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Experimental Allergic Encephalomyelitis (EAE) Peptides are active fragment of the myelin basic protein. By a cellmediated immune response, the peptide causes experimental allergic encephalomyelitis, which is an inflammatory demyelinating disease of the central nervous system. These peptides have been used as a model for studying multiple sclerosis (MS) due to the clinical and histopathological similarities of the inflammatory diseases affecting the central nervous system. Both Myelin PLP (PLP-3602-PI) and MOG (PMG-3660-PI) are antigenic peptides that induce EAE by binding to MHCII molecules on antigen presenting cells where they are recognized by class-II restricted T cells.

Discovery
Westall et al., in 1971 identified a peptide that causes experimental allergic encephalomyelitis 1,2. EAE is an autoimmune disease inducible by encephalitogenic helper T cells expressing Vβ8. Owhashi M et al., in 1997 examined the relationship between the stressor-induced alternation of clinical EAE and the induction of autoreactive T cells using Lewis rats 3.

Structural Characteristics
Belogurov AA et al demonstrated that autoantibodies (AAb) in multiple sclerosis (MS) reveal site-specific binding and cleavage toward myelin basic protein (MBP) epitope library. They have found several fragments of MBP immunodominant in terms of AAb binding and applied these peptides to DA rats with induced protracted relapsing EAE most closely related to MS. DA rats with EAE induced by syngenic spinal cord homogenate in complete Freund's adjuvant were treated by nasal route with human MBP 46–62, 81–102, 124–139, 147–170, and Copaxone®. MBP 124–139 and 147–170 displayed only mild therapeutic effects but MBP 46–62 significantly reduced EAE, reflected by lower clinical scores and shorter EAE duration compared to controls 4.  Three peptides overlapping the tryptophan region of bovine CNS myelin basic protein were synthesized by the solid phase procedure of Merrifield. These were the nonapeptide H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, the octapeptide H-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, and the heptapeptide H-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH. They were tested fro encephalitogenic activity in guinea pigs with either Freund's complete adjuvant containing M. tuberculosis or muramyl dipeptide in incomplete Freud's adjuvant at doses of 10 µg per animal. The results show that deletion of one or two residues from the amino-terminal end of the nonapeptide destroyed the ability of the shorter peptides to induce clinical but not histological signs of EAE 5. 

Mode of Action
Proteolipid protein (PLP) is the major protein of central nervous system (CNS) myelin. SJL(H-2S) mice immunized with a synthetic peptide corresponding to PLP residues 139-151 (HSLGKWLGHPDKF) develop acute EAE. A T cell line and 4 clones were derived from SJL/J mice were immunizied with this synthetic peptide. Severe clinical and histological EAE was induced by adoptive transfer of the peptide-specific T cell line and 3 of 4 T cell clones. The T cell line/clones all responded strongly to PLP peptide 139-151 in in vitro proliferative assays. However, two different reactivity patterns emerged when truncated PLP peptides 141-150 and 141-149 were tested, suggesting that more than 1 epitope may be present within the PLP 139-151 determinant. Two truncated PLP peptides were compared for the ability to induce EAE in vivo and proliferative responses in vitro. Immunization with PLP peptide 141-150 induced acute EAE in about 70% of mice tested, but PLP peptide 141-149 induced a comparatively mild form of EAE in 4 out of 9 mice tested. Lymph node cells from mice immunized with these peptides showed in vitro proliferative responses to each of the peptides, but the response to peptide 139-151 was always strongest. These combined in vivo and in vitro data further define the epitopes involved in PLP-induced EAE in SJL mice. Furthermore, the availability of multiple PLP-specific T cell clones will enable to study the diversity of the T cell repertoire to PLP 6.

