阅读说明：本技术 葎草花粉重组疫苗及其制备方法 (Humulus pollen recombinant vaccine and preparation method thereof ) 是由 周俊雄 尹佳 李欣 于 2019-03-22 设计创作，主要内容包括：本发明涉及一种重组混合葎草花粉变应原疫苗及其制备方法,用于有效诊断由葎草花粉诱发的变态反应疾病并对其进行特异性免疫治疗。本发明重组疫苗涵盖了15种葎草花粉变应原蛋白,可以有效诊断由葎草花粉诱发的变态反应疾病并对其进行特异性免疫治疗。(The invention relates to a recombinant mixed humulus pollen allergen vaccine and a preparation method thereof, which are used for effectively diagnosing allergic diseases induced by humulus pollen and carrying out specific immunotherapy on the allergic diseases. The recombinant vaccine of the invention covers 15 humulus pollen allergen proteins, and can effectively diagnose allergic diseases induced by humulus pollen and carry out specific immunotherapy on the allergic diseases.)

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 5.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 6.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 9.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 11.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 13.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 15.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 17.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 19.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 21.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 23.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 25.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 27.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 29.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 31.

A recombinant mixed humulus pollen allergen vaccine comprises an amino acid sequence shown in SEQ ID No. 33.

A recombinant mixed humulus pollen allergen vaccine comprising two or more proteins selected from the group consisting of the amino acid sequences set forth in seq id no: SEQ ID NO.5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 33.

A humulus pollen allergen protein which is a protein selected from the following amino acid sequences: SEQ ID NO.5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31 and 33

A therapeutic or diagnostic formulation comprising the humulus scandens pollen heavy allergen protein of claim 17.

The formulation of claim 18, wherein each protein in the formulation is obtained by in vitro recombinant expression, and the formulation is formulated to obtain the formulation.

The formulation of claim 18, wherein the formulation is a vaccine.

The formulation of claim 18, further comprising an adjuvant.

A gene encoding the protein of claim 17.

A vector comprising the gene of claim 22.

A host cell comprising the vector of claim 23.

A method for producing the protein of claim 17 by culturing the host cell of claim 24, inducing expression, and isolating the protein.

The method of claim 23, wherein said host cells are separately cultured, expression is induced, and said protein is isolated.

A method for constructing a Japanese hop pollen cDNA yeast expression library comprises the following steps:

(1) amplification of the fragment of interest attR1-Cm from pDEST-22^Rccd-attR2, and cloned to a target vector pPIC9K according to SnaBI and AvrII to obtain a final vector pPIC9K-attR1-Cm^R-ccd-attR2；

(2) Extracting total RNA of humulus pollen by a Trizol method;

(3) separating mRNA from the total RNA extracted in the step (2);

(4) constructing a DSN normalized Full-length cDNA library by using the mRNA obtained in the step (3) as a template and using a Superscript Full length library construction kit II;

(5) extracting and preparing the primary full-length homogenization library plasmid obtained in the step (4), recombining the primary homogenization library mixed plasmid to a yeast vector pPIC9K-attR1-CmR-ccd-attR2 through LR reaction, then transforming a recombination product into DH10B competent cells, and detecting the library capacity by using a resistant LB plate to obtain a full-length homogenization yeast expression library.

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. It is intended that all modifications or alterations to the methods, procedures or conditions of the present invention be made without departing from the spirit and substance of the invention.

Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

Example 1 construction of a DSN-normalized full-Length cDNA library

Construction of a DSN-normalized Full-length cDNA library was performed according to the Superscript Full length library construction kit II instructions. The method comprises the following steps:

vector transformation of pPICK 9K:

amplification of the fragment of interest (attR 1-Cm) from pDEST-22 (FIG. 3)^Rccd-attR2) and cloned according to SnaBI and AvrII onto a destination vector pPIC9K (FIG. 4) to obtain a final vector pPIC9K-attR1-Cm^R-ccd-attR2。

Extraction of total RNA:

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

mRNA isolation:

use of

MAG mRNA isolation Kit mRNA was isolated from the total RNA extracted in the second stage, and quality measurements of mRNA concentration and purity were performed using SDS-PAGE and Thermo nanodrop nucleic acid analyzer.

