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EBV BMLF1 (280-288) is an epitope of the early lytic BMLF1 peptide of Epstein-Barr virus. EBV BMLF1 peptide presented by HLA-A*02:01 is one of the most immunogenic T-cell targets from the EBV proteome. EBV BMLF1 (280-288) can be used for the isolation and the stimulation of antigen-specific T cells.\nApplications of EBV BMLF1 (280-288):\nCD8+ cells have an important role in controlling Epstein-Barr virus infection. Indeed, the expansion of infected B cells is controlled by CD8+ cytotoxic T cells. Therefore, EBV BMLF1 (280-288) is important in study to detect EBV-specific CD8+ T cells by flow cytometry, per example in peripheral blood mononuclear cells of infected patients. EBV BMLF1 (280-288) is also used to analyze and measure the immune response and cytokine production in PBMCs cells to the virus with an application of ELISPOT assay.

CEF11 是 EB 病毒 BMLF1 来源肽，用于研究裂解周期蛋白保守基序。其短序列支持 β 转角与溶剂可及性评估。研究者通过结构可塑性定义识别决定因素。该分子扩充 EB 病毒片段库。

CEF11 is a BMLF1-derived EBV peptide used to study conserved motifs in lytic-cycle proteins. Its short sequence supports β-turn and solvent-accessibility evaluation. Researchers assess structural plasticity to define recognition determinants. The molecule enhances EBV fragment libraries.

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.

Steven NM, et al. Immediate early and early lytic cycle proteins are frequent targets of the Epstein-Barr virus-induced cytotoxic T cell response. J. Exp. Med. May 1997; 185(9): 1605-1617
M.F.C. Callan, et al. J. Exp. Med., Volume 187, Number 9, May 4, 1998

多肽H2N-Gly-Leu-Cys-Thr-Leu-Val-Ala-Met-Leu-COOH的合成步骤：

1、合成CTC树脂：称取1.68g CTC Resin（如初始取代度约为0.64mmol/g）和1.29mmol 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3.23mmol 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3.23mmol Fmoc-Met-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6.45mmol DIPEA，3.06mmol 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-Met-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-Met-Leu-CTC Resin

Fmoc-Ala-Met-Leu-CTC Resin

H2N-Ala-Met-Leu-CTC Resin

Fmoc-Val-Ala-Met-Leu-CTC Resin

H2N-Val-Ala-Met-Leu-CTC Resin

Fmoc-Leu-Val-Ala-Met-Leu-CTC Resin

H2N-Leu-Val-Ala-Met-Leu-CTC Resin

Fmoc-Thr(tBu)-Leu-Val-Ala-Met-Leu-CTC Resin

H2N-Thr(tBu)-Leu-Val-Ala-Met-Leu-CTC Resin

Fmoc-Cys(Trt)-Thr(tBu)-Leu-Val-Ala-Met-Leu-CTC Resin

H2N-Cys(Trt)-Thr(tBu)-Leu-Val-Ala-Met-Leu-CTC Resin

Fmoc-Leu-Cys(Trt)-Thr(tBu)-Leu-Val-Ala-Met-Leu-CTC Resin

H2N-Leu-Cys(Trt)-Thr(tBu)-Leu-Val-Ala-Met-Leu-CTC Resin

Fmoc-Gly-Leu-Cys(Trt)-Thr(tBu)-Leu-Val-Ala-Met-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-Leu-Cys(Trt)-Thr(tBu)-Leu-Val-Ala-Met-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-Leu-Cys-Thr-Leu-Val-Ala-Met-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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