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CEF1, Influenza Matrix Protein M1 (58-66) 是源于甲型流感病毒 (influenza A virus) 的基质蛋白表位。
CEF1, Influenza Matrix Protein M1 (58-66) is an epitope derived from the matrix protein of the influenza A virus[1].

Portion of Influenza M

FluM1 (58-66) is a short part of the matrix protein of Influenza A virus which is the most abundant component of this enveloped virus localized under the viral lipid envelope. The GILGFVFTL epitope is highly conserved in Influenza A virus strains at a rate of 93% for 69 strains tested. Human Influenza epitopes may bind MHC molecules and then may be recognized by CD8+ cytotoxic T cells. Applications of FluM1 (58-66): FluM1 (58-66) is used to stimulate CTL responses in peripheral blood mononuclear cells (PBMCs). Then, ELISPOT assay is used to quantify peptide epitope specificity and IFN-γ releasing effector cells. FluM1 (58-66) has shown CTL responses qualified of immunodominant with restriction by HLA-A*02:01. It has also been detected CTL responses when FluM1 (58-66) is restricted by all HLA-C. Therefore, FluM1 (58-66) may help to understand the reaction of immune system against Influenza virus of each populations having different HLA type. FluM1 (58-66) has indeed prompted research to develop T-cell vaccine strategies capable of inducing specific CTL responses in patients upon immunization with Influenza M1 antigenic epitope. MVA-NP+M1 vaccine use GILGFVFTL epitope and is tested in clinical trial. Potential cross-reactivity with HIV-1 p17 Gag (77-85): Moreover, FluM1 (58-66) share similarities with HIV-1 p17 Gag (77-85) which can potentially show a cross-reactivity between these epitopes. It has been demonstrated a cross-reactivity and results suggest that immunity following infection by Influenza virus causes specific immune response to HIV-1 p17 Gag (77-85).

Definition

The CEF control peptides are 8-12 amino acids in length, with sequences derived from the human Cytomegalovirus, Epstein-Barr Virus and Influenza Virus1 These peptides are used in the stimulation of IFNg release from CD8+ T cells in individuals with defined HLA types1.  They are useful as positive control peptides in several cytokine assays such as Elispot.

Discovery

CEF peptides were first selected in 2002 based on their ability to recognize CD8+ T cells1.

Classification

They are derived from epitopes of viruses and hence have antigenic properties1.

Structural Characteristics

CEF peptides are 8-11 amino acids long with sequences: GILGFVFTL (Influenza A, HLA-A2), FMYSDFHFI (Influenza A, HLA-A2), CLGGLLTMV (EBV, HLA-A2), GLCTLVAML (EBV, HLA-A2), NLVPMVATV (HCMV, HLA-A2).

Mode of action

CEF peptides are effective epitopes for CD8+ T cells2.  They bind to these cells and trigger the production of IFNg.

Functions

CEF control peptides are used as positive control in Elispot assay that is used to investigate specific immune responses in various diseases including infections, cancer, allergies and autoimmune diseases2.  In this case the CEF peptides ensure that the cells under study are active and viable2.  Elispot is also useful in the development of vaccines especially for HIV where CEF peptides are used also as controls2.

References

1.     Currier JR, Kuta EG, Turk E, Earhart LB, Loomis-Price L, Janetzki S, Ferrari G, Birx DL, Cox JH (2002). A panel of MHC class I restricted viral peptides for use as a quality control for vaccine trial ELISPOT assays, J Immunol Methods, 260, 157-172.

2.     Gazagne A, Claret E, Wijdenes J, Yssel H, Bousquet F, Levy E, Vielh P, Scotte F, Goupil T, Fridman WH, Tartour E (2003). A Fluorospot assay to detect single T lymphocytes simultaneously producing multiple cytokines, J Immunol Methods, 283(1-2), 91-98.

多肽H2N-Gly-Ile-Leu-Gly-Phe-Val-Phe-Thr-Leu-COOH的合成步骤：

1、合成CTC树脂：称取1.84g CTC Resin（如初始取代度约为0.44mmol/g）和0.97mmol Fmoc-Leu-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2.43mmol 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-Leu-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-Leu-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2.43mmol Fmoc-Thr(tBu)-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4.86mmol DIPEA，2.31mmol 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-Thr(tBu)-Leu-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-Thr(tBu)-Leu-CTC Resin

Fmoc-Phe-Thr(tBu)-Leu-CTC Resin

H2N-Phe-Thr(tBu)-Leu-CTC Resin

Fmoc-Val-Phe-Thr(tBu)-Leu-CTC Resin

H2N-Val-Phe-Thr(tBu)-Leu-CTC Resin

Fmoc-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

H2N-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

Fmoc-Gly-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

H2N-Gly-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

Fmoc-Leu-Gly-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

H2N-Leu-Gly-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

Fmoc-Ile-Leu-Gly-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

H2N-Ile-Leu-Gly-Phe-Val-Phe-Thr(tBu)-Leu-CTC Resin

Fmoc-Gly-Ile-Leu-Gly-Phe-Val-Phe-Thr(tBu)-Leu-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-Gly-Ile-Leu-Gly-Phe-Val-Phe-Thr(tBu)-Leu-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-Gly-Ile-Leu-Gly-Phe-Val-Phe-Thr-Leu-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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