JPH0768273B2 - Anti-HIV peptide - Google Patents
Anti-HIV peptideInfo
- Publication number
- JPH0768273B2 JPH0768273B2 JP63303972A JP30397288A JPH0768273B2 JP H0768273 B2 JPH0768273 B2 JP H0768273B2 JP 63303972 A JP63303972 A JP 63303972A JP 30397288 A JP30397288 A JP 30397288A JP H0768273 B2 JPH0768273 B2 JP H0768273B2
- Authority
- JP
- Japan
- Prior art keywords
- leu
- ser
- gln
- lys
- thr
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 51
- 230000036436 anti-hiv Effects 0.000 title claims description 16
- 150000001413 amino acids Chemical group 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 8
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 21
- 210000004027 cell Anatomy 0.000 description 17
- 241000725303 Human immunodeficiency virus Species 0.000 description 16
- 102100036011 T-cell surface glycoprotein CD4 Human genes 0.000 description 12
- 108010041397 CD4 Antigens Proteins 0.000 description 8
- 235000001014 amino acid Nutrition 0.000 description 7
- VHYFNPMBLIVWCW-UHFFFAOYSA-N 4-Dimethylaminopyridine Chemical compound CN(C)C1=CC=NC=C1 VHYFNPMBLIVWCW-UHFFFAOYSA-N 0.000 description 6
- 229940079593 drug Drugs 0.000 description 6
- 239000003814 drug Substances 0.000 description 6
- 150000002148 esters Chemical class 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 230000002401 inhibitory effect Effects 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 239000011347 resin Substances 0.000 description 6
- 229920005989 resin Polymers 0.000 description 6
- AYFVYJQAPQTCCC-GBXIJSLDSA-N L-threonine Chemical compound C[C@@H](O)[C@H](N)C(O)=O AYFVYJQAPQTCCC-GBXIJSLDSA-N 0.000 description 4
- NQRYJNQNLNOLGT-UHFFFAOYSA-N Piperidine Chemical compound C1CCNCC1 NQRYJNQNLNOLGT-UHFFFAOYSA-N 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- MTCFGRXMJLQNBG-REOHCLBHSA-N (2S)-2-Amino-3-hydroxypropansäure Chemical compound OC[C@H](N)C(O)=O MTCFGRXMJLQNBG-REOHCLBHSA-N 0.000 description 3
- 125000003088 (fluoren-9-ylmethoxy)carbonyl group Chemical group 0.000 description 3
- HJBLUNHMOKFZQX-UHFFFAOYSA-N 3-hydroxy-1,2,3-benzotriazin-4-one Chemical compound C1=CC=C2C(=O)N(O)N=NC2=C1 HJBLUNHMOKFZQX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 208000029462 Immunodeficiency disease Diseases 0.000 description 3
- 101710125418 Major capsid protein Proteins 0.000 description 3
- 230000015572 biosynthetic process Effects 0.000 description 3
- 210000004899 c-terminal region Anatomy 0.000 description 3
- 230000002458 infectious effect Effects 0.000 description 3
- RLSSMJSEOOYNOY-UHFFFAOYSA-N m-cresol Chemical compound CC1=CC=CC(O)=C1 RLSSMJSEOOYNOY-UHFFFAOYSA-N 0.000 description 3
- 102000004196 processed proteins & peptides Human genes 0.000 description 3
- 108010043277 recombinant soluble CD4 Proteins 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 2
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 2
- 101710094648 Coat protein Proteins 0.000 description 2
- -1 Fmoc amino acid Chemical class 0.000 description 2
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 2
- 208000031886 HIV Infections Diseases 0.000 description 2
- 108010083930 HIV Receptors Proteins 0.000 description 2
- 102000006481 HIV Receptors Human genes 0.000 description 2
- 208000037357 HIV infectious disease Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 101710141454 Nucleoprotein Proteins 0.000 description 2
- 101710083689 Probable capsid protein Proteins 0.000 description 2
- IYYIVELXUANFED-UHFFFAOYSA-N bromo(trimethyl)silane Chemical compound C[Si](C)(C)Br IYYIVELXUANFED-UHFFFAOYSA-N 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000006482 condensation reaction Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- 125000000151 cysteine group Chemical class N[C@@H](CS)C(=O)* 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 208000033519 human immunodeficiency virus infectious disease Diseases 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 229960005486 vaccine Drugs 0.000 description 2
- VVQIIIAZJXTLRE-QMMMGPOBSA-N (2s)-2-amino-6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC[C@H](N)C(O)=O VVQIIIAZJXTLRE-QMMMGPOBSA-N 0.000 description 1
- ASOKPJOREAFHNY-UHFFFAOYSA-N 1-Hydroxybenzotriazole Chemical compound C1=CC=C2N(O)N=NC2=C1 ASOKPJOREAFHNY-UHFFFAOYSA-N 0.000 description 1
- FNQIGYRDLYROLW-UHFFFAOYSA-N 1-hydroxy-2h-1,2,3-benzotriazine Chemical compound C1=CC=C2N(O)NN=CC2=C1 FNQIGYRDLYROLW-UHFFFAOYSA-N 0.000 description 1
- YEDUAINPPJYDJZ-UHFFFAOYSA-N 2-hydroxybenzothiazole Chemical compound C1=CC=C2SC(O)=NC2=C1 YEDUAINPPJYDJZ-UHFFFAOYSA-N 0.000 description 1
- 229960000549 4-dimethylaminophenol Drugs 0.000 description 1
- 101710086578 Chaperone protein gp12 Proteins 0.000 description 1
- 101710177291 Gag polyprotein Proteins 0.000 description 1