4. Construction of a DSN normalized Full-length cDNA library was performed using the Superscript Full length library construction kit II:

(1) synthesizing the first strand of cDNA by using the mRNA obtained in the third stage as a template;

(2) performing RNaseI treatment on the cDNA obtained in the step (1), and enriching a chain cDNA with a 5' cap structure by using a cap antibody needle marked by biotin;

Biotin-DSN-attB2-Oligo(dT)Primer Sequence:(SEQ ID NO.1)

(3) PCR synthesizing a second cDNA chain by taking the first chain cDNA with the 5' cap structure in the step (2) as a template;

5' linker Sequences (SEQ ID NO.2, 3)

(4) Hybridizing the cDNA obtained in the step (3), and carrying out DSN homogenization treatment on cDNA double chains;

(5) performing linear PCR amplification on the cDNA subjected to DSN homogenization treatment in the step (4), and determining an optimal DSN treatment sample according to an SDS-PAGE electrophoresis result; then according to the previously determined processing sample and the cycle number thereof, carrying out PCR amplification on the sample by using a corresponding appropriate reference gene under the same condition, and ensuring that the electrophoresis result is consistent with the previous preliminary experiment;

(6) carrying out fractional separation and purification on the cDNA double strand;

(7) carrying out Gateway recombination reaction on the obtained cDNA obtained by the step (6) and a pDONR222 carrier (primers are shown in SEQ ID NO.34 and 35);

(8) electrotransformation of the recombinant product into DH10B competent cells to construct a cDNA library;

(9) the quality of the cDNA library was determined in a variety of ways: screening positive bacteria by using a kanamycin plate, and detecting the library capacity (the number of recombinant subclones) by a colony counting method; randomly picking 32 clones from each library, performing colony PCR, and performing electrophoretic detection on the proportion (recombination rate) of the recombinant clones and the average insert size; randomly picking 32 clones from each library, extracting plasmids for bidirectional sequencing, and judging whether the insert contains the full-length ORF or not through sequence analysis.

5. Construction and QC of full-length homogeneous yeast expression library

Extracting the primary full-length homogenization library plasmid obtained in the previous stage, and recombining the primary homogenization library mixed plasmid into a yeast vector pPIC9K-attR1-Cm through LR reaction^Rccd-attR2 (see SEQ ID NO.38), then the recombinant product was transformed into DH10B competent cells (see example 2) and tested for library size (number of recombinants clones) using a resistant LB plate assay, while 24 clones were randomly picked for each library, subjected to colony PCR, electrophoretically tested for recombinant clone proportion (recombination rate), and average insert size (see example 3).

Example 2: construction of a two-hybrid library of ppic9k Yeast

2.1 plasmid extraction of the Primary library

Take to comprise 5 × 10⁶～1×10⁷The library of positive clones was inoculated into 100mL of broth containing kanamycin (working concentration 50ug/mL) and cultured at 30 ℃ with shaking at 250rpm until OD600 was 1.0. Use ofHiPure Plasmid Filter Midiprep Kit extracts library plasmids.

2.2 library plasmid recombination

2.2.1 the resulting primary library plasmids were diluted to 300 ng/. mu.L

The ingredients were added as follows:

primary library plasmids (300 ng/. mu.L)	1μL
PPIC9K(300ng/μL)	1μL
LR Clonase II Mix	4μL
ddH 2O	14μL
Total volume	20μL

Mixing, and standing at 25 deg.C for 16-20 hr

2.2.2 adding 2 mul of Proreinase K into an LR recombination reaction system, and keeping the temperature at 37 ℃ for 15 min; 75 ℃ for 10 min;

2.2.3 Add 180. mu.L dd-water, 1. mu.L Glycogen (20 ug/. mu.L), 200. mu.L 5MNH4OAc, 1000. mu.L 100% ethanol, standing at-80 ℃ for 15 min; centrifuging at 4 deg.C and 14,000rpm for 25min, and removing supernatant;

2.2.4 adding 1ml 70% ethanol to wash the precipitate; centrifuging at 4 deg.C and 14,000rpm for 3min, and removing supernatant; repeating the steps;

2.2.5 air-drying the recombinant product at room temperature for 5-10 min;

2.2.6 repeatedly blowing and beating the recombinant protein for 30-40 times by using 8 mu L of TE Buffer to dissolve the recombinant product;

2.2.7 precooling a 1mm electric rotor at-80 ℃, adding the recombinant product and 50 mu L of electric-transfer competent cell DH10B into the electric rotor on ice, and standing on ice for 45 min;

