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Vaccine based on fusion expression of ferritin and pres1 antigen gene

A ferritin and vaccine technology, applied in the direction of fusion peptides, virus antigen components, hybrid peptides, etc., can solve problems that need to be improved

Active Publication Date: 2022-01-14
INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the effect of immune tolerance and activation of HBV-specific cellular immunity in patients with hepatitis B needs to be improved, so it is necessary to prepare a hepatitis B vaccine that can obtain significantly improved immunogenicity and induce a higher immune response

Method used

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  • Vaccine based on fusion expression of ferritin and pres1 antigen gene
  • Vaccine based on fusion expression of ferritin and pres1 antigen gene
  • Vaccine based on fusion expression of ferritin and pres1 antigen gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1. Construction and production of PRES1-Ferritin

[0048] Materials: 1. Slat species: DH5αe.coli, BL21 (DE3) PLYSS E.COLI sensitic cells were purchased from Beijing Fort-Biotechnology Co., Ltd. 2. Plasmid: PDEST14 ,. 3. Gene fragment: The Ferritin gene is synthesized by Suzhou Jinyi Biotechnology Co., Ltd., and the PRES1 gene is synthesized by Suzhou Jindixi Biotechnology Co., Ltd. according to the Genbank reference sequence.

[0049] method:

[0050] The Construction of the Pdest14-Pres1-Ferritin Expression vector. The primers with an enzyme-sized site were designed separately, and the PCR was used to amplify. The nucleic acid sequence of the amplified primers of the PRES1 peptide coding sequence is SEQ ID NO: 5 and SEQ ID NO: 6. The nucleic acid sequence of the amplified primers of the ferritin coding sequence is SEQ ID NO: 7 and SEQ ID NO: 8. The amplified PRES1 peptide coding sequence and the ferritin coding sequence were bisase digestion with the PDEST14 expres...

Embodiment 2

[0054] Example 2, PDEST14-PRES1-Ferritin Purification and Characterization

[0055] Coarse pure. The end of the induced end of the cell is resuspended with a buffer of 20 mm Tris-HCl, 50 mM NaCl, pH = 7.5, and the ultrasound crush is performed by 30% of the ultrasonic instrument. Each ultrasound 5S is cooled for 5 s, a total of 15 min, about 90 cycles. . The 12000 rpm was centrifuged for 15 min to discard the supernatant, and then the buffer was diverted by 20 mm Tris-HCl, 50 mM NaCl, pH = 9.5. After the continuous resuscitation of 12000 rpm, centrifuged for 15 minutes, collected. Impurities such as the precipitation were removed with 0.22 μm filter, and most of the proteins in the supernatant were used in this case.

[0056] 2. Gel filtration column purification. The superose6increase gel filtration column was balanced with 20 mM Tris-HCl, 50 mM NaCl, pH = 9.5 buffer, and 0.5 ml of protein coarse filtration after filtration, continued to use 20 mM Tris-HCl, 50 mMNACl, pH = 9.5 bu...

Embodiment 3

[0059] Example 3, antibody response detection caused by vaccines such as PRES1-Ferritin

[0060] Materials: Macchotra Peroxidase Marked Goat Anti-Mouse IgG purchased from Zhongzhou Jinqiao. TLR9 ligand murine CPG-B adjuventing (ODN1826, 5'-TccatgacgttcctgacgTT-3 ', all nucleotides were thiraminated) Synthesis by Shanghai Jierui Biological Engineering Co., Ltd. C57BL / 6 female mice (6-8 weeks) were purchased in Beijing Virong Lihua Experimental Animal Technology Co., Ltd.

[0061] method:

[0062] 1. Pres1-ferritin and other nanotonic immunized mice. The Pres1-Ferritin vaccine or 500 pmol Sc-PRES1-SC-ST-ST-Ferritin was subcutaneously immunized by a 500pmol sc-presnel-SC-ST-ST-ST-Ferritin control vaccine 2, 30 μg (according to the order). Body calculation) TLR9 ligand Cpg is adjuvant, diluted with PBS to 100 μl of immune system per mouse. Among them, the SC-PRES1 control vaccine refers to a vaccine comprising a Spycatcher-PRES1 linked protein; a PRES1-ST-ST-ferritin control vaccine...

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Abstract

The embodiment of this application discloses a hepatitis B vaccine. The hepatitis B vaccine includes: a fusion protein, the fusion protein includes the hepatitis B virus surface antigen peptide and ferritin, and the hepatitis B virus surface antigen peptide and ferritin are connected by a linker. The linker is a flexible linker. The ferritin is bacterial ferritin. The hepatitis B virus surface antigen peptides include preS1 peptides.

Description

Technical field [0001] The present application relates to the field of hepatitis B vaccine, and in particular, to the preparation of ferritin nanophyll vaccine. Background technique [0002] Hepatitis B is a disease that seriously endangers human health and widely circulating. At present, general vaccination is effective measures to control hepatitis B. The hepatitis B-PHHHH / PRES1) is an important part of the hepatitis B virus (HBV) outer membrane protein, which plays an important role in HBV infection with hepatocytes and body immune responses. PRES1 peptide in patients with chronic hepatitis B infection is low, but its immunogenicity is also low, and it is difficult to induce high antibody response. [0003] Current research currently reported hepatitis B virus vaccine mainly comprises any one or several antigen peptide segments of HBSAG, PRES1, PRES2 and HBCAG, plus some cytokines having immunogenous effects. However, the effects of immune tolerance and activating HBV-specif...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/29C07K19/00C12N15/62A61K39/39A61K47/64A61P31/20
CPCA61K39/12A61P31/20A61K47/644A61K39/39C07K14/005C07K14/195A61K2039/575A61K2039/55561C12N2730/10134C12N2730/10122C07K2319/00
Inventor 朱明昭周晓晓王文君
Owner INSITUTE OF BIOPHYSICS CHINESE ACADEMY OF SCIENCES
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