- 241000713772 Human immunodeficiency virus 1 Species 0.000 description 1
- 206010061598 Immunodeficiency Diseases 0.000 description 1
- 102100034343 Integrase Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004793 Polystyrene Substances 0.000 description 1
- 101710102575 Pre-neck appendage protein Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 1
- 229920005654 Sephadex Polymers 0.000 description 1
- 239000012507 Sephadex™ Substances 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 210000001744 T-lymphocyte Anatomy 0.000 description 1
- AYFVYJQAPQTCCC-UHFFFAOYSA-N Threonine Natural products CC(O)C(N)C(O)=O AYFVYJQAPQTCCC-UHFFFAOYSA-N 0.000 description 1
- 239000004473 Threonine Substances 0.000 description 1
- 101800001690 Transmembrane protein gp41 Proteins 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 210000004102 animal cell Anatomy 0.000 description 1
- 125000000637 arginyl group Chemical group N[C@@H](CCCNC(N)=N)C(=O)* 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 239000001569 carbon dioxide Substances 0.000 description 1
- 229910002092 carbon dioxide Inorganic materials 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000008614 cellular interaction Effects 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 229920001577 copolymer Polymers 0.000 description 1
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- MYRTYDVEIRVNKP-UHFFFAOYSA-N divinylbenzene Substances C=CC1=CC=CC=C1C=C MYRTYDVEIRVNKP-UHFFFAOYSA-N 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 238000002523 gelfiltration Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- NPZTUJOABDZTLV-UHFFFAOYSA-N hydroxybenzotriazole Substances O=C1C=CC=C2NNN=C12 NPZTUJOABDZTLV-UHFFFAOYSA-N 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 230000007813 immunodeficiency Effects 0.000 description 1
- 238000010166 immunofluorescence Methods 0.000 description 1
- 230000002163 immunogen Effects 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 210000002540 macrophage Anatomy 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 125000000538 pentafluorophenyl group Chemical group FC1=C(F)C(F)=C(*)C(F)=C1F 0.000 description 1
- 229920002223 polystyrene Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000010076 replication Effects 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 230000003612 virological effect Effects 0.000 description 1
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Peptides Or Proteins (AREA)
Description
【発明の詳細な説明】 〔産業上の利用分野〕 本発明は抗HIVペプチドに関するものである。DETAILED DESCRIPTION OF THE INVENTION [Field of Industrial Application] The present invention relates to an anti-HIV peptide.
本発明の抗HIVペプチドはヒト免疫不全ウエルス（HIV）
の感染を妨げる機能を有し、更に、HIV感染後の多該巨
細胞の形成も妨げる機能を有し、後天性ヒト免疫不全症
（エイズ）の治療または発症予防に使用することができ
るので、医療界に大きく貢献するものである。The anti-HIV peptides of the present invention are human immunodeficiency wealth (HIV)
Of the acquired human immunodeficiency disease (AIDS) can be used for the treatment or prevention of the onset of the human immunodeficiency disease (AIDS). It will greatly contribute to the medical world.
HIVによるエイズを治療または発症予防するための薬剤
としてはHIVが本来有し、そのウイルス粒子複製に必要
な逆転写酵素の阻害剤が実際に使用されてはいるが、正
常細胞に対する細胞毒性が強く、問題が多いとされてい
る。また、発症予防の目的にはワクチンが考えられる
が、HIVの外被蛋白質の抗原性が突然変異によって変化
するため、効果的なワクチンの開発は困難な状況にあ
る。Although HIV originally has a drug for treating or preventing the development of HIV-induced AIDS, an inhibitor of reverse transcriptase required for its viral particle replication is actually used, but its cytotoxicity against normal cells is strong. , There are many problems. Further, although a vaccine may be considered for the purpose of preventing onset, it is difficult to develop an effective vaccine because the antigenicity of the HIV coat protein is altered by mutation.
そこで、有効性がより高く、毒性のより低い薬剤の開発
が活発に行われているが、一群の有望な薬剤としてHIV
の細胞への結合または感染を阻害するものが考えられて
いる。Therefore, the development of more effective and less toxic drugs is being actively pursued, but as a group of promising drugs HIV
Are considered to inhibit the binding or infection of cells to cells.
その第１はHIVの外被蛋白質であるgp120やgp41に結合す
る抗体であり、該蛋白質を適当な動物に免疫して抗血清
やモノクローナル抗体として製することができる。しか
しながら、前述のように該蛋白質の抗原性は必ずしも一
定ではないため、変異を受けないアミノ酸配列に対する
抗体を見出すことが必要である上に、通常得られる抗体
は動物由来であるため、抗体もそのものが人体に対して
免疫原性を有し反復使用が不可能となる欠点がある。The first is an antibody that binds to HIV coat proteins gp120 and gp41, which can be produced as an antiserum or a monoclonal antibody by immunizing a suitable animal. However, as described above, since the antigenicity of the protein is not always constant, it is necessary to find an antibody against an amino acid sequence that is not mutated, and since the antibody that is usually obtained is of animal origin, the antibody itself is also However, it has the drawback that it is immunogenic to the human body and cannot be used repeatedly.