2.2.8 the voltage conditions on the TX ECM 630 electric converter were set as:

voltage of	2.0kV
Resistance (RC)	200 ohm
Capacity of	25uF

After electric shock, quickly adding 1mL of SOC culture medium into the electric revolving cup;

2.2.9 the electrotransfer is taken into a new 15mL centrifuge tube and is placed at 37 ℃ and cultured for 1 hour by shaking at 225-;

2.2.10 after the culture is finished, diluting 10 mu L of the bacterial liquid according to a ratio of 1:1000, and taking 50 mu L of a coating plate for library volume identification; adding glycerol into the rest culture until the final concentration is 20 percent, and storing at-80 ℃, thus obtaining the yeast library bacterial liquid.

Example 3 Yeast library quality identification

3.1 identification of library Capacity

After 10. mu.L of the stock solution of the transformed bacteria was diluted 1000-fold, 50. mu.L of the spread LB plate (containing the corresponding resistance) was taken out therefrom and cultured overnight at 37 ℃ for the next day to be counted.

CFU/mL-number of clones on plate/50 μ L × 1000 × 1 × 10³The number of clones on the plate was 310/50uL × 1000 × 1000uL ═ 6.2 × 10⁶cfu/mL

Total CFU of library (CFU/mL × total volume of library bacterial solution (mL) ═ 6.2 × 10⁶cfu/mL×3mL＝1.86×10⁷cfu

3.2 recombination Rate and insert Length identification

Randomly selecting 24 clones to carry out colony PCR identification (5 'AOX and 3' AOX sequences are shown as SEQ ID NO.36 and 37), and preparing the following reaction liquid:

ddH 2O	16.2μL
10×PCR Buffer	2.0μL
dNTP(10mM)	0.5μL
5’AOX(20uM)	0.5μL
3’AOX(20uM)	0.5μL
DNA Polymerase(5U/μL)	0.3μL
Total	20μL

the PCR reaction was carried out on an AB 2720PCR instrument under the following conditions:

TABLE 1, 24 library insert lengths and recombination rates

Cloning	Insertion size (kb)	Cloning	Insertion size (kb)	Cloning	Insertion size (kb)
1	0.6	9	0.9	17	1.3
2	1.0	10	1.0	18	1.5
3	1.2	11	1.3	19	2.0
4	1.5	12	0.7	20	1.0
5	1.4	13	2.0	21	1.1
6	1.4	14	1.0	22	1.2
7	1.5	15	1.1	23	1.1
8	1.2	16	1.0	24	1.5

Example 4 identification of recombinant protein Activity by double Membrane method

40 recombinant expression libraries were inoculated onto MM plates with sterile toothpicks and, at the same time, GS115 blank control bacteria transfected with pPIC9K plasmid without the peptide of interest were inoculated as control clones. After the clone with the diameter of about 2MM grows, carrying out moist heat sterilization on the NC membrane, then adhering the clone on the MM plate, marking, inverting, continuously culturing at 29 ℃, removing the NC membrane after the clone position is thoroughly wetted (about 3-4 days), and drying at 37 ℃ to form the solid-phase antigen spot. Blocking for 1h by BSA with the mass fraction of 1%, adding 15 patient mixed serum pools as primary antibody, washing for 3 times at 37 ℃ for 2h, and adding 1: the 1000 enzyme-labeled mouse anti-human IgE is washed for 3 times at 37 ℃ for 1h, ECL substrate is added for color development, the color of spots is observed, and the activity of the recombinant protein is determined. And single colony sequencing of corresponding transformants. 17 colonies showed positive results, see FIG. 5. The results of single colony sequencing are shown in Table 2, where Hum s3 was found in the NCBI database previously discovered by the applicant to be Hum s 3: the ADB97919.1cDNA sequences are completely consistent; hum SX2 is identical to the registered EU088286.1cDNA sequence in NCBI database, and is not listed in Table 2.

