGCATCGACTTCAGCGATATCACCGCGAACAGCTTCACCGTTCACTGGATCGCGCCACGTGCGACGATCACCGGCTATCGCATCCGCCATCACCCGGAACACTTTAGCGGTCGTCCACGCGAAGATCGCGTTCCGCATAGCCGCAATAGCATCACGCTGACCAATCTGACCCCGGGCACCGAATATGTTGTGAGCATCGTGGCGCTGAACGGCCGCGAAGAAAGCCCACTGCTGATTGGCCAGCAGAGCACCGTGAGTGATGGTGGCGGTGGCAGCAATATTGATCGCCCGAAAGGTCTGGCCTTCACGGATGTGGACGTGGACAGCATCAAAATCGCGTGGGAAAGCCCACAAGGCCAAGTTAGCCGCTACCGCGTGACCTATAGCAGCCCGGAAGATGGCATCCACGAACTGTTTCCGGCGCCGGATGGTGAAGAAGATACCGCGGAACTGCAAGGTCTGCGTCCGGGCAGCGAATACACGGTTAGCGTGGTGGCGCTACATGATGATATGGAAAGCCAGCCGCTAATAGGCACACAAAGCACAGCG.
組換えフィブロネクチンのアミノ酸配列は以下の通りである。
ＴＧＩＤＦＳＤＩＴＡＮＳＦＴＶＨＷＩＡＰＲＡＴＩＴＧＹＲＩＲＨＨＰＥＨＦＳＧＲＰＲＥＤＲＶＰＨＳＲＮＳＩＴＬＴＮＬＴＰＧＴＥＹＶＶＳＩＶＡＬＮＧＲＥＥＳＰＬＬＩＧＱＱＳＴＶＳＤＧＧＧＧＳＮＩＤＲＰＫＧＬＡＦＴＤＶＤＶＤＳＩＫＩＡＷＥＳＰＱＧＱＶＳＲＹＲＶＴＹＳＳＰＥＤＧＩＨＥＬＦＰＡＰＤＧＥＥＤＴＡＥＬＱＧＬＲＰＧＳＥＹＴＶＳＶＶＡＬＨＤＤＭＥＳＱＰＬＩＧＴＱＳＴＡ。
The amino acid sequence of recombinant fibronectin is as follows.
TGIDFSDITANSFTVHWIAPRATTIGYRIRHPEHFSSGRPREDRVPSHSRNSITLTNLTPGTEYVVSIVALNGREESPLLIGQQSTVSDGGGGSNIDRPKGLAFTDVDVDSIKIAWESPQGQVSRYRVSET
実施例２ 組換えフィブロネクチン変異体の発現、精製
（１）フィブロネクチンの発現菌株の調製
前記フィブロネクチンの発現菌株の調製過程は以下の通りである。
（１−１）大腸菌ＢＬ２１（ＤＥ３）コンピテントセルの調製：調製過程の詳細は、『分子クローニング実験ガイドライン』第３版（［米国］Ｊ．Ｓａｍｂｒｏｏｋ著、黄培堂訳）に記載されている。
（１−２）発現担体ｐＥＴ−２８ａ−Ｆｉｂｒｏｎｅｃｔｉｎを大腸菌ＢＬ２１（ＤＥ３）コンピテントセルへの転換する：転換過程の詳細は、『分子クローニング実験ガイドライン』第３版（［米国］Ｊ．Ｓａｍｂｒｏｏｋ著、黄培堂訳）に記載されている。
Example 2 Expression and purification of recombinant fibronectin mutant (1) Preparation of fibronectin-expressing strain The preparation process of the fibronectin-expressing strain is as follows.
(1-1) Preparation of Escherichia coli BL21 (DE3) competent cells: Details of the preparation process are described in "Molecular Cloning Experimental Guidelines", 3rd edition ([USA] by J. Sambrook, translated by Huang Yudo).
(1-2) Conversion of expression carrier pET-28a-fibronectin to Escherichia coli BL21 (DE3) competent cells: For details of the conversion process, refer to "Molecular Cloning Experimental Guidelines", 3rd edition ([USA] J. Sambrook, It is described in (Translated by Huangyuan).