第２の試みとしては細胞上のHIV受容体であるCD4分子に
着目し、これに対する抗体を投与することにより細胞を
被覆し、HIVの感染から免れさせようとする考え方であ
る。この場合はHIVの結合を妨げることはできるが、同
時に正常細胞の機能に影響を及ぼすことは避けられな
い。The second attempt is to focus on the CD4 molecule, which is the HIV receptor on cells, and to administer an antibody against it to coat the cells and prevent them from being infected with HIV. In this case, it is possible to prevent the binding of HIV, but at the same time, it is inevitable to affect the function of normal cells.
そこで、抗体を使用する上での問題点を解決するために
HIVの受容体であるCD4分子そのものを治療に応用しよう
という考えが提案された。可溶性のCD4分子はHIVのgp12
0に結合し感染の伝播を防ぐ効果を示し、クラスII特異
的なＴ細胞相互作用やマクロファージの機能など正常な
細胞機能を阻害することはないと言われている。このよ
うな可溶性CD4分子が遺伝子工学的に作成されている。
（Hussey,R.E.ら。Nature第331巻78頁、1988年）また、
CD4分子上にあってHIVのgp120と結合する部分の検索を
行われている。Therefore, in order to solve the problems in using antibodies
The idea of applying the CD4 molecule itself, which is an HIV receptor, to therapy has been proposed. Soluble CD4 molecule is HIV gp12
It is said that it binds to 0, exhibits an effect of preventing the spread of infection, and does not inhibit normal cell functions such as class II-specific T cell interaction and macrophage function. Such a soluble CD4 molecule has been prepared by genetic engineering.
(Hussey, RE et al. Nature 331, 78, 1988)
A part of the CD4 molecule that binds to HIV gp120 is being searched.
（発明が解決しようとする問題点） 本発明はCD4分子を構成するアミノ酸鎖のうちHIVの感染
伝播を阻害し得る、より短いアミノ酸鎖、すなわち抗HI
Vペプチドを提供するものである。(Problems to be Solved by the Invention) The present invention relates to a shorter amino acid chain of the amino acid chains constituting the CD4 molecule, which is capable of inhibiting the transmission of HIV infection, that is, anti-HI.
It provides a V peptide.
本発明は、Pro−Leu−Ile−Ile−Lys−Asn−Leu−Lys−
Ile−Glu−Asp−Ser−Asp−Thr−Tyr−Ile−Cys−Glu−
Val−Glu−Aps−Gln−Lys−Glu−Glu−Val−Gln−Leu−
Leu−Val−Phe−Gly−Leu−Thr−Ala−Asn−Ser−Asp−
Thr−His−Leu−Leu−Gln−Gly−Gln−Ser−Leu−Thr−
Leu−Thr−Leu−Glu−Ser−Pro−Pro−Gly−Ser−Ser−
Pro−Ser−Val−Gln−Cys で示されるアミノ酸鎖より成る、抗HIV活性を有するペ
プチドに関するものである。The present invention is Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-
Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys-Glu-
Val-Glu-Aps-Gln-Lys-Glu-Glu-Val-Gln-Leu-
Leu-Val-Phe-Gly-Leu-Thr-Ala-Asn-Ser-Asp-
Thr-His-Leu-Leu-Gln-Gly-Gln-Ser-Leu-Thr-
Leu-Thr-Leu-Glu-Ser-Pro-Pro-Gly-Ser-Ser-
The present invention relates to a peptide having an anti-HIV activity, which consists of an amino acid chain represented by Pro-Ser-Val-Gln-Cys.
本発明の抗HIVペプチドは完全分子型である可溶性CD4よ
りも著じるしく短いが、gp120との結合にあずかる部分
のみを有するペプチドとなっており、不必要な反応に関
わる可能性は少く、特異性が高く、より有効なものと考
えられる。Although the anti-HIV peptide of the present invention is significantly shorter than soluble CD4 which is a complete molecular form, it is a peptide having only a part involved in binding to gp120, and is unlikely to be involved in an unnecessary reaction. It has high specificity and is considered to be more effective.
また、本発明の抗HIVペプチド用いれば、より有効な薬
剤をデザインしていく上でも、化学的方法などによる修
飾が容易となる。例えばアミノ酸の一部をＤ体にするな
どしてプロテアーゼ抵抗性とすることもできるし、適当
な薬剤を結合して、より効率的な抗HIV薬を創製するこ
とも可能となる。Further, when the anti-HIV peptide of the present invention is used, modification by a chemical method or the like becomes easy in designing a more effective drug. For example, it is possible to make a part of the amino acid D-form to make it protease resistant, and it is also possible to bind a suitable drug to create a more efficient anti-HIV drug.
次に本発明を詳細に説明する。Next, the present invention will be described in detail.
本発明者らはCD4分子をコードしているDNA塩基配列（Ma
ddon,P.J.et al.,Cell,47,333−348,1986）により決定
されているアミノ酸配列に基き、CD4分子の部分ペプチ
ドを合成した。合成は、Fmocアミノ酸を使用した固相法
（Sheppard,R.C.et al,J.Chem.Soc.,Chem.Comm.,165−1
66,1985）によって行った。即ち、まずそれぞれの部分
ペプチドのＣ−末端に相当するFmocアミノ酸ペンタフル
オロフェニル（Pfp）エステルをジメチルホルムアミド
中で４−ジメチルアミノピリジン存在下、ｐ−アルコキ
シベンジルアルコール樹脂に結合させ、次いで結合すべ
き別のFmocアミノ酸Pfpエステルと縮合反応により結合
させた。The present inventors have constructed a DNA nucleotide sequence (Ma
A partial peptide of the CD4 molecule was synthesized based on the amino acid sequence determined by Ddon, PJ et al., Cell, 47 , 333-348, 1986). The synthesis was carried out by the solid phase method using Fmoc amino acid (Sheppard, RC et al, J. Chem. Soc., Chem. Comm., 165-1).
