Artificial antibody produced using partial sequence of enolase protein originated from plasmodium falciparum, and method for producing same

一种序列、抗原的技术，应用在抗体效价检查材料领域，能够解决抗原肽确认试验困难等问题

Inactive Publication Date: 2017-08-01
JAPAN AGENCY FOR MEDICAL RES & DEV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, it was found that the confirmation test for the synthesized antigen peptide by mass analysis, GPC analysis, etc. was difficult ( figure 1 )

Method used

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  • Artificial antibody produced using partial sequence of enolase protein originated from plasmodium falciparum, and method for producing same
  • Artificial antibody produced using partial sequence of enolase protein originated from plasmodium falciparum, and method for producing same
  • Artificial antibody produced using partial sequence of enolase protein originated from plasmodium falciparum, and method for producing same

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0233] (Example 1) Synthesis method of antigenic peptide (I') and antigenic peptide (II') The synthesis diagram is shown below. (E 5 - AD22-PG: serial number 6; E 5 -AD22-PGC: serial number 7; K-E 4 -C: serial number 8; K-E 4 -CK: serial number 9)

[0234] [chemical 8]

[0235] Example of Novel Antigen Threat (I') (AD22map2-SS) Manufacturing Schematic

[0236]

[0237] [chemical 9]

[0238] Example of Novel Antigen Threat (II') (AD22map2pal-SS) Manufacturing Schematic

[0239]

[0240] Initially, monomers of each antigenic peptide (I') and antigenic peptide (II') (AD22map2 and AD22map2pal, respectively) were synthesized according to the usual Fmoc peptide synthesis method. In the Fmoc peptide synthesis method, the resin Fmoc-βAla-PEGresin to which the first amino acid was introduced in advance according to a conventional method was used (the amount of Fmoc amino acid introduced into the resin here is 1 eq). 2% DBU / DMF (or 30% piperidine / DMF) was used in the depro...

Embodiment 2

[0244] (Example 2) Analysis data of antigenic peptide (I') (AD22map2-SS)

[0245] Appearance: white freeze-dried body

[0246] Amino acid analysis value: (hydrolysis condition: 6M HClaq.(with Phenol)110℃, 22hrs)

[0247] Asp(28) 28.00, Thr(8) 7.78, Ser(8) 7.21, Glu(36) 35.62, Gly(4) 4.02, Ala(4) 4.00, Cys(2) 1.36, Leu(4) 4.03, Tyr( 8) 8.00, Lys(10) 10.07, NH3(16) 18.08, Pro(8) 8.18, Phe(8)+β-Ala(2) 10.01.

[0248] Purity (HPLC): 96.3% ( image 3 )

[0249] Analysis conditions: column (Column), Zorbax 300SB-C18 (4.6x150mm); eluant (Eluant), 10-60% MeCN / 0.1% TFA (25 minutes); temperature (Temp.) 50 ° C; flow rate (Flow rate ): 1.0ml / min; Detector, 220nm; Load, 4μL (0.28mg / 0.560mL 50% AcOH).

[0250] ESI-MS: MW=15249.3 (theoretical value 15249.3) ( Figure 4 )

[0251] Measuring condition device: HP 1100series LC / MSD manufactured by Agilent Technologies

[0252] Sample concentration: 1nmol / 5micro-L

[0253] Dilution solvent: 50% MeCN / H 2 O: 1N NH 3 aq.=95:5

Embodiment 3

[0254] (Example 3) Analysis data of antigenic peptide (II') (AD22map2pal-SS)

[0255] Appearance: white freeze-dried body

[0256] Amino acid analysis value: (hydrolysis condition: 6M HClaq. (phenol) 110°C, 22hrs)

[0257] Asp(28) 27.96, Thr(8) 7.75, Ser(8) 7.21, Glu(36) 35.70, Gly(4) 4.01, Ala(4) 4.00, Cys(2) 1.90, Leu(4) 4.04, Tyr( 8) 7.81, Lys(12) 11.99, NH3(16) 17.21, Pro(8) 8.20, Phe(8)+β-Ala(2) 9.98

