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Therapeutic HBV DNA vaccines, method for preparing the same and application thereof

A DNA vaccine and vaccine technology, applied in botany equipment and methods, biochemical equipment and methods, applications, etc., can solve the problems of DNA shedding, tedious preparation of gold particles, short duration of immune response, etc., and achieve the goal of improving the immune effect Effect

Active Publication Date: 2008-01-02
广州广药益甘生物制品股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In clinical trials of HBV DNA vaccines, the use of gene guns to bombard the gold particles coated with plasmid DNA into the skin can combine effective antigen presentation and recognition. The preparation of particles is cumbersome and the coated DNA will fall off with the increase of temperature.

Method used

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  • Therapeutic HBV DNA vaccines, method for preparing the same and application thereof

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Experimental program
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Effect test

Embodiment 1

[0062] Construction of therapeutic HBV DNA vaccine

[0063] The therapeutic HBV DNA vaccine includes a DNA vaccine plasmid (abbreviated as pS2.S) whose target gene is the protein preS2+S in the HBV envelope and an adjuvant plasmid (abbreviated as pIIF) whose target gene is human IL2 and IFN-γ.

[0064] We used the eukaryotic expression vector pVAX1 purchased from Invitrogen. According to the requirements of the vector, we added the KOZAK series when designing upstream primers, that is, ANNATGG, -3 is A, +4 is G, to enhance the expression of the target gene. Upstream primer P1 of pS2.S: 5`-GG AAGCT`T AGG ATG GGC TGG CTG CAG AGC CTG-3`, downstream primer P2: 5`-GG GAATT`CTTA AAT GTA TAC CCA AAG ACA A-3', HindIII and EcoRI sites were introduced into the two primers respectively. Using the pcDNA3.1 / preS2.S plasmid as a template, using pfu high-fidelity polymerase, the target fragment was amplified by PCR at 95°C for 5min, 94°C for 50sec, 55°C for 30sec, 72°C for 90sec, 30cycle...

Embodiment 2

[0067] In Vitro Identification of Double Plasmid of Therapeutic HBV DNA Vaccine

[0068] Extract the vaccine plasmid pS2.S and the adjuvant plasmid pIIF according to the instructions of the Endofree Plasmid Mega Kit, and refer to Lipofectamine, the liposome transfection reagent of Invitrogen Company TM 2000 operating instructions are carried out. Cultivate COS-7 cell lines in RPMI1640 medium containing 10% newborn calf serum (FBS) according to conventional methods, digest adherent cells with trypsin 24 hours before transfection, resuspend in serum-containing RPMI1640 medium, press 3× 10 5 Cells / well were transplanted in 6-well culture plate, 37°C, 5% CO 2 After culturing, when the adherent cells grow to 90-95% confluence, take 4 μg of plasmid pS2.S, adjuvant pIIF, pcDNA3.1 / preS2.S plasmid, pcDNA3.1 / hIL2IFNγ plasmid for transfection, and set up untransfected cells at the same time. The transfected cell group and the empty plasmid transfected cell group were used as controls....

Embodiment 3

[0074] Preparation of Double Plasmid of Therapeutic HBV DNA Vaccine

[0075] The preparation process of the HBV DNA vaccine: the preparation method of the vaccine plasmid (pS2.S) and the adjuvant plasmid (pIIF) are the same, but they are produced separately. The fermentation, cracking and purification process are as follows:

[0076] Fermentation: adopt BIOSTAT DL 70 fermenter (Germany B.Braun), with Yeast Extract 8g / L, Tryptone16g / L, NaCL 4g / L, glycerol 24ml / L, NaCl 2 HPO 4 h 2 O 7.08g / L, KH 2 PO 4 12H 2 0 1.58g / L; appropriate amount of trace amino acid as the base medium, with Yeast Extract 100g / L+Tryptone 50g / L+glycerol 400ml / L+MgSO 4 2g / L+an appropriate amount of amino acid is a feed medium fed at a constant rate, with 10% inoculum size, cultured at a constant temperature of 37°C, and the pO2 is controlled below 35%, and the OD600 value of the bacterial density is detected online to calculate the plasmid content. After fermenting for 10 hours, the bacteria were ha...

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Abstract

The invention relates to a kind of HBV bi-plasmid DNA vaccine, the preparation method and its application. Said HBV bi-plasmid DNA vaccine comprises vaccine plasmid pS2. S and its adjuvant plasmid pIIF, the eucaryon expression vector is pVAX1. The plasmid is transfected to Africa green monkey kidney cell(COS-7), and the detected target gene expression is higher. The compatibility of bi-plasmid is carried out with ratio being 1: 1 after crack and purification, the Balb / c mouse is injected with volume impulse technique and it is detected that the HBV DNA vaccine can induce body to generate higher HBsAg antibody performance and special cell immunity response. The invention also relates to the application of said HBV DNA vaccine for medicine preparation to treat hepatitis B.

Description

field of invention [0001] The invention relates to the construction, preparation and application of a novel therapeutic vaccine. Specifically, the present invention relates to the construction, preparation and application of therapeutic HBV DNA vaccine. Background technique [0002] Persistent infection with hepatitis B virus (HBV) is widespread worldwide. HBV infection can cause acute and chronic hepatitis and severe hepatitis, and is closely related to liver cirrhosis and liver cancer. It is an infectious disease that seriously endangers human health. At present, there are about 350 million chronic hepatitis B virus infections in the world, 75% of which are distributed in the Asia-Pacific region. There are more than 120 million chronic asymptomatic infections in my country, and the prognosis of chronic hepatitis B is poor. The current antiviral drugs are mainly nucleoside analogs and recombinant interferon drugs, but the effect is not very satisfactory. Therefore, there...

Claims

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Application Information

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IPC IPC(8): A61K39/29A61K48/00A61P1/16A61P31/20C12N15/51
Inventor 陈光明饶桂荣罗显荣杨富强莫国玉陈阳述
Owner 广州广药益甘生物制品股份有限公司
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