66, 1985). That is, first, Fmoc amino acid pentafluorophenyl (Pfp) ester corresponding to the C-terminal of each partial peptide should be bound to p-alkoxybenzyl alcohol resin in the presence of 4-dimethylaminopyridine in dimethylformamide, and then bound. It was coupled by condensation reaction with another Fmoc amino acid Pfp ester.
また、スレオニンとセリンについては１−オキソ−２−
ヒドロキシ−ジヒドロ−ベンゾトリアジン（DHBT）エス
テルを用いて導入した。各アミノ酸のカップリング反応
終了後、ペプチドの結合した樹脂を20％ピペリジン・ジ
メチルホルムアミド混液で処理してＮ末端Fmoc基を除去
し、次いでｍ−クレゾール存在下、室温にてTFA−チオ
アニソールを作用させて脱保護をすると同時に、目的と
する部分ペプチドを樹脂より回収した、また、アルギニ
ン残基を含むペプチドはトリメチルシリルブロミド（TM
SBr）−チオアニソール/TFAで処理を行った。For threonine and serine, 1-oxo-2-
It was introduced using hydroxy-dihydro-benzotriazine (DHBT) ester. After completion of the coupling reaction of each amino acid, the peptide-bonded resin was treated with a 20% piperidine / dimethylformamide mixed solution to remove the N-terminal Fmoc group, and then TFA-thioanisole was allowed to act at room temperature in the presence of m-cresol. At the same time, the target partial peptide was recovered from the resin, and the peptide containing the arginine residue was trimethylsilyl bromide (TM).
Treatment with SBr) -Thioanisole / TFA.
本発明者らは上述の方法によりCD4分子の部分ペプチド
を合成したが、その方法はこれに限られるわけではなく
他の化学合成法、例えばBocアミノ酸を用いる方法や、
当該部分ペプチドに対応するDNAを得て、これを適当な
ベクターに挿入し、動物細胞や微生物で発現させて目的
とする部分ペプチドを得ても良いのである。The present inventors have synthesized a partial peptide of the CD4 molecule by the above method, but the method is not limited to this, other chemical synthesis methods, for example, a method using Boc amino acid,
It is also possible to obtain the DNA corresponding to the partial peptide, insert this into an appropriate vector, and express it in animal cells or microorganisms to obtain the desired partial peptide.
本発明者らはこのようにして得たCD4分子の部分ペプチ
ドを、HIV感受性の細胞株MT−４とHIVの感染系に添加し
て本発明でいう抗HIV活性、すなわちHIV感染による細胞
の死滅や変性を阻害する活性を有するペプチドを選択し
本発明の抗HIVペプチドを得るに至ったのである。The present inventors have added the partial peptide of the CD4 molecule thus obtained to the infection system of HIV-sensitive cell lines MT-4 and HIV to have anti-HIV activity in the present invention, that is, kill cells by HIV infection. Therefore, the anti-HIV peptide of the present invention was obtained by selecting a peptide having an activity of inhibiting denaturation.
尚、本ペプチドはそのＮ末端、Ｃ末端にシステインが存
在する。従って本ペプチドの安定性を保つためには遊離
のSH基を保護しておく必要がある。この目的のために通
常はアセトメチル基が使用される。The peptide has cysteines at its N-terminal and C-terminal. Therefore, in order to maintain the stability of this peptide, it is necessary to protect the free SH group. The acetomethyl group is usually used for this purpose.
実施例１ ペプチドＣ末端のシステインをその誘導体Fmoc−Cys（A
cm）−opfp活性エステル（1mmol）及び触媒としてDMAP
（４−ジメチルアミノピリジン）（0.2mmol）を用い、
ジメチルホルムアミド（DMF）中、ｐ−alkoxy benzyl a
lchol Resin（0.2mmol）（0.35meq OH/g,Polystyrene 1
％ Divinyl benzene Copolymer.国産化学（株））上に
エステル結合により導入した。Example 1 Peptide C-terminal cysteine was used as a derivative Fmoc-Cys (A
cm) -opfp active ester (1 mmol) and DMAP as catalyst
(4-dimethylaminopyridine) (0.2 mmol) was used,
P-alkoxy benzyl a in dimethylformamide (DMF)
lchol Resin (0.2mmol) (0.35meq OH / g, Polystyrene 1
% Divinyl benzene Copolymer. Kokusan Kagaku Co., Ltd. was introduced by an ester bond.