Functions
Cell mediated autoimmune response, EAE is a cell mediated autoimmune response directed against autologous central nervous system myelin 7.
Sensitization with myelin basic protein (MBP), a major protein constituent of central nervous system compact myelin, emulsified in complete Freund's adjuvant, produce the full clinical and histological picture of EAE in a wide variety of animal.
The encephalitogenic determinants responsible for EAE induction are species-specific. That is, different sequences of amino acid residues located at unique positions within the MBP molecule are critical for the induction of EAE in each mammalian species 8.
Stressor-induced suppression of clinical EAE is not simply because of the failure of induction of autoreactive T cells, nor localization of the autoreactive T cells in the central nervous system 3.
Species-specific immune response, the capacity of MBP in complete Freund's adjuvant to induce an encephalitogenic immune response against autologous central nervous system myelin in a given mammalian species appears to be dictated by latent, species-specific immune response genes which presumably encode antigen-receptor molecules recognizing specific MBP sequences 1.

References

Westall FC, Robinson AB, Caccam J, Jackson J, Ylar EH, (1971). Essential chemical requirements for induction of allergic encephalomyelitis. Nature, 229(5279):22-24.
Shapira R, Chou FC, McKneally S, Urban E, Kibler RF (1971). Biologicl activity and synthesis of an encephalitogenic determinant. Science, 173(998):736-738.
Owhashi M, Shouzui Y, Arita H (1997). Stress down-regulates experimental allergic encephalomyelitis (EAE) but permits activation and localization of autoreactive vβ8.2+ T Cells.  International Journal of Neuroscience, 89(3-4):177-188.
Belogurov AA Jr, Zargarova TA, Turobov VI, Novikova NI, Favorova OO, Ponomarenko NA, Gabibov AG  (2009). Suppression of ongoing experimental allergic encephalomyelitis in DA rats by novel peptide drug, structural part of human myelin basic protein 46–62.   Autoimmunity, 42(4):362-364.
Levit S, Powers JM, Milek D, Brostoff SW (1980). Peptide length requirement for experimental allergic encephalomyelitis in guinea pigs. Neurochem Res., 5(1):37-42.
 Kuchroo VK, Sobel RA, Yamamura T, Greenfield E, Dorf ME, Lees MB (1991). Induction of Experimental Allergic Encephalomyelitis by Myelin Proteolipid-Protein-Specific T Cell Clones and Synthetic Peptides. Pathobiology, 59(5):305-312.
Paterson PY (1982). Molecular and cellular determinants of neuroimmunologic inflammatory disease. Fed. Proc. Fed. Am. Soc. Exp. Biol., 41:2569-2576.
Hashim GA (1978). Myelin basic protein: structure, function and antigenic determinants. Immunol. Rev., 39:60-107.

多肽H2N-Tyr-Gly-Ser-Leu-Pro-Gln-Lys-Ala-Gln-Arg-Pro-Gln-Asp-Glu-Asn-COOH的合成步骤：

1、合成CTC树脂：称取2.98g CTC Resin（如初始取代度约为0.85mmol/g）和3.04mmol Fmoc-Asn(Trt)-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7.6mmol 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-Asn(Trt)-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-Asn(Trt)-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7.6mmol Fmoc-Glu(OtBu)-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15.2mmol DIPEA，7.22mmol 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-Glu(OtBu)-Asn(Trt)-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-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Ser(tBu)-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Ser(tBu)-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Gly-Ser(tBu)-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

H2N-Gly-Ser(tBu)-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-CTC Resin

Fmoc-Tyr(tBu)-Gly-Ser(tBu)-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-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-Tyr(tBu)-Gly-Ser(tBu)-Leu-Pro-Gln(Trt)-Lys(Boc)-Ala-Gln(Trt)-Arg(Pbf)-Pro-Gln(Trt)-Asp(OtBu)-Glu(OtBu)-Asn(Trt)-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-Tyr-Gly-Ser-Leu-Pro-Gln-Lys-Ala-Gln-Arg-Pro-Gln-Asp-Glu-Asn-COOH。结构图见产品结构图。

切割液选择：1）TFA：H2O=95%：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，甲基化修饰肽等。

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