TABLE 2 sequencing results of positive single colonies

Remarking: the registration number of NCBI is temporarily unpublished

Example 4 double-Membrane method for rapidly screening high-expression strains of methanol Yeast

Selecting transformants from the MD plate, respectively inoculating the transformants into 96-well culture plates, wherein the culture wells are YPD liquid (1% yeast extract, 2% peptone, 2% glucose by mass fraction, 2.5mg/mL G418) added with G418, culturing at 29 ℃ for 5-6 d, inoculating the grown colonies into another new plate added with G418 with the same mass concentration, and culturing at 29 ℃ for 5-6 d; inoculating the grown colony to a new plate added with 4.0mg/mL G418, culturing at 29 ℃ for 5-6 d, inoculating the grown colony to another new plate added with G418 with the same mass concentration, and culturing at 29 ℃ for 5-6 d. The bacterial liquid to be screened in each of 2.5mg/mL G418 and 4.0mg/mL G418 plates is streaked and coated on YPD-G418 plates with corresponding mass concentration, and cultured at 29 ℃ until single colony appears. 60 transformants on G418 plates of different mass concentrations were randomly picked with sterile toothpicks and inoculated onto MM plates (mass fraction 1.34% YNB, 4X 10)^-5% biotin, 0.5% methanol and 1.5% agar) and, as a control clone, a GS115 blank which had been transfected with the pPIC9K plasmid which contained no peptide of interest was inoculated. After the clone with the diameter of about 2MM grows, carrying out moist heat sterilization on the NC membrane, then adhering the clone on the MM plate, marking, inverting, continuously culturing at 29 ℃, removing the NC membrane after the clone position is thoroughly wetted (about 3-4 days), and drying at 37 ℃ to form the solid-phase antigen spot. Blocking for 1h by BSA with the mass fraction of 1%, adding rabbit anti-pig ZP3 antibody, washing for 3 times at 37 ℃ for 2h, adding 1: washing with 500 enzyme-labeled goat anti-rabbit IgG at 37 deg.C for 1 hr for 3 times, and adding DAB-H₂O₂The substrate was developed and the color of the spots was observed to be light. The corresponding clones showing the darkest spot color (strong positive) and the corresponding clones showing the positive spot color on the NC membrane were picked up, induced by conventional shake flask culture according to the Invitrogen company manual, and the supernatant was taken for conventional SDS-PAGE and Western Blotting identification.

Example 5 amplification of culture, inducible expression and purification

Inoculating corresponding yeast strain in BMG culture medium, culturing at 30 deg.C overnight, transferring into fermenter, culturing with ammonia water at pH of 6.0 and dissolved oxygen at 60%, and performing high density culture. Thereafter, the cells were cultured in BMM medium to maintain the methanol concentration at 1% and induced to express for 3-4 days. The supernatant was collected by centrifugation, and PMSF was added as appropriate as a protease inhibitor. Fixing Ni column and waste liquid collecting vessel, flowing out 20% ethanol in the column, adding PBS 1CV to clean the Ni column. And (3) loading the clear solution on the column, controlling the flow rate to be 150cm/h, and properly adopting an injector needle and a pipette head to control the flow rate to receive a flow-through liquid sample, wherein the sample is used as the flow-through sample. And (3) preparing a 25mM imidazole solution 1-1.5CV from PBS to clean the Ni column, and inoculating a sample of the impurity washing solution. And (3) preparing 200mM imidazole solution by using PBS (phosphate buffer solution) for eluting the target protein about 1CV, and collecting the target protein into a centrifugal tube, wherein the sample is the purified target protein. Adding TEV Protease into the newly purified target protein solution containing high-concentration imidazole, dialyzing at 4 ℃ to remove imidazole and carrying out enzyme digestion, thereby finally obtaining the target allergen protein.

Example 6 ELISA binding inhibition assay

The IgE binding inhibition assay can evaluate 2 antigens or antigen mixtures containing similar epitopes. This example was used to evaluate the similarity (cross-reactivity) between recombinant mixed humulus pollen allergen vaccines (in this example, different recombinant allergens were mixed in equal proportions) and natural allergen infusions in IgE binding epitopes to assess the feasibility of immunotherapy using the mixed recombinant humulus pollen allergen vaccine instead of the humulus pollen natural allergen infusions.

1 coating: coating buffer (Na)₂CO₃ 0.16g，NaHCO₃0.293g, 100ml, pH9.6), and coating in 96-well enzyme-labeled plate (A-D rows, 1-12 columns) at 5 μ g/well, and sealing at 4 deg.C overnight.

2, washing the plate: discard the liquid, PBST wash plate 3 times, each time 3min, absorbent paper absorb the liquid in the dry hole.

3, sealing: blocking solution 1% BSA-PBST was added to the wells at 200. mu.l/well, and the mixture was allowed to stand at room temperature for 2 hours.

4, washing the plate: discarding the liquid, washing the plate for 3 times, 3min each time, and absorbing the liquid in the pores with absorbent paper.