使用したFmoc−アミノ酸誘導体（ミリジエン製）の側鎖
の保護は、Asp（OBut）、Glu（OBut）、Thr（But）、Se
r（But）、Lyr（Bot）、Lys（Boc）、His（Boc）、Arg
（Mtr）、Cys（Acm）で、ペプチド鎖伸長時の各縮合反
応は、Thr.Serを除きすべてPfp（ペンタフルオロフェニ
ル）活性エステル体（2.5eq）を用い触媒としてHOBT
（１−ハイドロキシベンゾトリアゾール）0.2mmolの存
在下DMF中で行なった。一方Ser.ThrはDHBT（3,4−ジハ
イドロ−３−ハイドロキシ−４−オキソ−1,2,3−ベン
ゾトリアジン）エステルを用いた。The side chains of the used Fmoc-amino acid derivative (manufactured by Millidiene) were protected by Asp (OBu t ), Glu (OBu t ), Thr (Bu t ), Se.
r (Bu t ), Lyr (Bo t ), Lys (Boc), His (Boc), Arg
With (Mtr) and Cys (Acm), each condensation reaction during peptide chain extension uses Pfp (pentafluorophenyl) active ester (2.5 eq) except Thr.Ser, and HOBT as a catalyst.
It was carried out in DMF in the presence of 0.2 mmol of (1-hydroxybenzotriazole). On the other hand, Ser.Thr used DHBT (3,4-dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine) ester.
各々のFmoc基の除去は、20％ピペリジのDMF溶液を用い
た。更に各々のcoupling reactionは、ニンヒドリンで
モニターした。The removal of each Fmoc group used a 20% piperidi DMF solution. In addition, each coupling reaction was monitored with ninhydrin.
すべてのCouplng反応終了後、Ｎ末端に残ったFmoc基を2
0％ピペリジン/DMFにより除去し、NHZ−基をフリーと
し、その後、すべての他の保ご基の除去と、樹脂からペ
プチドを切り出すために、TFA−thioanisoleでｍ−cres
ole存在下で室温３時間処理し、グラスフィルターでろ
過し、樹脂を除き、ろ液を室温で濃縮し、これにether
を加えpowderを得た。これを、集め、適当なBufferに溶
解し、Sephadex G−25にかけ、0.5N AcOHで溶出させ、
脱塩、精製を行なった。一方Argを含むペプチドは、TMS
Br−thioanisole/TEAでｍ−cresole存在下０℃60min処
理し、以後同様に処理した。ゲルろ過後のペプチドは更
にHPLCで精製した。After completion of all Couplng reactions, the remaining Fmoc group at the N-terminus was
Removal with 0% piperidine / DMF to free the NH Z -group, then m-cres with TFA-thioanisole to remove all other retention groups and cleave the peptide from the resin.
in the presence of ole at room temperature for 3 hours, filtered through a glass filter to remove the resin, and concentrate the filtrate at room temperature.
Was added to obtain a powder. This was collected, dissolved in an appropriate buffer, applied to Sephadex G-25, and eluted with 0.5N AcOH,
It was desalted and purified. On the other hand, the peptide containing Arg is TMS
It was treated with Br-thioanisole / TEA in the presence of m-cresole at 0 ° C. for 60 minutes, and thereafter treated in the same manner. The peptide after gel filtration was further purified by HPLC.
HPLCの分析条件は0.1％TFA中アセトニトリル22％→32％
の濃度勾配（40分）でYMC−PACK R−ODS−５（4.6×250
mm）を流速1ml/分で流し、230nmでモニターした、目的
とする本発明の抗HIVペプチド（部分ペプチド70−132）
はリテンション・タイム26.85分で溶出した。収率は約
4.6％であった。また、この部分ペプチドのアミノ酸分
析の結果は理論値とほぼ一致した。HPLC analysis conditions are 0.1% acetonitrile in TFA 22% → 32%
YMC-PACK R-ODS-5 (4.6 x 250)
mm) at a flow rate of 1 ml / min and monitored at 230 nm, the anti-HIV peptide of the present invention (partial peptide 70-132).
Elutes with a retention time of 26.85 minutes. Yield is about
It was 4.6%. The result of amino acid analysis of this partial peptide was almost in agreement with the theoretical value.
実施例2.部分ペプチドの抗HIV活性 HIV−１（HTLV−III B）が既に持続感染しているMOLT−
４細胞の培養液をHIV液とし、これを10倍段階希釈した
後、合成した16個のペプチド（1mg/ml）それぞれと室温
で30分保持した。次にHIV感受性であるMT−４細胞１×1
06cells−mlと1:1（v/v）で混ぜ（全量0.2ml）、RPMI−
1640−10％FCS培養液で炭酸ガス培養器中、37℃で４日
間保持した。Example 2. Anti-HIV activity of partial peptides HIV-1 (HTLV-III B) MOLT- already persistently infected
A culture solution of 4 cells was used as an HIV solution, which was serially diluted 10-fold, and then held with each of the 16 synthesized peptides (1 mg / ml) at room temperature for 30 minutes. Next, HIV-sensitive MT-4 cells 1 x 1
Mix with 0 6 cells-ml at 1: 1 (v / v) (total volume 0.2 ml), RPMI-
The cells were maintained at 37 ° C. for 4 days in a carbon dioxide incubator with 1640-10% FCS culture solution.
感染価の測定はHIVのgag蛋白質p18に対するモノクロー
ナル抗体を用いた免疫蛍光法で行った。The infectious titer was measured by an immunofluorescence method using a monoclonal antibody against HIV gag protein p18.
表１に示すように部分ペプチド70−132（CD4分子のＮ末
端より数え、70番目から132番目のアミノ酸鎖により成
るペプチド）に抗HIV活性が認められた。As shown in Table 1, anti-HIV activity was observed in the partial peptide 70-132 (a peptide consisting of the 70th to 132nd amino acid chains counted from the N-terminal of the CD4 molecule).
尚、感染価（TCID50）は値の小さいものほど抗HIV活性
が高い。The smaller the infectious titer (TCID 50 ) is, the higher the anti-HIV activity is.