（２）フィブロネクチン変異体の発現誘導及び可溶性分析
具体的な操作過程は以下の通りである。
ステップ（１）で得られた発現菌株ｐＥＴ−２８ａ−Ｆｉｂｒｏｎｅｃｔｉｎをカナマイシンの含有量が５０μｇ／ｍＬのＬＢ培地１０ｍＬに接種し、３７℃、１８０ｒｐｍで培養し、ＯＤ６００＝０．８であるときにＩＰＴＧを加え、最終濃度が１ｍＭ、３７℃で４時間発現を誘導した後、５０００ｇ、４℃で１０分間遠心分離して菌体を回収した。菌体をＴｒｉｓ−ＨＣｌ（ｐＨ８．０，０．１５ｍｏｌ／ＬＮａＣｌ）緩衝液２０ｍｍｏｌ／Ｌに再懸濁し、高圧（８００ｂａｒ）で細胞を均一に破砕し、１８０００×ｇ、４℃で３０分間遠心分離し、上清及び沈殿物は、別々に後続きのＳＤＳ−ＰＡＧＥ電気泳動（５％濃縮ゲル、１２％分離ゲル）及びＷｅｓｔｅｒｎ ｂｌｏｔ分析のためにサンプルを保留した。
(2) Expression induction and solubility analysis of fibronectin mutant The specific operation process is as follows.
The expression strain pET-28a-Fibronectin obtained in step (1) was inoculated into 10 mL of LB medium having a kanamycin content of 50 μg / mL, cultured at 37 ° C. and 180 rpm, and IPTG when OD600 = 0.8. Was added, and expression was induced at a final concentration of 1 mM and 37 ° C. for 4 hours, and then the cells were collected by centrifugation at 5000 g and 4 ° C. for 10 minutes. The cells were resuspended in 20 mmol / L of Tris-HCl (pH 8.0, 0.15 mol / LNaCl) buffer, the cells were uniformly disrupted at high pressure (800 bar) and centrifuged at 18000 xg, 4 ° C. for 30 minutes. The supernatant and precipitate were separately reserved for subsequent SDS-PAGE electrophoresis (5% concentrated gel, 12% isolated gel) and Western blot analysis.
（３）フィブロネクチン変異体の振とうフラスコでの発酵及び可溶性タンパク質の精製
ステップ（１）で得られたフィブロネクチン変異体の発現菌株をカナマイシンの含有量が５０μｇ／ｍＬのＬＢ培地１Ｌにそれぞれ接種し、前記発現条件で振とうフラスコで発酵して誘導した。４℃、６０００×ｇで１０分間遠心分離して菌体を回収してから、菌体の沈殿物をＮＴＡ−１０緩衝液に１：１０の体積比で再懸濁し、高圧（１０００ｂａｒ）で細胞を均一に破砕した。４℃、２５０００×ｇで３０分間遠心分離して上清を回収した。
(3) Fermentation of fibronectin mutant in a shaking flask and purification of soluble protein The strain expressing the fibronectin mutant obtained in step (1) was inoculated into 1 L of LB medium having a kanamycin content of 50 μg / mL. It was induced by fermentation in a shaking flask under the above expression conditions. After centrifuging at 4 ° C. and 6000 xg for 10 minutes to collect the cells, the cell precipitate was resuspended in NTA-10 buffer at a volume ratio of 1:10, and the cells were subjected to high pressure (1000 bar). Was uniformly crushed. The supernatant was collected by centrifugation at 25,000 × g at 4 ° C. for 30 minutes.
上清を、ベッドボリュームが２０ｍＬのＮｉ−ＮＴＡアフィニティークロマトグラフィーカラムに加え、０．６ｍＬ／ｍｉｎの流速で、ベースラインに戻るようにＮＴＡ−０緩衝液で洗浄し、１ｍＬ／ｍｉｎの流速で、ＮＴＡ−４０緩衝液で不純物タンパク質を洗浄し、ＮＴＡ−８０緩衝液で不純物タンパク質を洗浄し、ＮＴＡ−２５０緩衝液で目的タンパク質を溶出した。精製されたフィブロネクチン変異体タンパク質を、Ｓｅｐｈａｄｅｘ Ｇ−２５分子篩でイミダゾールを溶出して３ＫＤａ限外濾過チューブで濃縮し、精製されたフィブロネクチン変異体の純度をＳＤＳ−ＰＡＧＥ電気泳動により評定した。 The supernatant was added to a Ni-NTA affinity chromatography column with a bed volume of 20 mL, washed with NTA-0 buffer at a flow rate of 0.6 mL / min to return to baseline, and at a flow rate of 1 mL / min. The impurity protein was washed with NTA-40 buffer, the impurity protein was washed with NTA-80 buffer, and the target protein was eluted with NTA-250 buffer. The purified fibronectin variant protein was eluted with imidazole on Sephadex G-25 molecular sieves and concentrated on a 3KDa ultrafiltration tube, and the purity of the purified fibronectin variant was assessed by SDS-PAGE electrophoresis.
