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Insulin β Chain Peptide (15-23)，也成为 INS，是胰岛相关 T 细胞识别的胰岛素衍生肽。Insulin β Chain Peptide (15-23) 四聚体染色了 INS 反应性 CTL 克隆 G9C8，但该四聚体和阴性对照四聚体 (TUM) 均未染色 NOD 或 8.3-TCRαβ 转基因 NOD 小鼠脾脏 CD8+ T 细胞。
Insulin β Chain Peptide (15-23), also known as INS, is an insulin-derived peptide recognized by islet-associated T cells. The Insulin β Chain Peptide (15-23) tetramer stained the INS-reactive CTL clone G9C8, but neither this tetramer nor the negative control tetramer (TUM) stained the splenic CD8+ T cells from NOD or 8.3-TCRαβtransgenic NOD mice[1][2].

Peptide H-LYLVCGERG-OH is a Research Peptide with significant interest within the field academic and medical research. Recent citations using H-LYLVCGERG-OH include the following: During the early prediabetic period in NOD mice, the pathogenic CD8+ T-cell population comprises multiple antigenic specificities TP DiLorenzo, SM Lieberman , T Takaki, S Honda - Clinical , 2002 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S1521661602952988 Peripheral proinsulin expression controls low-avidity proinsulin-reactive CD8 T cells in type 1 diabetes TC Thayer, JA Pearson, E De Leenheer , SJ Hanna - Diabetes, 2016 - Am Diabetes Assochttps://diabetesjournals.org/diabetes/article-abstract/65/11/3429/17484 Convective-heating thermal decomposition/digestion of peptides and proteins on surfaces R Zhou, F Basile - Journal of Analytical and Applied Pyrolysis, 2017 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0165237016307987 Convective-Heating Thermal Decomposition/Digestion R Zhou, F Basile - Digestion, 2014 - academia.eduhttps://www.academia.edu/download/111737285/j.jaap.2017.07.00420240223-1-9015as.pdf A Single L/D-Substitution at Q4 of the mInsA2-10 Epitope Prevents Type 1 Diabetes in Humanized NOD Mice M Zhang, Y Wang, X Li, G Meng, X Chen - Frontiers in , 2021 - frontiersin.orghttps://www.frontiersin.org/articles/10.3389/fimmu.2021.713276/full Targeted suppression of autoreactive CD8+ T-cell activation using blocking anti-CD8 antibodies M Clement , JA Pearson, S Gras , HA Van Den Berg - Scientific reports, 2016 - nature.comhttps://www.nature.com/articles/srep35332 Functional inhibition related to structure of a highly potent insulin-specific CD8 T cell clone using altered peptide ligands LG Petrich de Marquesini - European journal of , 2008 - Wiley Online Libraryhttps://onlinelibrary.wiley.com/doi/abs/10.1002/eji.200737762 Proteomic identification of MHC class I-associated peptidome derived from non-obese diabetic mouse thymus and pancreas L Wang, X Li, S Yang, X Chen, J Li, S Wang - Journal of , 2023 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S1874391922002706 Peptide-specific cytotoxicity of T lymphocytes against glutamic acid decarboxylase and insulin in type 1 diabetes mellitus K Kimura, T Kawamura, S Kadotani, H Inada - Diabetes research and , 2001 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0168822700002254 The development of a sub/supercritical fluid chromatography based purification method for peptides K Govender , T Naicker , S Baijnath , HG Kruger - of Pharmaceutical and , 2020 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0731708520314254 Proinsulin Expression Shapes the TCR Repertoire but Fails to Control the Development of Low-Avidity Insulin-Reactive CD8+ T Cells JA Pearson, TC Thayer, JE McLaren, K Ladell - Diabetes, 2016 - Am Diabetes Assochttps://diabetesjournals.org/diabetes/article-abstract/65/6/1679/35164 Analysis of structure and function relationships of an autoantigenic peptide of insulin bound to H-2Kd that stimulates CD8 T cells in insulin-dependent diabetes FS Wong , AK Moustakas , L Wen - Proceedings of the , 2002 - National Acad Scienceshttps://www.pnas.org/doi/abs/10.1073/pnas.072037299 Targeting proinsulin to local immune cells using an intradermal microneedle delivery system; a potential antigen-specific immunotherapy for type 1 diabetes F Arikat, SJ Hanna, RK Singh, L Vilela, FS Wong - Journal of controlled , 2020 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0168365920301218 Adoptive transfer of autoimmune diabetes using immunodeficient nonobese diabetic (NOD) mice E De Leenheer , FS Wong - Type-1 Diabetes: Methods and Protocols, 2016 - Springerhttps://link.springer.com/protocol/10.1007/7651_2015_294 MCS-18, a novel natural plant product prevents autoimmune diabetes C Seifarth, L Littmann, Y Resheq, S Rössner - Immunology , 2011 - Elsevierhttps://www.sciencedirect.com/science/article/pii/S0165247811001325 Antigen-dependent immunotherapy of non-obese diabetic mice with immature dendritic cells C Haase, L Yu, G Eisenbarth - Clinical & Experimental , 2010 - academic.oup.comhttps://academic.oup.com/cei/article-abstract/160/3/331/6428884 Presentation of Antigen by Endothelial Cells and Chemoattraction Are Required for Homing of Insulin-specific CD8+ T Cells AY Savinov , FS Wong , AC Stonebraker - The Journal of , 2003 - rupress.orghttps://rupress.org/jem/article-abstract/197/5/643/39754 Contribution of Fas to diabetes development AY Savinov , A Tcherepanov - Proceedings of the , 2003 - National Acad Scienceshttps://www.pnas.org/doi/abs/10.1073/pnas.0237359100 Progression of autoimmune diabetes driven by avidity maturation of a T-cell population A Amrani, J Verdaguer , P Serra, S Tafuro, R Tan - Nature, 2000 - nature.comhttps://www.nature.com/articles/35021081