部分ペプチド 感染価（TCID50） Control 106.33 １−69 ＞106.50 ８−25 106.33 18−69 105.50 26−47 105.33 47−69 105.50 70−132 101.50 86−132 103.66 96−110 103.50 111−132 ＞106.50 120−161 ＞106.50 132−139 105.66 161−180 105.00 190−197 106.00 318−334 106.33 このCD4ペプチド70−132は、量に比例して抗HIV増殖阻
止活性が認められた（図1a）。更に、多核巨細胞形成阻
止活性が認められた（図1b）。多核巨細胞形成能は、MO
LT−４細胞と、HIVに持続感染したMOLT−４細胞を10:1
の割合で混合し、24時間、37℃保持した後判定した。Partial peptide Infectious titer (TCID 50 ) Control 10 6.33 1-69> 10 6.50 8-25 10 6.33 18-69 10 5.50 26-47 10 5.33 47-69 10 5.50 70-132 10 1.50 86-132 10 3.66 96-110 10 3.50 111-132> 10 6.50 120-161> 10 6.50 132-139 10 5.66 161-180 10 5.00 190-197 10 6.00 318-334 10 6.33 This CD4 peptide 70-132 is anti-HIV proliferation in proportion to the amount. Inhibitory activity was observed (Fig. 1a). Furthermore, a multinucleated giant cell formation inhibitory activity was observed (Fig. 1b). The ability to form multinucleated giant cells is MO
10: 1 between LT-4 cells and MOLT-4 cells persistently infected with HIV
The mixture was mixed at a ratio of, and kept at 37 ° C. for 24 hours, and then judged.
ここでいう部分ペプチドのアミノ酸配列は以下に示す通
りである。The amino acid sequence of the partial peptide referred to here is as shown below.
〔部分ペプチド１−69〕 Ｈ−Gln−Vly−Asn−Lys−Val−Val−Leu−Gly−Lys−L
ys−Gly−Asp−Thr−Val−Glu−Leu−Thr−Cys−Thr−A
la−Ser−Gln−Lys−Lys−Ser−Ile−Gln−Phe−His−T
rp−Lys−Asn−Ser−Asn−Gln−Ile−Lys−Ile−Leu−G
ly−Asn−Gln−Gly−Ser−Phe−Leu−Thr−Lys−Gly−P
ro−Ser−Lys−Leu−Asn−Asp−Arg−Ala−Asp−Ser−A
rg−Arg−Ser−Leu−Trp−Asp−Gln−Gly−Asn−Phe−O
H 〔部分ペプチド８−25〕 Ｈ−Gly−Lys−Lys−Gly−Asp−Thr−Val−Glu−Leu−T
hr−Cys−Thr−Ala−Ser−Gln−Lys−Lys−Ser−OH 〔部分ペプチド18−69〕 Ｈ−Cys−Thr−Ala−Ser−Gln−Lys−Lys−Ser−Ile−G
ln−Phe−His−Trp−Lys−Asn−Ser−Asn−Gln−Ile−L
ys−Ile−Leu−Gly−Asn−Gln−Gly−Ser−Phe−Leu−T
hr−Lys−Gly−Pro−Ser−Lys−Leu−Asn−Asp−Arg−A
la−Asp−Ser−Arg−Arg−Ser−Leu−Trp−Asp−Gln−G
ly−Asn−Phe−OH 〔部分ペプチド26−47〕 Ｈ−Ile−Gln−Phe−His−Trp−Lys−Asn−Ser−Asn−G
ln−Ile−Lys−Ile−Leu−Gly−Asn−Gln−Gly−Ser−P
he−Leu−Thr−OH 〔部分ペプチド47−69〕 Ｈ−Thr−Lys−Gly−Pro−Ser−Lys−Leu−Asn−Asp−A
rg−Ala−Asp−Ser−Arg−Arg−Ser−Leu−Trp−Asp−G
ln−Gly−Asn−Phe−OH 〔部分ペプチド70−132〕 Ｈ−Pro−Leu−Ile−Ile−Lys−Asn−Leu−Lys−Ile−G
lu−Asp−Ser−Asp−Thr−Tyr−Ile−Cys−Glu−Val−G
lu−Asp−Gln−Lys−Glu−Glu−Val−Gln−Leu−Leu−V
al−Phe−Gly−Leu−Thr−Ala−Asn−Ser−Asp−Thr−H
is−Leu−Leu−Gln−Gly−Gln−Ser−Leu−Thr−Leu−T
hr−Leu−Thr−Leu−Glu−Ser−Pro−Pro−Gly−Ser−S
er−Pro−Ser−Val−Gln−Cys−OH 〔部分ペプチド86−132〕 Ｈ−Cys−Glu−Val−Clu−Asp−Gln−Lys−Clu−Glu−V
al−Gln−Leu−Leu−Val−Phe−Gly−Leu−Thr−Ala−A
sn−Ser−Asp−Thr−His−Leu−Leu−Gln−Gly−Gln−S
er−Leu−Thr−Leu−Thu−Ler−Glu−Ser−Pro−Pro−G
ly−Ser−Ser−Pro−Ser−Val−Gln−Cys−OH 〔部分ペプチド96−110〕 Ｈ−Gln−Leu−Leu−Val−Phe−Gly−Leu−Thr−Ala−A
sn−Ser−Asp−Thr−His−Leu−OH 〔部分ペプチド111−132〕 Ｈ−Leu−Gln−Gly−Gln−Ser−Leu−Thr−Leu−Thr−L
eu−Glu−Ser−Pro−Pro−Gly−Ser−Ser−Pro−Ser−V
al−Gln−Cys−OH 〔部分ペプチド120−161〕 Ｈ−Leu−Glu−Ser−Pro−Pro−Gly−Ser−Ser−Pro−S
er−Val−Gln−Cys−Arg−Ser−Pro−Arg−Gly−Lys−A
sn−Ile−Gln−Gly−Gly−Lys−Thr−Leu−Ser−Val−S
er−Gln−Leu−Glu−Leu−Gln−Asp−Ser−Gly−Thr−T
