Method of preparing dust mite allergen vaccine with nano particle carrier

A nanoparticle and a technology for applying nanoparticle, applied in the field of new vaccine invention, to achieve the effect of reducing hypersensitivity and simple production process

Inactive Publication Date: 2005-12-14
SHENZHEN UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The present invention has not been reported both at home and abroad

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0012] Example 1: Preparation of recombinant dust mite allergen Der f2 by genetic engineering technology

[0013] Gene engineering recombinant expression plasmid recombinant pET-24a(+) / Der f2 was constructed by the research group of Professor Liu Zhigang, School of Life Sciences, Shenzhen University. Transform the plasmid into E coli Bl21(DE3), pick a single colony and inoculate it in 20ml LB containing 50ug / ml kanamycin, shake the bacteria overnight at 37°C and 200rpm. Inoculate in 1L LB / kam culture medium with 2% inoculum amount, shake the bacteria at 260rpm until the OD600 is 0.6, add IPTG to make the final concentration 0.2mM, induce for 5 hours, centrifuge at 5000g 4°C for 10min, remove the supernatant , wash the pellet once with 1×PBS, resuspend the bacteria in 40ml 1×PBS / 5mM EDTA, add lysozyme to a final concentration of 0.5mg / ml, DTT to a final concentration of 10mM, PMSF to a final concentration of 1mmol / L, Triton X -100 to a final concentration of 1%, and after shak...

Embodiment 2

[0055] Example 2: Preparation of recombinant dust mite allergen Der p2 by genetic engineering technology

[0056] Gene engineering recombinant expression plasmid recombinant pET-24a(+) / Der p2 was constructed by the research group of Professor Liu Zhigang, School of Life Sciences, Shenzhen University. Other preparation methods are the same as in Example 1.

Embodiment 3

[0057] Example 3: Preparation of dust mite allergen nanoparticles

[0058] Dissolve 50mg of PLGA in 1ml of DCM, add 100μl of recombinant allergen Derp2 / Der f2 solution (100mg / ml), sonicate for 1 minute (40w), milky white colostrum can be seen, add 2ml of 2% PVA solution, and sonicate again for 1 minute , to form a double emulsion, then transfer the double emulsion to 50ml ddH2O (pH4.6), stir at room temperature for more than 4 hours to completely evaporate the organic solvent, collect the precipitate after centrifuging and washing three times (5000rpm×15min), redisperse it with ddH2O, and place Freeze-dry at a low temperature in a freeze dryer for 24 hours, collect the dried allergen-PLGA nanoparticle powder, and store at 4°C.

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PUM

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Abstract

The present invention relates to method of preparing dust mite allergen vaccine with nanometer particle as carrier. Dust mite is one of the main allergen causing allergic diseases. The present invention applies biodegradable nanometer PLGA particle as the allergen carrier to coat recombinant dust mite allergen Der p2/Der f2 in preparing nanometer level dust mite allergen vaccine. Specifically, by means of gene engineering technology, recombinant Der p2/Der f2 protein is prepared through cloning and expression in colibacillus and PLGA coated nanometer particle is prepared through re-emulsifying and solvent volatizing process. The form, average size, coating rate, medicine carrying amount and extracorporeal accumulation and release curve of the nanometer particle are obtained, and test shows that Der p2/Der f2-PLGA nanometer particle has certain curative effect on allergic asthma.

Description

technical field [0001] The invention relates to the field of new vaccine inventions, in particular to a method for preparing dust mite allergen vaccines using nanoparticles as carriers. Background technique [0002] Allergic diseases (such as allergic asthma, allergic rhinitis, etc.) are clinically common and frequently-occurring diseases. According to the 2000 WHO / IAACI report, the incidence and mortality of asthma are on the rise in recent years. According to the epidemiological statistics of asthma among adolescents in some cities in the country, the incidence rate of asthma is 3.3%-5.1%, far higher than 1% 10 years ago; at least 10 million children in my country suffer from asthma. [0003] Allergic diseases are caused by various contact or inhalation allergens, most of which are proteins, polypeptides, sugars, and synthetic substances such as drugs. Dust mites and house dust mites are strong allergens, which can cause systemic allergic reactions including allergic asth...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K39/00
Inventor 刘志刚冉丕鑫邢苗钟南山吉坤美高波喻海琼夏立新白羽
Owner SHENZHEN UNIV
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