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Therapeutic hepatitis B vaccine

A hepatitis B and vaccine technology, applied in the direction of antibody medical ingredients, peptide preparation method, digestive system, etc., can solve the problem of reduced ability of transgenic mice to resist infection, and achieve the effect of eliminating virus and inhibiting HBV replication

Active Publication Date: 2014-03-19
北京热休生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Studies have shown that gp96 plays a crucial role in the immunological function of Toll-like receptors (TLRs) in APC, and the loss of TLRs in gp96 gene-deficient DC cells can trigger innate immunity, resulting in transgenic mice resisting infection significantly reduced ability

Method used

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  • Therapeutic hepatitis B vaccine
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1, Verification of the expression ability of HBsAg gene, HBcAg gene and gp96 gene

[0031] 1. Construction of recombinant plasmids

[0032] Prepare the DNA shown in Sequence 2 of the sequence listing and insert it between the EcoR I and Xho I restriction sites of the mammalian expression vector pcDNA3.1 (purchased from Invitrogen, product number V790-20) to obtain the recombinant plasmid pcDNA-gp96 .

[0033] The DNA shown in Sequence 2 of the sequence listing was prepared and inserted between the EcoR I and Xho I restriction sites of pEGFP-N1 to obtain the recombinant plasmid pEGFP-N1-gp96.

[0034] The DNA shown in Sequence 4 of the sequence listing was prepared and inserted between the EcoR I and Xho I restriction sites of the mammalian expression vector pcDNA3.1 to obtain the recombinant plasmid pcDNA-HBcAg (pcDNA-HBc).

[0035] The DNA shown in sequence 6 of the sequence listing was prepared and inserted between the EcoR I and Xho I restriction sites of t...

Embodiment 2

[0041] The preparation of embodiment 2, HBcAg

[0042] 1. Prepare the DNA shown in sequence 4 and insert it between the BamH1 and Xho1 restriction sites of the prokaryotic expression vector pGEX (purchased from GE, product number 28-9546-48, with a GST tag) to obtain a recombinant plasmid pGEX-HBcAg.

[0043] 2. The recombinant plasmid pGEX-HBcAg was introduced into Escherichia coli DH5α (purchased from Tiangen Biochemical Technology Company, product number CB101-03) to obtain recombinant bacteria.

[0044] 3. Inoculate the recombinant bacteria in LB medium, cultivate to OD ≈ 0.6, add IPTG to a final concentration of 0.5 mM, and continue to cultivate at 37°C for 4 hours.

[0045] 4. The collected cells were sonicated in an ice bath (200W, broken for 4s and stopped for 6s, 3 cycles of 99 times), and centrifuged at 12000 rpm for 20min at 4°C. Collect the supernatant.

[0046] 5. The supernatant was subjected to affinity chromatography at 4°C. The carrier was Glutathione-Sepha...

Embodiment 3

[0053] Embodiment 3, the preparation of recombinant gp96 protein (rgp96)

[0054] 1. Construction of recombinant plasmid pHFMDZ-R1L2GAmy-gp96

[0055] 1. Extract the mRNA of human liver cancer cell HepG2, reverse transcribe and synthesize cDNA.

[0056] 2. Perform PCR amplification using the cDNA in step 1 as a template to obtain a PCR amplification product.

[0057] Upstream primer: 5'-CCGgaattcATGGACGATGAAGTTGATG-3';

[0058] Downstream primer: 5'-CCGctcgagCTATTAGAATTCATCTTTTTCAGCTGTAG-3'.

[0059] 3. Double-digest the PCR amplification product with restriction endonucleases EcoRI and XhoI, and recover the digested product.

[0060] 4. The plasmid pHFMDZ-R1A (purchased from Invitrogen, product number V20520) was double digested with restriction endonucleases EcoRI and XhoI, and the vector backbone was recovered.

[0061] 5. Ligate the digested product of step 3 with the vector backbone of step 4 to obtain the recombinant plasmid pHFMDZ-R1-gp96.

[0062] Sequencing resul...

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Abstract

The invention discloses a therapeutic hepatitis B vaccine. Active components of the therapeutic hepatitis B vaccine comprise a protein gp96, a hepatitis B surface antigen and a hepatitis B core protein. The protein gp96 has a sequence shown in the sequence 1 of the sequence table. The hepatitis B surface antigen has a sequence shown in the sequence 5 of the sequence table. The hepatitis B core protein has a sequence shown in the sequence 3 of the sequence table. The active components also comprise a plasmid pcDNA-gp96 containing a coding gene of the protein gp96, a plasmid pcDNA-HB containing a coding gene of the hepatitis B surface antigen and a plasmid pcDNA-HBc containing a coding gene of the hepatitis B core protein. The therapeutic hepatitis B vaccine provided by the invention can effectively inhibit hepatitis B virus (HBV) replication, can eliminate viruses infecting the liver and has a very important value to hepatitis B prevention and treatment.

Description

technical field [0001] The invention relates to a hepatitis B therapeutic vaccine. Background technique [0002] Although effective preventive HBV vaccines can be vaccinated, there are still 350 million HBV carriers in the world, and 93 million people are chronically infected with HBV in China alone, and about 15-40% of infected patients may develop life-threatening diseases such as liver disease. Chronic HBV infection seriously endangers public health such as cirrhosis, liver failure and hepatocellular carcinoma. Existing treatments for chronic hepatitis B patients include IFN-α and nucleosides or nucleoside analogs (such as lamivudine, adefovir, and entecavir), which are costly, partially effective, and mutagenic Increased HBV drug resistance. Therefore, there is an urgent need to develop new therapeutic vaccines to complement or replace existing antiviral treatment options. Since the clearance of HBV virus is mainly mediated by virus-specific CTL and helper T cell resp...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/29C07K1/18A61P1/16A61P31/20
Inventor 孟颂东王赛锋李扬李星辉刘振邱立鹏闫晓丽陈立钊胡坤
Owner 北京热休生物技术有限公司
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