rp−Thr−Cys−OH 〔部分ペプチド132−139〕 Ｈ−Cys−Arg−Ser−Pro−Arg−Gly−Lys−Asn−OH 〔部分ペプチド161−180〕 Ｈ−Cys−Thr−Val−Leu−Gln−Asn−Gln−Lys−Lys−V
al−Glu−Phe−Lys−Ile−Asp−Ile−Val−Val−Leu−A
la−OH 〔部分ペプチド190−197〕 Ｈ−Lys−Lys−Glu−Gly−Glu−Gln−Val−Glu−OH 〔部分ペプチド318−334〕 Ｈ−Ser−Leu−Lys−Leu−Glu−Asn−Lys−Glu−Ala−L
ys−Val−Ser−Lys−Arg−Glu−Lys−Ala−OH[Partial peptide 1-69] H-Gln-Vly-Asn-Lys-Val-Val-Leu-Gly-Lys-L
ys-Gly-Asp-Thr-Val-Glu-Leu-Thr-Cys-Thr-A
la-Ser-Gln-Lys-Lys-Ser-Ile-Gln-Phe-His-T
rp-Lys-Asn-Ser-Asn-Gln-Ile-Lys-Ile-Leu-G
ly-Asn-Gln-Gly-Ser-Phe-Leu-Thr-Lys-Gly-P
ro-Ser-Lys-Leu-Asn-Asp-Arg-Ala-Asp-Ser-A
rg-Arg-Ser-Leu-Trp-Asp-Gln-Gly-Asn-Phe-O
H [partial peptide 8-25] H-Gly-Lys-Lys-Gly-Asp-Thr-Val-Glu-Leu-T
hr-Cys-Thr-Ala-Ser-Gln-Lys-Lys-Ser-OH [partial peptide 18-69] H-Cys-Thr-Ala-Ser-Gln-Lys-Lys-Ser-Ile-G
ln-Phe-His-Trp-Lys-Asn-Ser-Asn-Gln-Ile-L
ys-Ile-Leu-Gly-Asn-Gln-Gly-Ser-Phe-Leu-T
hr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-Arg-A
la-Asp-Ser-Arg-Arg-Ser-Leu-Trp-Asp-Gln-G
ly-Asn-Phe-OH [partial peptide 26-47] H-Ile-Gln-Phe-His-Trp-Lys-Asn-Ser-Asn-G
ln-Ile-Lys-Ile-Leu-Gly-Asn-Gln-Gly-Ser-P
he-Leu-Thr-OH [partial peptide 47-69] H-Thr-Lys-Gly-Pro-Ser-Lys-Leu-Asn-Asp-A
rg-Ala-Asp-Ser-Arg-Arg-Ser-Leu-Trp-Asp-G
ln-Gly-Asn-Phe-OH [partial peptide 70-132] H-Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys-Ile-G
lu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys-Glu-Val-G
lu-Asp-Gln-Lys-Glu-Glu-Val-Gln-Leu-Leu-V
al-Phe-Gly-Leu-Thr-Ala-Asn-Ser-Asp-Thr-H
is-Leu-Leu-Gln-Gly-Gln-Ser-Leu-Thr-Leu-T
hr-Leu-Thr-Leu-Glu-Ser-Pro-Pro-Gly-Ser-S
er-Pro-Ser-Val-Gln-Cys-OH [partial peptide 86-132] H-Cys-Glu-Val-Clu-Asp-Gln-Lys-Clu-Glu-V
al-Gln-Leu-Leu-Val-Phe-Gly-Leu-Thr-Ala-A
sn-Ser-Asp-Thr-His-Leu-Leu-Gln-Gly-Gln-S
er-Leu-Thr-Leu-Thu-Ler-Glu-Ser-Pro-Pro-G
ly-Ser-Ser-Pro-Ser-Val-Gln-Cys-OH [partial peptide 96-110] H-Gln-Leu-Leu-Val-Phe-Gly-Leu-Thr-Ala-A
sn-Ser-Asp-Thr-His-Leu-OH [partial peptide 111-132] H-Leu-Gln-Gly-Gln-Ser-Leu-Thr-Leu-Thr-L
eu-Glu-Ser-Pro-Pro-Gly-Ser-Ser-Pro-Ser-V
al-Gln-Cys-OH [partial peptide 120-161] H-Leu-Glu-Ser-Pro-Pro-Gly-Ser-Ser-Pro-S
er-Val-Gln-Cys-Arg-Ser-Pro-Arg-Gly-Lys-A
sn-Ile-Gln-Gly-Gly-Lys-Thr-Leu-Ser-Val-S
er-Gln-Leu-Glu-Leu-Gln-Asp-Ser-Gly-Thr-T
rp-Thr-Cys-OH [partial peptide 132-139] H-Cys-Arg-Ser-Pro-Arg-Gly-Lys-Asn-OH [partial peptide 161-180] H-Cys-Thr-Val-Leu- Gln-Asn-Gln-Lys-Lys-V
al-Glu-Phe-Lys-Ile-Asp-Ile-Val-Val-Leu-A
la-OH [partial peptide 190-197] H-Lys-Lys-Glu-Gly-Glu-Gln-Val-Glu-OH [partial peptide 318-334] H-Ser-Leu-Lys-Leu-Glu-Asn- Lys-Glu-Ala-L
ys-Val-Ser-Lys-Arg-Glu-Lys-Ala-OH
図1aは、実施例２においてCD4ペプチド70−132の量に比
例して抗HIV増殖阻止活性が認められた図で、図1bは同
じく多核巨細胞形成阻止活性が認められた図である。FIG. 1a shows that anti-HIV growth inhibitory activity was observed in proportion to the amount of CD4 peptide 70-132 in Example 2, and FIG. 1b shows that multinucleated giant cell formation inhibitory activity was similarly observed.