図１に示すように、ｐＥＴ−２８ａ−Ｆｉｂｏｎｅｃｔｉｏｎ／ＢＬ２１工学細菌（ｅｎｇｉｎｅｅｒｉｎｇ ｂａｃｔｅｒｉａ）は１ｍＭ ＩＰＴＧによって発現を誘導した後、遠心分離して菌体を回収し、菌体を高圧で均一に破砕して遠心分離し、１２％のＳＤＳ−ＰＡＧＥ電気泳動によってフィブロネクチンの発現状況を分析し、その結果は、フィブロネクチンは主に菌体を破砕して遠心分離した沈殿物に存在することを示し、最適化されていない組換えフィブロネクチン変異体は主に封入体の形態で発現されることを示し、図３において、１は貫通ピーク、２は４０ｍＭイミダゾール溶出ピーク、３は８０ｍＭイミダゾール溶出ピーク、４は２５０ｍＭイミダゾール溶出ピークを表し、図４から、翻訳一時停止理論に従って最適化された組換えフィブロネクチン変異体は主に菌体を破砕して遠心分離した上清に存在することがわかり、それが主に可溶性の形態で発現されることを示した。 As shown in FIG. 1, the expression of pET-28a-Fibination / BL21 engineering bacteria was induced by 1 mM IPTG, then centrifuged to collect the cells, and the cells were uniformly crushed at high pressure. Centrifugated and analyzed for fibronectin expression by 12% SDS-PAGE electrophoresis, the results showed that fibronectin was predominantly crushed and present in the centrifuged precipitate and was optimized. It is shown that the non-recombinant fibronectin mutant is expressed mainly in the form of an encapsulater, in which 1 is a penetration peak, 2 is a 40 mM imidazole elution peak, 3 is an 80 mM imidazole elution peak, and 4 is a 250 mM imidazole elution peak. Representing peaks, FIG. 4 shows that recombinant fibronectin variants optimized according to translational arrest theory are predominantly present in the supernatant of crushed and centrifuged cells, which are predominantly soluble forms. It was shown to be expressed in.
（４）フィブロネクチン変異体の不溶性部分の精製
ステップ（２）で得られたフィブロネクチンの発現菌体を高圧で均一に破砕して遠心分離して沈殿物を得て、さらに以下のステップを実行した。
（４−１）封入体を０．４％のデオキシコール酸ナトリウムで洗浄し、１００００ｒｐｍ、４℃で遠心分離し、遠心沈殿物を得た。
（４−２）沈殿物を８Ｍ尿素含有ＮＴＡ−０緩衝液に溶解し、１００００ｒｐｍ、４℃で遠心分離し、溶解上清を得た。
（４−３）溶解上清をベッドボリュームが２０ｍＬのＮｉ−ＮＴＡアフィニティークロマトグラフィーカラムに加え、流速０．６ｍＬ／ｍｉｎで、ベースラインに戻るように８Ｍ尿素含有ＮＴＡ−０緩衝液で洗浄し、流速１ｍＬ／ｍｉｎで、８Ｍ尿素含有ＮＴＡ−４０緩衝液で不純物タンパク質を洗浄した。
（４−４）次に、カラム上のその場でのリフォールディング（ｒｅｆｏｌｄｉｎｇ）方法によって封入体タンパク質をリフォールディングし、最後にＮＴＡ−２５０緩衝液で目的タンパク質を溶出し、精製されたフィブロネクチン変異体のタンパク質をＳｅｐｈａｄｅｘ Ｇ−２５分子篩でイミダゾールを溶出し、精製されたフィブロネクチン変異体の純度をＳＤＳ−ＰＡＧＥ電気泳動により評定した。
(4) Purification of insoluble portion of fibronectin mutant The fibronectin-expressing cells obtained in step (2) were uniformly crushed at high pressure and centrifuged to obtain a precipitate, and the following steps were further performed.
(4-1) The inclusion body was washed with 0.4% sodium deoxycholate and centrifuged at 10000 rpm and 4 ° C. to obtain a centrifugal precipitate.
(4-2) The precipitate was dissolved in NTA-0 buffer containing 8M urea and centrifuged at 10000 rpm and 4 ° C. to obtain a dissolved supernatant.
(4-3) The dissolution supernatant was added to a Ni-NTA affinity chromatography column having a bed volume of 20 mL, washed with an 8M urea-containing NTA-0 buffer solution at a flow rate of 0.6 mL / min so as to return to the baseline. Impurity proteins were washed with 8M urea-containing NTA-40 buffer at a flow rate of 1 mL / min.