5 inhibitor sample preparation:

diluting natural herba Humuli Scandentis allergen extractive solution to 5 μ g/ml as first dilution, arranging at A3-12, B3-12 (multiple wells), diluting at 3 × multiple ratio, 50 μ l/well;

the recombinant mixed humulus allergen vaccine was diluted to 0.05. mu.g/ml as a first dilution, placed at C3-12, D3-12 (duplicate wells), diluted at 3X fold ratio, 50. mu.l/well.

6, preparing a diluent of a negative serum pool and a positive serum pool:

diluting the positive serum pool (15 cases of humulus allergic patient serum are mixed in equal proportion) 1: and 5, diluting. 50 μ l/well of A2-A12, B2-12, C2-12, D2-12.

Dilutions negative serum pools (healthy control 5, mixed in equal proportions) 1: and 5, diluting. Add 50. mu.l/well to A1-D1.

Then 50. mu.l/well of 1% BSA-PBST diluent was added to A1-D1, A2-D2 to make the serum titers of the negative and positive controls equal to those of A3-A12, B3-12, C3-12, and D3-12.

7 primary antibody incubation: incubate plate 37 ℃ overnight at 4 ℃ after 2 h.

8, washing the plate: discarding the liquid, washing the plate for 3 times, 3min each time, and blotting the liquid in the pores with water paper.

Incubation of biotin-labeled goat anti-human IgE antibodies (secondary antibodies): the antibody was diluted in diluent 1:1000, 100. mu.l per well, at room temperature for 1 h.

10, washing the plate: discarding the liquid, washing the plate for 3 times, 3min each time, and blotting the liquid in the pores with water paper.

11 incubation of horseradish enzyme-labeled streptavidin: diluent 1:500 diluted secondary antibody, 100. mu.l per well, room temperature 0.5 h.

12, washing the plate: discarding the liquid, washing the plate for 3 times, 3min each time, and blotting the liquid in the pores with water paper.

13, color development: adding color development solution, 100ul per well, and keeping away from light at room temperature for 5 min.

14, terminating: stop solution was added at 50ul per well and the 450nm reading was taken over 30 minutes.

In data analysis, the Log dilution is plotted on the abscissa and the inhibition ratio on the ordinate. OD_{Negative control}＝(A1+B1+C1+D1)/4；OD_{Positive control}(a2+ B2+ C2+ D2)/4; taking the mean value of multiple wells for the OD value of each competitive inhibition dilution; inhibition per dilution of competitive inhibition [% ] or_{Positive control}-OD_{Mean value of dilution})/(OD_{Positive control}-OD_{Negative control}) 100%. A3-A12, B3-12 was used to investigate the gradient dilutionThe natural humulus pollen allergen immersion liquid has competitive inhibition effect on the reaction of the natural humulus pollen allergen coated on the enzyme label plate and serum IgE antibodies of a patient allergic to the humulus pollen; the competitive inhibition effect of the recombinant humulus pollen allergen vaccine diluted in a gradient manner on the reaction of natural humulus pollen allergen coated on an ELISA plate and serum IgE antibodies of a patient allergic to humulus pollen is examined by C3-12 and D3-12.

As shown in FIG. 6, in the present example, the native hop pollen allergen (5. mu.g/ml) was 97.5% inhibited against the coated native hop pollen allergen at the first dilution, while the recombinant hybrid hop vaccine (0.05. mu.g/ml) was 69% inhibited against the coated native hop pollen allergen. The result shows that the recombinant mixed humulus vaccine has similarity of 69%/97.5% ═ 70.8% with the natural humulus pollen allergen, which indicates that the similarity between the two is higher and the cross reaction is large, and the recombinant mixed humulus vaccine can basically replace the natural humulus pollen immersion liquid in the aspect of human IgE antigen recognition; in addition, the recombinant mixed humulus vaccine only needs 1% (0.05 mu g/ml/5 mu g/ml) of the natural humulus pollen immersion liquid when reaching the titer of 70.8% of the natural humulus pollen immersion liquid, which indicates that the recombinant mixed humulus vaccine has higher biological activity because the non-allergen components in the humulus pollen are removed under the same concentration condition. The recombinant mixed humulus vaccine has clear components, the content of each component is convenient to accurately control, and single-component purification and combination are easy, so the recombinant mixed humulus vaccine represents the development direction of allergen precision molecular diagnosis and treatment.