───────────────────────────────────────────────────── フロントページの続き (72)発明者 藤井 信孝 京都府京都市左京区吉田下阿達町69―29 京都大学薬学部薬品製造学教室内 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Nobutaka Fujii 69-29 Yoshida Shimoda-cho, Sakyo-ku, Kyoto City, Kyoto Prefecture Kyoto University Faculty of Pharmaceutical Sciences
Claims (1)
−Ile−Glu−Asp−Ser−Asp−Thr−Tyr−Ile−Cys−Glu
−Val−Glu−Aps−Gln−Lys−Glu−Glu−Val−Gln−Leu
−Leu−Val−Phe−Gly−Leu−Thr−Ala−Asn−Ser−Asp
−Thr−His−Leu−Leu−Gln−Gly−Gln−Ser−Leu−Thr
−Leu−Thr−Leu−Glu−Ser−Pro−Pro−Gly−Ser−Ser
−Pro−Ser−Val−Gln−Cys で示されるアミノ酸鎖より成る、抗HIV活性を有するペ
プチド。1. Pro-Leu-Ile-Ile-Lys-Asn-Leu-Lys
-Ile-Glu-Asp-Ser-Asp-Thr-Tyr-Ile-Cys-Glu
-Val-Glu-Aps-Gln-Lys-Glu-Glu-Val-Gln-Leu
-Leu-Val-Phe-Gly-Leu-Thr-Ala-Asn-Ser-Asp
-Thr-His-Leu-Leu-Gln-Gly-Gln-Ser-Leu-Thr
-Leu-Thr-Leu-Glu-Ser-Pro-Pro-Gly-Ser-Ser
-A peptide having an anti-HIV activity, which comprises an amino acid chain represented by Pro-Ser-Val-Gln-Cys.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63303972A JPH0768273B2 (en) | 1988-12-02 | 1988-12-02 | Anti-HIV peptide |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP63303972A JPH0768273B2 (en) | 1988-12-02 | 1988-12-02 | Anti-HIV peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JPH02152989A JPH02152989A (en) | 1990-06-12 |
| JPH0768273B2 true JPH0768273B2 (en) | 1995-07-26 |
Family
ID=17927494
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP63303972A Expired - Lifetime JPH0768273B2 (en) | 1988-12-02 | 1988-12-02 | Anti-HIV peptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JPH0768273B2 (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111732632A (en) * | 2020-07-16 | 2020-10-02 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
Families Citing this family (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03254693A (en) * | 1990-03-06 | 1991-11-13 | Calpis Food Ind Co Ltd:The | Antigen for preparation of anti-idiotype antibody, anti-idiotype antibody and its production |
| WO1994000488A1 (en) * | 1992-06-23 | 1994-01-06 | Sumitomo Pharmaceuticals Company, Limited | Anti-hiv peptide or peptide derivative |
| US6090388A (en) * | 1998-06-20 | 2000-07-18 | United Biomedical Inc. | Peptide composition for prevention and treatment of HIV infection and immune disorders |
| EP1460088A1 (en) | 2003-03-21 | 2004-09-22 | Biotest AG | Humanized anti-CD4 antibody with immunosuppressive properties |
| WO2008092905A2 (en) | 2007-02-01 | 2008-08-07 | Boehringer Ingelheim International Gmbh | Specific activation of a regulatory t cell and its use for treatment of asthma, allergic disease, autoimmune disease, graft rejection and for tolerance induction |
| EP2260057B1 (en) | 2008-03-13 | 2016-11-23 | Biotest AG | Anti-CD4 antibody dosage regimen for treating autoimmune disease |
| RU2540013C2 (en) | 2008-03-13 | 2015-01-27 | Биотест Аг | Agent for treating disease |
| GB0920944D0 (en) | 2009-11-30 | 2010-01-13 | Biotest Ag | Agents for treating disease |
-
1988
- 1988-12-02 JP JP63303972A patent/JPH0768273B2/en not_active Expired - Lifetime
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111732632A (en) * | 2020-07-16 | 2020-10-02 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
| CN111732632B (en) * | 2020-07-16 | 2021-12-21 | 台州吉诺生物科技有限公司 | Synthesis method of linaclotide |
Also Published As
| Publication number | Publication date |
|---|---|
| JPH02152989A (en) | 1990-06-12 |
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