Nanoparticle vaccine preparation containing recombinant hepatitis B surface antigen and preparation method thereof

A hepatitis B surface antigen and nanoparticle technology, applied in the field of life science, can solve the problems of inability to induce mucosal immunity and cellular immunity, and achieve the effect of enhancing ability, enhancing immune response, and enhancing uptake

Active Publication Date: 2016-02-03
HEBEI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] The purpose of the present invention is to provide a nanoparticle vaccine preparation containing recombinant hepatitis B surface antigen, which overcomes many problems caused by the injection of aluminum adjuvanted hepatitis B vaccine and the shortcomings of not being able to induce mucosal immunity and cellular immunity

Method used

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  • Nanoparticle vaccine preparation containing recombinant hepatitis B surface antigen and preparation method thereof
  • Nanoparticle vaccine preparation containing recombinant hepatitis B surface antigen and preparation method thereof
  • Nanoparticle vaccine preparation containing recombinant hepatitis B surface antigen and preparation method thereof

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preparation example Construction

[0038] Preparation of encapsulated FITC-HSA nanoparticles: In the process of preparing nanoparticles, FITC-HSA is directly dissolved in the inner water phase instead of HSA, and the process remains unchanged to obtain encapsulated FITC-HSA nanoparticles

[0039] Nanoparticle uptake by macrophages: Seed macrophages in 96-well cell culture plates (approximately 5×10 5 cells / mL), 200 μL per well, cultured until the cells adhered to the wall, then added 200 μl of encapsulated FITC-HSA nanoparticle suspension, and the concentration of nanoparticles was set at 100 μg / ml. After incubating together for 12 hours, discard all the supernatant, wash twice with PBS, add cell lysate (PBS buffer containing 1% TritonX-100 and 2% SDS) to each well, and immerse in ice for 30 minutes to fully break up the macrophages , using a fluorescence spectrophotometer to measure the fluorescence intensity at 525nm to study the effect of mannan and chitosan modification on the uptake of nanoparticles by mac...

Embodiment 1

[0043] Sodium phosphate buffer solution (pH7.4) containing human serum albumin, HBsAg, sodium bicarbonate and PVA is used as the inner water phase. In this mixed solution, the concentration of HBsAg is 1mg / ml, and the concentration of human serum albumin is 5mg / ml , the concentration of sodium bicarbonate is 2.5mg / ml, and the concentration of PVA is 5.0mg / ml. Take 5.0ml of this solution and add it to 10ml containing 600mg polylactic acid-polyglycolic acid block copolymers with molecular weights of 9.0kDa, 13kDa, 23kDa, 43kDa and 87kDa (the mass ratio of polylactic acid to polyglycolic acid (PLA:PLG) is 75:25) in dichloromethane solution, the mixture was emulsified at a colostrum speed of 17,500 rpm for 2 min in a high-speed homogenizer to obtain a stable W / O colostrum. Then slowly inject the primary emulsion into 20ml of sodium phosphate buffer (pH7.0) containing 20mg / mlNaCl and 25mg / mlPVA, and emulsify at 17500 rpm for 10 minutes to form a W / O / W emulsion , add 20ml of phosph...

Embodiment 2

[0047] Sodium phosphate buffer solution (pH7.0) containing human serum albumin, HBsAg, sodium bicarbonate and PVA is used as the inner water phase. In this mixed solution, the concentration of HBsAg is 1mg / ml, and the concentration of human serum albumin is 5mg / ml , the concentration of sodium bicarbonate is 1.25mg / ml, and the concentration of PVA is 20.0mg / ml. Take 5.0ml of this solution and add it to 20ml of dichloromethane solution containing 1000mg2.3kDa polylactic acid-polyglycolic acid block copolymer (where the mass ratio of polylactic acid to polyglycolic acid (PLA:PLG) is 85:15). The mixture was emulsified with a high-speed homogenizer at a speed of 17,500 rpm for 10 minutes to obtain a stable W / O colostrum. Then slowly inject the primary emulsion into 20ml of sodium phosphate buffer (pH7.0) containing 100mg / mlNaCl and 10mg / mlPVA, and emulsify at 9500 rpm for 20 minutes to form a W / O / W emulsion , add 20ml of phosphate (pH7.4) buffer solution containing 100mg / mlNaCl t...

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Abstract

The invention provides a nanoparticle vaccine preparation containing a recombinant hepatitis B surface antigen. The preparation comprises the following components: a core which is composed of human serum albumin, the recombinant hepatitis B surface antigen and sodium bicarbonate; an encapsulated layer which is a polylactic acid-polyglycolic acid segmented copolymer for encapsulating the core; an external layer which is composed of chitosan or mannan or a mixture thereof for coating. The preparation method comprises the following steps: (1) adding the recombinant hepatitis B surface antigen, the human serum albumin and sodium bicarbonate are added into a phosphate buffered solution containing polyvinyl alcohol to obtain an internal water phase; (2) dissolving the polylactic acid-polyglycolic acid segmented copolymer into an organic solvent for preparing an oil phase, adding the internal water phase obtained in the step (1) into the oil phase, and stirring at a high speed for forming a W/O primary emulsion; (3) adding the W/O primary emulsion into an external water phase solution containing polyvinyl alcohol, stirring at a high speed for forming a W/O/W complex emulsion, stirring at a low speed, carrying out a centrifugal washing, collecting nanoparticles, carrying out a vacuum freeze drying to obtain the finished product nanoparticle vaccine preparation containing the recombinant hepatitis B surface antigen.

Description

technical field [0001] The present invention relates to a kind of biological preparation, especially a kind of polylactic acid-polyglycolic acid block copolymer (PLGA) which can be immunized in nasal cavity, has pH value sensitivity and antigen presenting cell targeting, and encapsulates hepatitis B surface antigen A nanoparticle vaccine preparation belongs to the technical field of life sciences. Background technique [0002] Hepatitis B is a worldwide infectious disease that seriously threatens human health. It is a disease caused by hepatitis B virus (HBV) infection. Currently, hepatitis B vaccination is one of the most effective measures to prevent and control HBV transmission. The most commonly used hepatitis B vaccine is an aluminum-adjuvanted hepatitis B vaccine that is purified, inactivated, and adsorbed by adding aluminum adjuvant to the hepatitis B surface antigen (HBsAg) obtained by genetic recombination technology. In order to achieve a durable and effectiv...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/29A61K9/16A61K47/42A61K47/34A61K47/36A61P1/16A61P31/20
Inventor 贺进田李泽熊芳芳王改珍
Owner HEBEI NORMAL UNIV
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