Example 7 basophil activation assay

The recombinant humulus pollen allergen vaccine prepared by the invention performs a basophil activation test on whole blood of a humulus pollen allergic patient, thereby verifying that the recombinant humulus pollen allergen vaccine has the biological functions of allergic reaction of binding sIgE antibody and activating basophil degranulation. The specific immune therapeutic vaccine is also proved to have the characteristics of the specific immune therapeutic vaccine theoretically. Therefore, it has a function as a recombinant allergen vaccine.

The test principle is as follows: the reaction of allergens with patient's whole blood cells can mimic the allergic process in humans: that is, specific IgE antibodies bind to the cell surface via bridging with the corresponding allergen, activating the intracellular signaling cascade leading to activated degranulation of basophils (to which CCR3 is persistently expressed, being its specific marker). During this degranulation, the intracellular complexes affect the transmembrane protein CD63(gp53), which is expressed on the cell surface and exposed to the extracellular matrix, and thus can rely on flow cytometry principles (labeling of basophils with anti-human chemokine receptor CCR 3-phycoerythrin (anti-CCR3-PE), labeling of activated basophils with anti-human CD63 monoclonal antibody fluorescein isothiocyanate (anti-CD63-FITC), non-specific cell activator fMLP as a positive quality control), and judging whether a control is allergic to a particular allergen as a percentage change in degranulation of basophils. The method comprises the following steps: selecting healthy control and Japanese hop allergy patients, taking EDTA (ethylene diamine tetraacetic acid) anticoagulated whole blood samples, adding stimulation buffer solution (negative control), recombinant Japanese hop pollen allergen vaccine mixture (the components are mixed in equal proportion and diluted to 2 x 10-7mg/ml) and fMLP (positive quality control) as activators of basophils into the whole blood, adding anti-CD63-FITC and anti-CCR3-PE for dyeing, and detecting by an up-flow cytometer within 48 h. As a result: see fig. 7. When the negative control, the recombinant humulus pollen allergen vaccine and the positive control are respectively basophil activators in the health control, the basophil activation rates are respectively less than 15 percent, less than 15 percent and more than or equal to 15 percent, and when the negative control, the recombinant humulus pollen allergen vaccine and the positive control are respectively basophil activators in the humulus pollen allergic patients, the basophil activation rates are respectively less than 15 percent, more than or equal to 15 percent and more than or equal to 15 percent. And (4) conclusion: the recombinant humulus pollen allergen vaccine can effectively combine an sIgE antibody and activate basophilic granulocyte degranulation, so that the recombinant humulus pollen allergen vaccine has antigenicity and corresponding biological functions, can be effectively applied to specific diagnosis of a humulus pollen allergic patient, can also be used as a potential therapeutic vaccine, and has development and application prospects.

Example 8 evaluation of recombinant Mixed Humulus pollen allergen vaccine immunotherapy against mouse allergic asthma model