[0258] Purity (HPLC): 96.4% ( Figure 5 )

[0259] Analysis conditions: column (Column), Zorbax 300SB-C18 (4.6x150mm); eluant (Eluant), 30-80% MeCN / 0.1% TFA (25 minutes); temperature (Temp.) 50 ° C; flow rate (Flow rate ), 1.0ml / min; Detector: 220nm; Load, 8μL (0.32mg / 0.320mL 50% AcOH).

[0260] ESI-MS: MW=15982.2 (theoretical value 15982.5) ( Image 6 )

[0261] Measuring condition device: HP 1100series LC / MSD manufactured by Agilent Technologies

[0262] Sample concentration: 1nmol / 5micro-L

[0263] Dilution solvent: 50% MeCN / H 2 O: 1N NH 3 aq.=95:5

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Abstract

Provided is a physiologically active peptide which comprises a partial amino acid sequence of enolase originated from Plasmodium falciparum and which has a molecular structure that meets the standards set for the production of GMP. A peptide having such a structure that two peptide molecules each comprising (i) an amino acid sequence A01-Ala-Ser-Glu-Phe-Tyr-Asn-Ser-Glu-Asn-Lys-Thr-Tyr-Asp-Leu-Asp-Phe-Lys-Thr-Pro-Asn-Asn-Asp-A02 (SEQ ID NO: 1) or (ii) an amino acid sequence A03-Ala-Ser-Glu-Phe-Tyr-Asn-Ser-Glu-Asn-Lys-Thr-Tyr-Asp-Leu-Asp-Phe-Lys-Thr-Pro-Asn-Asn-Asp-Lys-Ser-Leu-Val-Lys-Thr-A04 (SEQ ID NO: 2) are lined to a liker peptide represented by (iii) Lys-A05-Cys-A06 in such a manner that the two peptide molecules can be arranged in a biantennary form through amide bonds between carboxyl terminal of the two peptide molecules and two amino groups in a Lys residue in the linker peptide. Preferably, the peptide has a dimerized structure, in which the peptides are dimerized through an S-S bond formed between a Cys residue in the sequence for the linker peptide in one of the peptides and a Cys residue in the sequence for the linker peptide in the other of the peptides. (In the amino acid sequences (i), (ii) and (iii), each of A01 to A06 represents a molecule composed of an arbitrary number, including 0, of amino acid residues.)

Description

technical field [0001] The present invention relates to a novel physiologically active peptide and its use. In addition, the present invention also relates to an antigen for immunization, a pharmaceutical composition, and a material for testing antibody titers that inhibit the proliferation of Plasmodium by utilizing immune reactions with humans and other animals, and is characterized in that it has enolase derived from Plasmodium part of the amino acid sequence. Background technique [0002] [Prior Art and Its Problems] [0003] (1) The present conditions of malaria as infectious disease: [0004] The WHO World Health Organization presumes in the latest report that malaria is prevalent in 103 countries in the world, and 207 million people were sick and 627,000 people died in 2012 (Non-Patent Document 1). Almost all cases (80%) and deaths (90%) were reported from the African region. Further, 77% of the dead were children under 5 years old. Compared with 2000, the number...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/445A61K38/00A61P33/06A61P37/04G01N33/53C12N9/88
CPCA61K38/00C07K14/445C12N9/88G01N33/53C12Y402/01011A61P33/06A61P37/04Y02A50/30A61K47/646A61K9/1647A61K9/1658A61K9/1682A61K39/015A61K2039/575A61K2039/6068G01N33/543G01N33/56905G01N33/6854G01N2333/445G01N2333/988G01N2800/52
Inventor 奥浩之狩野繁之矢野和彦
Owner JAPAN AGENCY FOR MEDICAL RES & DEV
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