专肽生物_定制多肽合成服务_多肽合成定制服务_多肽合成厂家_多肽合成公司

这种15聚体的微生物肽是（CFP）10蛋白的片段，其由结核分枝杆菌10kDa培养滤液分泌，刺激CD4+和CD8+细胞产生IFN-γ和CTL活性，这些细胞来自表达多种MHC II类和I类分子的人。

This 15-mer microbial peptide is fragment of (CFP)10 protein, which is secreted from mycobacterium tuberculosis 10-kDa culture filtrate stimulate IFN-γ production and CTL activity by CD4+ and CD8+ cells, from persons expressing multiple MHC class II and class I molecules. The capacity of CFP10(71-85) to stimulate IFN-gamma production and CTL activity by CD4(+) and CD8(+) cells from persons expressing a spectrum of MHC molecules suggests that this peptide is an excellent candidate for inclusion in a subunit antituberculosis vaccine.

Definition

Bacterial peptides are protein fragments which are either part of a bacterium or produced by a bacteria1.

Classification

Different classes of peptides are produced by bacteria. Some examples include, antibiotics, enterotoxins, flagellar proteins, lipoproteins and various enzymes1.

Structural Characteristics

Structural characteristics of some bacterial peptides are described below-

A)      Malaria merozoite surface peptide (MSP-1): It is synthesized as a large precursor on the surface of the bacterium Plasmodium falciparum.  Proteolytic cleavage results in the production of a 19 KDa product whose tertiary structure is maintained by disulphide bridges2.

B)     Giardia variable surface protein: This peptide is the specific conserved region of the Giardia variable surface proteins (VSPs) that are cysteine rich zinc finger proteins. VSPs differ in size and sequence, they are characterized by this highly conserved C-terminal membrane spanning region, a hydrophilic cytoplasmic tail with a conserved five amino acid CRGKA signature sequence3,4.

C)    P.falciparum liver stage antigen 3: The protein is 200Kda and is highly conserved among parasites from different geographic regions5.

Mode of action

A)     MSP-1 is known to trigger antibody response by CD4 helper T cells. It is likely that these cells bind to the C-terminal domain of MSP-12.

B)     VSPs have a conserved hydrophilic amono acid trail that is palmitoyted by palmityl tranferases upon which they are activated3,4.

C)    P. falciparum liver stage antigen 3 is a potent antigen that is recongnized by T cells5.

Functions

A)     MSP-1 is a vaccine candidate for Plasmodium falciparum infection. It triggers a CD-4 T cell response2.

B)     VSPs are necessary for survival in the environment and host infection3,4.

C)    P.falciparum stage antigen 3 is also a good candidate vaccine as it activates both T and B cell responses5.

References

1.     Gitai Z (2005). "The new bacterial cell biology: moving parts and subcellular architecture". Cell, 120 (5): 577–86.

2.     Stuart JQ and Jean L (2001). Different regions of the malaria merozoite surface protein 1 of Plasmodium chabaudi elicit distinct T-cell and antibody isotype responses. Infect Immun, 69(4): 2245–2251.

3.     Davids BJ, Reiner DS, Birkeland SR, Preheim SP, Cipriano MJ, McArthur AG, Gillin FD (2006). A New Family of Giardial Cysteine-Rich Non-VSP Protein Genes and a Novel Cyst Protein. PLoS ONE, 20,1:e44.

4.     Touz MC, Conrad JT, Nash TE (2005). A novel palmitoyl acyl transferase controls surface protein palmitoylation and cytotoxicity in Giardia lamblia. Mol Microbiol., 58 (4), 999-1011.

5.     Jean-Pierre S, Blanca LP, Karima B, Pierra D, Pierra D (2001). DNA Immunization by Plasmodium falciparum liver-stage antigen 3 induces protection against Plasmodium yoelii Sporozoite challenge. Infect Immun., 69, 1202–1206.

多肽H2N-Glu-Ile-Ser-Thr-Asn-Ile-Arg-Gln-Ala-Gly-Val-Gln-Tyr-Ser-Arg-COOH的合成步骤：

1、合成CTC树脂：称取1.25g CTC Resin（如初始取代度约为0.81mmol/g）和1.22mmol Fmoc-Arg(Pbf)-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3.04mmol 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-Arg(Pbf)-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-Arg(Pbf)-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3.04mmol Fmoc-Ser(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6.08mmol DIPEA，2.89mmol 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-Ser(tBu)-Arg(Pbf)-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-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Ser(tBu)-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Ser(tBu)-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Ile-Ser(tBu)-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

H2N-Ile-Ser(tBu)-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-CTC Resin

Fmoc-Glu(OtBu)-Ile-Ser(tBu)-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-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-Glu(OtBu)-Ile-Ser(tBu)-Thr(tBu)-Asn(Trt)-Ile-Arg(Pbf)-Gln(Trt)-Ala-Gly-Val-Gln(Trt)-Tyr(tBu)-Ser(tBu)-Arg(Pbf)-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-Glu-Ile-Ser-Thr-Asn-Ile-Arg-Gln-Ala-Gly-Val-Gln-Tyr-Ser-Arg-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。

以上所有内容，为专肽生物原创内容，请勿发布到其他网站上。