(4-4) Next, the encapsulated protein is refolded by an in-situ refolding method on the column, and finally the target protein is eluted with NTA-250 buffer to purify the fibronectin mutant. Protein was eluted with imidazole on a Sephadex G-25 molecular sieve and the purity of the purified fibronectin variant was assessed by SDS-PAGE electrophoresis.
具体的な試験結果は図５及び図６に示すように、ＳＤＳ−ＰＡＧＥ電気泳動の結果によると、精製された可溶性及び封入体で発現される組換えフィブロネクチン変異体の純度が９５％を超えることを示し、これにより、本発明に係る組換えフィブロネクチンの純度は非常に高いことがわかった。 Specific test results, as shown in FIGS. 5 and 6, according to the results of SDS-PAGE electrophoresis, the purity of the recombinant fibronectin mutant expressed in purified soluble and inclusion bodies exceeds 95%. From this, it was found that the purity of the recombinant fibronectin according to the present invention is very high.
実施例３ 組換えフィブロネクチン変異体の細胞増殖活性の促進の測定
具体的な測定過程は以下の通りである。
（１）ＢＡＬＢ／ｃ ３Ｔ３細胞を９６ウェル細胞培養プレート（５０００個細胞／ウェル）に接種し、３７℃、５％のＣＯ２細胞インキュベーターで２４時間培養した。
（２）ＤＭＥＭ無血清培地に変更し、１２時間培養し続けた。
（３）組換えフィブロネクチン変異体（可溶性で発現される精製成分、封入体で発現される精製成分）及びＰＢＳ（陰性対照群）をそれぞれ加え、４８〜７２時間培養し続けた。
（４）各ウェルにＣＣＫ−８試薬１０μＬを加え、３７℃、５％のＣＯ２細胞インキュベーターで２時間インキュベートした後、取り出した。
（５）マイクロプレートリーダーを用いて、９６ウェルプレートの４５０ｎｍ及び６３０ｎｍでの吸光値を読み取り、６３０ｎｍを参照波長として、４５０ｎｍでの吸光度を測定し、測定結果を記録した。
相対細胞増殖促進率＝（実験群４５０ｎｍでの吸光値−陰性対照群４５０ｎｍでの吸光値）／陰性対照群４５０ｎｍでの吸光値×１００％。
Example 3 Measurement of promotion of cell proliferation activity of recombinant fibronectin mutant The specific measurement process is as follows.
(1) BALB / c 3T3 cells were inoculated into a 96-well cell culture plate (5000 cells / well) and cultured at 37 ° C. in a 5% CO 2 cell incubator for 24 hours.
(2) The medium was changed to DMEM serum-free medium, and the cells were continuously cultured for 12 hours.
(3) Recombinant fibronectin mutants (soluble purified components expressed, purified components expressed in inclusion bodies) and PBS (negative control group) were added, respectively, and the cells were cultured for 48 to 72 hours.
(4) 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated at 37 ° C. in a 5% CO 2 cell incubator for 2 hours and then taken out.
(5) Using a microplate reader, the absorbance values at 450 nm and 630 nm of the 96-well plate were read, the absorbance at 450 nm was measured with 630 nm as a reference wavelength, and the measurement results were recorded.
Relative cell proliferation promotion rate = (Absorption value at 450 nm in the experimental group-Absorption value at 450 nm in the negative control group) / Absorption value at 450 nm in the negative control group x 100%.
具体的な試験結果は図７に示すように、大腸菌では、可溶性及び封入体で発現される組換えフィブロネクチン変異体は、いずれも良好な細胞増殖の促進効果を有し、本発明では、そのヌクレオチド配列を最適化した後、可溶性で発現される組換えフィブロネクチン変異体の細胞増殖の促進効果は、封入体で発現される組換えフィブロネクチン変異体の細胞増殖の促進効果よりも優れている。 Specific test results are shown in FIG. 7. In Escherichia coli, the recombinant fibronectin mutants expressed in soluble and inclusion bodies both have a good cell proliferation promoting effect, and in the present invention, the nucleotides thereof. After optimizing the sequence, the effect of promoting cell proliferation of the recombinant fibronectin mutant expressed in a soluble form is superior to the effect of promoting cell proliferation of the recombinant fibronectin mutant expressed in inclusion bodies.
