Humulus pollen recombinant vaccine and preparation method thereof

A technology of pollen and humulus, which is applied in the field of recombinant vaccines for the treatment of humulus pollinosis and its preparation, can solve the problems of unclear expiration date, unstable composition and concentration, spontaneous precipitation of products, etc., and is suitable for large-scale industrial production, Effects of improved diagnostic sensitivity, good batch-to-batch consistency

Active Publication Date: 2022-05-06
PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The allergens currently used in the diagnosis of allergic diseases and specific immunotherapy are still crude extracts, which have many problems: (1) The composition is complex, including major and minor allergens, non-sensitizing or toxic protein components and Other macromolecules and small molecules; (2) Potential safety hazards, which are easily contaminated by exogenous toxic substances or pathogenic microorganisms during the production of crude leachate, which affects its safety; (3) Its composition and concentration are unstable, and it is difficult to Carry out standardization of allergens; (4) spontaneous precipitation of the product; (5) long-term use during treatment may cause severe IgE-mediated allergic reactions in patients with hypersensitivity constitution, which may cause local or systemic adverse reactions, severe cases Can cause death; (6) Production decline caused by raw material storage problems; (7) Product expiration date cannot be clearly defined

Method used

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  • Humulus pollen recombinant vaccine and preparation method thereof
  • Humulus pollen recombinant vaccine and preparation method thereof
  • Humulus pollen recombinant vaccine and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1 Construction of DSN normalized full-length cDNA library

[0047] The construction of the DSN normalized full-length cDNA library was carried out according to the instructions of the Superscript Full length library construction kit II. Proceed as follows:

[0048] 1. Transformation of pPICK9K vector:

[0049] from pDEST-22( image 3 ) to amplify the target fragment (attR1-Cm R -ccd-attR2) and cloned into the destination vector pPIC9K according to SnaBI and AvrII ( Figure 4 ) to obtain the final vector pPIC9K-attR1-Cm R -ccd-attR2.

[0050] 2. Extraction of total RNA:

[0051] Total RNA was extracted by Trizol method, and the concentration and purity of total RNA were determined by SDS-PAGE and Thermo nanodrop nucleic acid analyzer.

[0052] 3. mRNA isolation:

[0053] use The MAG mRNA isolation Kit isolates mRNA from the total RNA extracted in the second stage, and uses SDS-PAGE and Thermo nanodrop nucleic acid analyzer to detect the quality of mRNA ...

Embodiment 2

[0073] Example 2: Construction of ppic9k yeast two-hybrid library

[0074] 2.1 Plasmid extraction of primary library

[0075] Fetch contains the 5 x 10 6 ~1×10 7 Inoculate 100 mL of broth culture containing kanamycin (working concentration: 50 ug / mL) with the library bacteria liquid of each positive clone, and shake the bacteria at 250 rpm at 30°C until the OD600 is 1.0. use HiPurePlasmid Filter Midiprep Kit extracts library plasmids.

[0076] 2.2 Library plasmid recombination

[0077] 2.2.1 Dilute the obtained primary library plasmid to 300ng / μL

[0078] Add the ingredients according to the table below:

[0079] Primary library plasmid (300ng / μL) 1μL PPIC9K (300ng / μL) 1μL LR Clonase II Mix 4μL wxya 2 o

14μL total capacity 20 μL

[0080] After mixing, place at 25°C for 16-20 hours

[0081] 2.2.2 Add 2 μL of Proreinase K to the LR recombination reaction system, 37°C, 15min; 75°C, 10min;

[0082] 2.2.3 Add 180 μL dd-water...

Embodiment 3

[0092] Example 3 Yeast library quality identification

[0093] 3.1 Identification of library capacity

[0094] Take 10 μL of the transformed bacterial stock solution and dilute it 1000 times, take out 50 μL of the LB plate (with the corresponding resistance) and culture it overnight at 37°C for counting the next day.

[0095] CFU / mL=number of clones on the plate / 50μL×1000 times×1×10 3 μL=The number of clones on the plate 310 / 50uL×1000×1000uL=6.2×10 6 cfu / mL

[0096] Total library CFU=CFU / mL×total volume of library bacterial solution (mL)=6.2×10 6 cfu / mL×3mL=1.86×10 7 cfu

[0097] 3.2 Identification of recombination rate and insert length

[0098] 24 clones were randomly selected for colony PCR identification (5'AOX and 3'AOX sequences are shown in SEQ ID NO.36 and 37), and the following reaction solution was prepared:

[0099] wxya 2 o

16.2μL 10×PCR Buffer 2.0 μL dNTP (10mM) 0.5μL 5'AOX (20uM) 0.5μL 3'AOX (20uM) 0.5μL DNA...

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Abstract

The invention relates to a recombinant mixed humulus pollen allergen vaccine and a preparation method thereof, which are used for effectively diagnosing and performing specific immunotherapy on allergic diseases induced by humulus pollen. The recombinant vaccine of the invention covers 15 kinds of humulus pollen allergen proteins, can effectively diagnose allergic diseases induced by humulus pollen and perform specific immunotherapy on them.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to various humulus pollen allergens, a recombinant vaccine for treating humulus pollinosis through injection desensitization and a preparation method thereof. Background technique [0002] Allergic diseases are one of the major global health problems. More than 25% of the population in industrialized countries is troubled by allergic asthma, allergic rhinoconjunctivitis and allergic dermatitis, among which allergic asthma is the most common. Inhalation of allergenic pollen is the most important factor in triggering allergic asthma and other respiratory allergic diseases. Surveys such as VRTA-L.A found that nearly 50% of patients with allergic diseases were allergic to grass pollen. According to this year's domestic survey, the prevalence of allergies in the Chinese population is increasing year by year along with the improvement of economic and living standards. [0003] ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/36A61K39/35C12N15/29
CPCA61K39/35A61K39/36
Inventor 周俊雄尹佳李欣
Owner PEKING UNION MEDICAL COLLEGE HOSPITAL CHINESE ACAD OF MEDICAL SCI
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