The purpose is as follows: (1) establishing an asthma mouse model of mixed recombinant humulus pollen allergen allergy; (2) asthma mice were specifically immunotherapeutically sensitized with a humulus pollen allergen recombinant mixed vaccine (containing 15 humulus allergenic proteins described in table 2, and two allergenic protein molecules Hum s3 and Hum SX2 obtained in examples 4 and 5 described herein). The method comprises the following steps: (1) establishing an asthma mouse model with mixed recombinant humulus pollen allergen allergy by using a method of subcutaneous sensitization and atomization excitation after neck; (2) mice successfully modeled were given a specific immunotherapy for a period of time to asthma mice allergic to mixed recombinant humulus pollen allergens using the retrocervical subcutaneous injection method. As a result: (1) the research finds that the sRAW value of the model group is obviously improved compared with that of the control group, and particularly when the concentration of the acetylcholine is 25mg/ml and 50mg/ml (p is less than 0.01), the establishment of the mouse asthma model is proved to be successful. After specific immunotherapy, the sRAW value of the treatment group is reduced compared with that of the model group, particularly when the concentration of acetylcholine is 25mg/ml and 50mg/ml (p is less than 0.01), and the results prove that the immunotherapy of the mouse asthma model by using the recombinant mixed humulus pollen allergen vaccine reduces and improves the airway hyperreactivity of mice. (2) The total number of inflammatory cells, the number of neutrophils, the number of lymphocytes, the number of mononuclear cells and the number of eosinophils in the model group are obviously increased compared with those in the control group (P < 0.01); the severe inflammatory reaction of the airway of the model group mouse is shown, the infiltration of inflammatory cells is mainly EOS, the EOS% is obviously increased (P is less than 0.01), and the successful modeling is shown. After specific immunotherapy, the total number of inflammatory cells, the number of neutrophils, the number of lymphocytes and the number of eosinophils in a treatment group are all obviously reduced (P is less than 0.01) compared with that in a model group, and the EOS% is also obviously reduced (P is less than 0.01), so that the immunotherapy effectively inhibits the airway inflammation of mice and reduces the cell infiltration. (3) Researches show that the levels of total IgE and mixed recombinant humulus pollen allergen sIgE of a model group mouse are obviously higher than those of a control group mouse (P is less than 0.01), and the experiment proves that the modeling is successful and the mouse can be induced to generate a large amount of IgE. After specific treatment, the expression level of total IgE and mixed recombinant humulus pollen allergen sIgE in a treatment group is obviously reduced relative to that in a model group (P is less than 0.01), and the successful immunotherapy inhibits the IgE production. In addition, the expression level of sIgG of the mixed recombinant humulus pollen allergen vaccine in the treatment group is obviously increased relative to that in the model group (P <0.01), and the success of immunotherapy is proved to induce the generation of sIgG antibody. (4) Lung tissue HE staining revealed massive infiltration of inflammatory cells in the airways and peripheral blood vessels of the lungs of the mice in the model group, forming a multi-layered cell ring with airway thickening and edema. No special abnormality was observed in the control group. After specific immunotherapy, inflammatory cell infiltration and edema of lung tracheas and peripheral blood vessels of the mice are obviously relieved; the AB-PAS staining of lung tissue found: the airway wall of the model group is widely dyed into bluish purple, and dispersed bluish purple can be seen in the airway, which indicates that a large amount of mucus substances are generated; the hyperplastic goblet cells in the airway epithelium account for > 75% of the total airway epithelium. No obvious abnormalities were seen in the control group, and hyperplastic goblet cells in airway epithelium accounted for < 25% of the total airway epithelial cells. The treated airway wall segment was stained bluish purple with hyperplastic goblet cells in the airway epithelium accounting for < 50% of the total airway epithelium. And (4) conclusion: (1) successfully establishing an asthma mouse model allergic to the mixed recombinant humulus pollen allergen vaccine; (2) the specific immunotherapy of asthma mice allergic to the hybrid recombinant humulus pollen allergen vaccine is effective.

Example 9 recombinant vaccine against humulus pollen for personalized therapy

The purpose is as follows: the single and mixed humulus pollen recombinant molecular vaccine is evaluated to be repeatedly injected subcutaneously to give immune protection reaction and toxic reaction to SD rats, the single and mixed humulus pollen recombinant vaccine is evaluated to cause humoral immune response related to human bodies in animal bodies, and animal test data in pharmacology and safety aspects are provided for clinical test and clinical application of individual molecular therapy of the humulus pollen recombinant vaccine.

The method comprises the following steps: healthy SPF-grade SD rats are selected, and the rats are half male and half female. The groups were randomly divided into 17 groups of 12 according to body weight and sex. The groups #1 to # 15 are humulus single-molecule recombinant vaccine administration treatment groups, which respectively correspond to recombinant proteins of Hum SC1 to 15 of the present invention (yeast-induced, expressed, purified, lyophilized, respectively, corresponding to table 2 above); the number #16 group is a humulus hybrid recombinant vaccine administration treatment group, and is formed by mixing the recombinant proteins of Hum SC1-15 in equal proportion; the No.17 group is vehicle control group (0.2% -0.4% phenol, 4.5-5.5g/L sodium chloride, 0.025% -0.035% human serum albumin). The administration route is as follows: subcutaneous injection. Administration concentration and volume: groups #1-15, each injection was 0.05ug/ml 1.0 ml. Group #16, each injection was 0.06ug/ml 1.0ml (corresponding to the sum of doses of Hum SC1-15 humulus scandens recombinant sensitized protein 0.0015 ug/ml 1.0 ml). The administration frequency is as follows: the administration is performed 2 times per week at 2-3 days intervals. Continuous dosing for 6 months, recovery period: stopping the drug and recovering for 1 month. The following examinations were carried out on the experimental animals throughout the experiment: general symptom observation, injection site irritation, body weight, and food intake examination. Animals were euthanized at 6 months after continuous dosing and at the end of 1 month of withdrawal during recovery (3 animals/sex/group), animals were examined for gross anatomy and histopathology, and serum IgG2a levels were measured to assess antigen immunity. Rat serum IgG2a content was measured using the Rat IgG2a ELISA Kit ((Jianglai biology)).