Amrani A, et al. Progression of autoimmune diabetes driven by avidity maturation of a T-cell population. Nature. 2000;406(6797):739-742. : https://pubmed.ncbi.nlm.nih.gov/10963600/
Takaki T, et al. Requirement for both H-2Db and H-2Kd for the induction of diabetes by the promiscuous CD8+ T cell clonotype AI4. J Immunol. 2004;173(4):2530-2541. : https://pubmed.ncbi.nlm.nih.gov/15294969/

多肽H2N-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-COOH的合成步骤：

1、合成CTC树脂：称取2.09g CTC Resin（如初始取代度约为0.82mmol/g）和2.06mmol Fmoc-Gly-OH于反应器中，加入适量DCM溶解氨基酸（需要注意，此时CTC树脂体积会增大好几倍，避免DCM溶液过少），再加入5.14mmol DIPEA（Mw：129.1,d：0.740g/ml），反应2-3小时后，可不抽滤溶液，直接加入1ml的HPLC级甲醇，封端半小时。依次用DMF洗涤2次，甲醇洗涤1次，DCM洗涤一次，甲醇洗涤一次，DCM洗涤一次，DMF洗涤2次（这里使用甲醇和DCM交替洗涤，是为了更好地去除其他溶质，有利于后续反应）。得到  Fmoc-Gly-CTC Resin。结构图如下：

2、脱Fmoc：加3倍树脂体积的20%Pip/DMF溶液，鼓氮气30分钟，然后2倍树脂体积的DMF 洗涤5次。得到 H2N-Gly-CTC Resin 。（此步骤脱除Fmoc基团，茚三酮检测为蓝色，Pip为哌啶）。结构图如下：

3、缩合：取5.14mmol Fmoc-Arg(Pbf)-OH 氨基酸，加入到上述树脂里，加适当DMF溶解氨基酸，再依次加入10.28mmol DIPEA，4.88mmol HBTU。反应30分钟后，取小样洗涤，茚三酮检测为无色。用2倍树脂体积的DMF 洗涤3次树脂。（洗涤树脂，去掉残留溶剂，为下一步反应做准备）。得到Fmoc-Arg(Pbf)-Gly-CTC Resin。氨基酸：DIPEA：HBTU：树脂=3：6：2.85：1（摩尔比）。结构图如下：

4、依次循环步骤二、步骤三，依次得到

H2N-Arg(Pbf)-Gly-CTC Resin

Fmoc-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

H2N-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

Fmoc-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

H2N-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

Fmoc-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

H2N-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

Fmoc-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

H2N-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

Fmoc-Leu-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

H2N-Leu-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

Fmoc-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

H2N-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

Fmoc-Leu-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin

以上中间结构，均可在专肽生物多肽计算器-多肽结构计算器中，一键画出。

最后再经过步骤二得到 H2N-Leu-Tyr(tBu)-Leu-Val-Cys(Trt)-Gly-Glu(OtBu)-Arg(Pbf)-Gly-CTC Resin，结构如下：

5、切割：6倍树脂体积的切割液（或每1g树脂加8ml左右的切割液），摇床摇晃 2小时，过滤掉树脂，用冰无水乙醚沉淀滤液，并用冰无水乙醚洗涤沉淀物3次，最后将沉淀物放真空干燥釜中，常温干燥24小试，得到粗品H2N-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-COOH。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：5%、TFA：H2O=97.5%：2.5%

2）TFA：H2O：TIS=95%：2.5%：2.5%

3）三氟乙酸：茴香硫醚：1，2-乙二硫醇：苯酚：水=87.5%：5%：2.5%：2.5%：2.5%

（前两种适合没有容易氧化的氨基酸，例如Trp、Cys、Met。第三种适合几乎所有的序列。）

6、纯化冻干：使用液相色谱纯化，收集目标峰液体，进行冻干，获得蓬松的粉末状固体多肽。不过这时要取小样复测下纯度 是否目标纯度。

7、最后总结：

杭州专肽生物技术有限公司（ALLPEPTIDE https://www.allpeptide.com）主营定制多肽合成业务，提供各类长肽，短肽，环肽，提供各类修饰肽，如：荧光标记修饰（CY3、CY5、CY5.5、CY7、FAM、FITC、Rhodamine B、TAMRA等），功能基团修饰肽（叠氮、炔基、DBCO、DOTA、NOTA等），同位素标记肽（N15、C13），订书肽（Stapled Peptide）,脂肪酸修饰肽（Pal、Myr、Ste），磷酸化修饰肽（P-Ser、P-Thr、P-Tyr），环肽（酰胺键环肽、一对或者多对二硫键环），生物素标记肽，PEG修饰肽，甲基化修饰肽

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