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Deoxy nucleotide containing CpG single chain as adjuvant of hepatitis B virus vaccine

A hepatitis B virus, deoxynucleotide technology, applied in the direction of antiviral agents, medical preparations containing active ingredients, sugar derivatives, etc. and other problems to achieve the effect of prolonging the time limit of immune response, reducing the number of immunizations, and enhancing immunogenicity

Inactive Publication Date: 2006-06-07
CHANGCHUN HUAPU BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The main mechanism of this vaccine to prevent hepatitis B is to induce the body to produce and secrete protective antibody IgG1. Although this antibody can neutralize the extracellular virus, it cannot completely remove the latent hepatitis B virus in infected cells. often leads to the hiding of hepatitis B virus in the patient's body

Method used

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  • Deoxy nucleotide containing CpG single chain as adjuvant of hepatitis B virus vaccine
  • Deoxy nucleotide containing CpG single chain as adjuvant of hepatitis B virus vaccine
  • Deoxy nucleotide containing CpG single chain as adjuvant of hepatitis B virus vaccine

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] The design of embodiment 1CpG single-stranded deoxynucleotide

[0036] The design sequence is as follows:

[0037] (G)n(L)nX1X2CGY1Y2(M)n(G)n

[0038] X1=A, T, G; X2=A, T; Y1=A, T; Y2=A, T, C; L, M=A, T, C, G; n is 0-6

[0039] 5'-ggggTCgTTCgTCgTTgggggg-3' (SEQ ID NO: 1) [121]

[0040] 5'-ggggATAACgTTgCgggggg-3' (SEQ ID NO: 2) [143]

[0041] 5'-ggggTgCAACgTTCAgggggg-3' (SEQ ID NO: 3) [402]

[0042] 5'-ggggTCCTACgTAggAgggggg-3' (SEQ ID NO: 4) [123]

[0043] 5'-ggggTCCATgACgTTCCTgAAgggggg-3' (SEQ ID NO: 5) [603]

[0044] 5'-gggggACgTCgCCgggggggg-3' (SEQ ID NO: 6) [118]

[0045] 5'-ggATCCgTACgCATgggggg-3' (SEQ ID NO: 7) [320]

[0046] 5'-ggggggAATCgATTCgggggg-3' (SEQ ID NO: 8) [154]

[0047] 5'-gggATgCATCgATgCATCgggggg-3' (SEQ ID NO: 9) [464]

[0048] 5'-ggTgCgACgTCgCAgggggg-3' (SEQ ID NO: 10) [471]

[0049] 5'-gggACgTACgTCgggggg-3' (SEQ ID NO: 11) [390]

[0050] 5'-gggggATCgACgTCgATCgggggg-3' (SEQ ID NO: 12) [322]

[0051] 5'-ggCgATCgATCgATCggggggg-3' (SEQ ID ...

Embodiment 2

[0232] Synthesis of embodiment 2CpG single-stranded deoxynucleotides

[0233] The solid-phase phosphoramidite triester method is used to synthesize DNA fragments. This method has the advantages of high efficiency and rapidity, and has been widely used in DNA chemical synthesis.

[0234] DNA chemical synthesis is different from enzymatic DNA synthesis. The enzymatic DNA synthesis process extends from the 5'→3' direction, while DNA chemical synthesis starts from the 3' end. Concrete reaction steps are as follows:

[0235] 1. Deprotection group

[0236] Use trichloroacetic acid to remove the protective group DMT (dimethoxytrityl) of the nucleotide attached to CPG (Controlled Pore Glass) to obtain a free 5'-hydroxyl terminal for the next condensation reaction use.

[0237] 2. Activation

[0238] The phosphoramidite-protected nucleotide monomer is mixed with tetrazolium activator and enters the synthesis column to form phosphoramidite tetrazolium active intermediate (its 3'-end...

Embodiment 3

[0248] Embodiment 3 detects hepatitis B virus antibody with ELISA method

[0249] 1. Reagents

[0250] 1. Preparation of HBsAg (without aluminum adjuvant, Beijing Institute of Biological Products) vaccine: 1 mg of HBsAg protein lyophilized powder was dissolved in 1 ml of PBS to prepare an application solution (1 mg / ml).

[0251] 2. HRP-horse anti-mouse secondary antibody: Beijing Dingguo Biotechnology Co., Ltd.

[0252] 3. PBS: 1000ml

[0253] NaCl………………………………… 8g (Beijing Chemical Plant)

[0254] KCl………………………………… 0.2g (Beijing Chemical Plant)

[0255] Na 2 HPO 4 12H 2 O………………………… 2.9g (Beijing Chemical Plant)

[0256] K H 2 PO4…………………………………… 0.2g (Beijing Chemical Plant)

[0257] After fully dissolving with 800ml of ultrapure water, adjust the pH to 7.2-7.4 with HCl or NaOH, and set the volume to 1000ml

[0258] 4. Coating solution: 100ml

[0259] PBS…………………………………80ml

[0260] 50% Glutaraldehyde…………………… 1.6ml (Beijing Chemical Plant)

[0261] After fully dissolved,...

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Abstract

The present invention provides one kind of adjuvant, which includes at least one singe-stranded deoxynuceotide containing one or several CpG dinucleotides. Applying the adjuvant with hepatitis B vaccine can enhance the immune effect of the hepatitis B vaccine obviously.

Description

field of invention [0001] The present invention relates to a single-stranded deoxynucleotide containing CpG dinucleotide, in particular to a single-stranded deoxynucleotide containing CpG dinucleotide as a hepatitis B virus vaccine adjuvant, and the present invention also relates to a single-stranded deoxynucleotide containing CpG dinucleotide CpG dinucleotide The sequence of single-stranded deoxynucleotides. Background of the invention [0002] Hepatitis B is caused by the hepatitis B virus (HBV) in the liver inflammation Damage is a serious disease that endangers the health of the global population. By 2004, 400,000,000 people worldwide had been infected with hepatitis B virus (Lin KW, Kirchner JT.Hepatitis B.Am Fam Physician, 2004 Jan 1, 69(1):75-82.), among the infected Among them, Asians account for the majority, and the carrier rate of hepatitis B virus in our country is as high as 10%. Inoculation of hepatitis B surface antigen (HbsAg) genetically engineered vaccin...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07H21/00A61K39/29A61K39/39A61P1/16A61P31/14
Inventor 王丽颖包木胜周晓静于永利
Owner CHANGCHUN HUAPU BIOTECHNOLOGY CO LTD
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