As a result: (1) general symptom observations in animals: abnormal symptoms in terms of respiration, movement, eyelid signs, stool, urethra, skin, hair, salivary secretion, and the like, as well as dying and dead toxic reactions associated with the test article were not seen in each group of animals throughout the duration of the experiment. (2) Injection site irritation: and (4) visual observation: during the 6-month administration period and the recovery period, no local reactions such as congestion, edema, induration, necrosis and the like occurred on the injection part of each group of rats. (3) Body weight and food intake: the weight average of male rats and female rats in each group is increased after 6 months of administration and 1 month of withdrawal, and the difference is not statistically significant (P is more than 0.05) compared with animals in a CN control group. (4) Histopathological examination results: each animal dissects the parenchymatous viscera in general, and the parenchymatous viscera has normal shape and color, uniform texture and no lumps. The mucosa of the hollow viscera is intact and the color is normal. As a result of microscopic observation, no pathological changes related to the test article were observed in the examined organs (except subcutaneous tissues at the administration site) of the animals at the end of the administration and at the end of the recovery period. (5) After 6 months of administration and 1 month of withdrawal, the total IgG2a (total IgG2a, i.e., tgg 2a) of male and female rats in each group did not change significantly, but the content of specific IgG2a (specific IgG2a, i.e., sgg 2a, such as Hum SC1 sgg 2a, Hum SC2 sgg 2a, Hum SC3 sgg 2a … … and Hum SC15 sgg 2a) in each group of humulus allergen was statistically different from that in the control group (P <0.05), and the content of sIgG2a in the group of humulus recombinant mixed vaccine (Hum SC1-15 mixed in equal amount) was statistically different from that in the control group (P <0.05), as shown in fig. 8a and fig. 8 b.

And (4) conclusion: according to the humulus single-molecule allergen recombinant vaccine Hum SC1-15 and the humulus allergen mixed recombinant vaccine (the humus SC1-15 has uniform component content), under the scheme of the embodiment, the toxicological changes related to related single-molecule vaccines, mixed vaccines and solvent reference products are not seen in the results of general symptom observation, weight, ingestion, organ weight, organ coefficient, general anatomical examination and the like of rats, and the recombinant single-molecule vaccine and the mixed vaccine have good safety. After the rats receive immunotherapy, the monomolecular vaccine and the mixed vaccine both generate IgG2a antibody with higher titer and immunoprotection (in human immunotherapy, the high-titer IgG4 antibody is generated and competitively combined with IgE to reduce allergic inflammatory reaction), and after the rats are stopped for 1 month in the recovery period, the protective IgG2a antibody still continuously exists, so that the immunotherapy of the single and mixed humulus recombinant allergen vaccine has long-term curative effect and protective effect and good safety.

Example 10 humulus pollen recombinant vaccine personalized treatment regimen

10.1 accurate diagnosis

Prick test is carried out on patients by using prick liquid of all potential allergens of humulus pollen, and physiological saline is used as a negative control, and histamine is used as a positive control. All patients were discontinued from the anti-allergic drugs and hormones for 1 week before skin test. The prick part of skin is made by selecting the curved side of forearm, disinfecting with alcohol, and pricking 1 allergen every 3cm on skin. Observing histamine reaction after 8min of pricking, and observing allergen reaction after 15-20 min of pricking. The allergen fraction of humulus pollen of the patient was confirmed by comparing the allergen wheal range with the standard histamine wheal range.

10.2 personalized treatment

According to the diagnosis result of the Japanese hop pollen allergen prick test of a patient, the components and the proportion of the recombinant vaccine are determined. The purified allergen is prepared according to the needs of patients and is added with adjuvants to prepare various recombinant vaccines with different concentrations, and the recombinant vaccines are repeatedly injected or sublingually taken or repeatedly contacted with the patients through other administration routes, the dosage is from small to large, the concentration is from low to high, after the maintenance dosage is reached, the enough treatment course is maintained, so that the tolerance of the patients to the allergen is improved, and when the allergen is contacted again, the allergic phenomenon is reduced or no longer generated.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.