実施例４ 組換えフィブロネクチン変異体の細胞接着活性の促進の測定
前記細胞接着活性の測定の具体的な過程は以下の通りである。
（１）組換えフィブロネクチン変異体のタンパク質溶液の濃度は２０μｇ／ｍｌであり、９６ウェル細胞培養プレートの各ウェルにサンプル溶液５０μＬを加え、３７℃で２時間静置し、対照ウェルにＰＢＳ５０μＬを加えた。
（２）ＢＡＬＢ／ ３Ｔ３細胞をトリプシン消化し、カウントし、各ウェルに５×１０４個の細胞を加え、３７℃、ＣＯ２インキュベーターで２時間培養した。
（３）ＰＢＳで３回洗浄し、接着していない細胞を取り除いた後、ＤＭＥＭ培地２００μＬを加えた。
（４）各ウェルにＣＣＫ−８試薬１０μＬを加え、３７℃、５％のＣＯ２細胞インキュベーターで２時間インキュベートした後、取り出した。
（５）マイクロプレートリーダーを用いて、９６ウェルプレートの４５０ｎｍ及び６３０ｎｍでの吸光値を読み取り、６３０ｎｍを参照波長として、４５０ｎｍでの吸光度を測定し、測定結果を記録した。
（６）細胞接着促進率＝（実験群４５０ｎｍでの吸光値−陰性対照群４５０ｎｍでの吸光値）／陰性対照群４５０ｎｍでの吸光値×１００％。
Example 4 Measurement of promotion of cell adhesion activity of recombinant fibronectin mutant The specific process of measurement of the cell adhesion activity is as follows.
(1) The concentration of the protein solution of the recombinant fibronectin mutant is 20 μg / ml, 50 μL of the sample solution is added to each well of the 96-well cell culture plate, the mixture is allowed to stand at 37 ° C. for 2 hours, and 50 μL of PBS is added to the control well. rice field.
(2) BALB / 3T3 cells were trypsin-digested, counted, 5 × 10 4 cells were added to each well, and the cells were cultured at 37 ° C. in a CO 2 incubator for 2 hours.
(3) After washing with PBS three times to remove non-adhered cells, 200 μL of DMEM medium was added.
(4) 10 μL of CCK-8 reagent was added to each well, and the mixture was incubated at 37 ° C. in a 5% CO 2 cell incubator for 2 hours and then taken out.
(5) Using a microplate reader, the absorbance values at 450 nm and 630 nm of the 96-well plate were read, the absorbance at 450 nm was measured with 630 nm as a reference wavelength, and the measurement results were recorded.
(6) Cell adhesion promotion rate = (absorption value at 450 nm in the experimental group-absorption value at 450 nm in the negative control group) / absorption value at 450 nm in the negative control group × 100%.
具体的な試験結果は図８、図９に示すように、細胞接着実験の結果によると、可溶性及び封入体で発現される組換えフィブロネクチン変異体は、いずれも良好な細胞接着の促進効果を有し、本発明では、そのヌクレオチド配列を最適化した後、組換えフィブロネクチン変異体は主に可溶性の形態で発現され、最終により強い細胞接着の促進効果を有する。 Specific test results are shown in FIGS. 8 and 9, and according to the results of cell adhesion experiments, both soluble and recombinant fibronectin mutants expressed in inclusion bodies have a good effect of promoting cell adhesion. However, in the present invention, after optimizing the nucleotide sequence, the recombinant fibronectin mutant is expressed mainly in a soluble form and finally has a stronger cell adhesion promoting effect.
なお、上記の実施例は、本発明の技術案を説明するために使用されるものに過ぎず、本発明の保護範囲を限定するものではなく、最適の実施例を参照しながら本発明を詳細に説明したが、当業者は、本発明の技術案の本質及び範囲から逸脱することなく、本発明の技術案に対して修正又は同等の置換を行うことができることを理解すべきである。 It should be noted that the above examples are merely used to explain the technical proposal of the present invention, do not limit the scope of protection of the present invention, and describe the present invention in detail with reference to the optimum examples. As described above, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical proposal of the present invention without departing from the essence and scope of the technical proposal of the present invention.
Claims (5)
前記翻訳一時停止して最適化する過程は、フィブロネクチン変異体の最後の２０コドンのうちの翻訳速度の速いコドンを翻訳速度の遅いコドンに置き換えることである、ことを特徴とする調製方法。 The method for preparing a recombinant fibronectin mutant according to claim 1, which is obtained by suspending translation and optimizing the fibronectin mutant .
The translation process of optimizing temporary stopped, prepared wherein the der Ru, it can replace the fast codons rate of translation of the last 20 codons of fibronectin variants slow rate of translation codon.
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