WO2019179510A1 - Recombinant humulus japonicus pollen vaccine and preparation method thereof - Google Patents

Abstract

The present invention relates to a recombinant, mixed Humulus japonicus pollen allergens-based vaccine and a preparation method thereof. The present invention is used to effectively diagnose an allergic disease induced by Humulus japonicus pollen and used in specific immunotherapy therefor. The recombinant vaccine of the present invention contains 15 Humulus japonicus pollen allergen proteins, and therefore can be used to effectively diagnose an allergic disease induced by Humulus japonicus pollen and used in specific immunotherapy therefor.

Description

Valerian pollen recombinant vaccine and preparation method thereof Technical field

The invention belongs to the field of biotechnology, and particularly relates to a plurality of psyllium pollen allergens and a recombinant vaccine for treating valerian hay fever by injection desensitization and a preparation method thereof.

Background technique

Allergic diseases are one of the major global health problems. More than 25% of the population in industrialized countries suffer from allergic asthma, allergic rhinoconjunctivitis and allergic dermatitis, with allergic asthma being the most common. Inhaled allergenic pollen is the most important factor in causing allergic asthma and other respiratory allergic diseases. VRTA-L.A and other surveys found that nearly 50% of patients with allergic diseases are allergic to grass pollen. According to this year's domestic survey, the prevalence of allergic reactions in China has been increasing year by year with the improvement of economic living standards.

HumμLus japonicus is mainly distributed in the temperate and subtropical regions of the northern hemisphere, and is reported in the Far East. In addition to the distribution of Xinjiang and Qinghai provinces (including Taiwan), there are also distributions in East Asian countries (North Korea, Japan, Russia, Vietnam) and Europe, the southeastern United States, and western Canada. In the first national airborne allergic pollen survey in the 1980s, 53 of the 79 pollen collection points were collected for psyllium pollen, second only to Artemisia pollen, and its distribution range was basically covered. In the whole country, the peak of dissemination is in August and September, and the peak of daily spread is from 2 to 4 in the afternoon.

In recent years, it has been found that valerian pollen has become an important sensitization of Artemisia pollen in summer and autumn in China and East Asia. In 1990, the study on the main sensitized pollen in summer and autumn in Beijing showed that 68% of the patients with hay pollen skin test in summer and autumn were ≥++, and the positive rate of serum sIgE was 59%. In 1993, Sun Xiuzhen reported that the patients with bronchial asthma in Xi'an had a skin test with valerian pollen infusion, and 54% of patients had a positive reaction, second only to valerian and Artemisia pollen. In 2002, Wang Qingju and other investigations showed that in the inhaled allergen skin test of children with allergic asthma in Yimeng Mountain, the positive rate of summer and autumn pollen was 26.64%, of which the positive rate of alfalfa pollen was 50.77%, accounting for 13.52% of the total number of respondents. The third place in the Yimeng mountain area. Epidemiological investigations on allergenic pollen were also conducted in Chongqing, Guiyang, Zibo and other places. It was found that 葎草草 pollen ranked second in allergenic pollen in the surveyed areas. It has been reported that the Liaoning area is the area with the highest distribution of alfalfa in China, and the hay fever caused by alfalfa pollen has also ranked second in all tested pollen. In 2007, Yao Lina et al. studied the pollen exposure in Beijing and found that the autumn airborne pollen in Beijing was mainly composed of the genus Heliconia and Artemisia pollen, with a composition ratio of 51% and 31% respectively. The main sensitized pollen day in the pollen season The average concentration and the highest concentration of the genus Hemerocallis were higher than the Artemisia pollen. In Korea, 6.1% to 14% of patients with asthma, rhinitis and conjunctivitis are allergic to psyllium pollen. In the group of apple-growing farmers in Korea, the pollen is allergic to 12%.

For allergen specific immunotherapy (SIT) for alfalfa pollen, an in vivo diagnostic preparation and a therapeutic preparation for valerian pollen are required. In this study, through the construction of the cDNA expression library of valerian pollen and the bioinformatics analysis of large-scale sequencing, the potential allergenic protein disease was screened for expression and purification, and the recombinant allergen vaccine was obtained.

The allergens currently used for the diagnosis of allergic diseases and specific immunotherapy are still crude leachates, and there are many problems: (1) complex components, which contain major secondary allergens, non-sensitizing or toxic protein components and Other macromolecules, small molecular substances; (2) safety hazards, which are easily contaminated by exogenous toxic substances or pathogenic microorganisms during the production of crude leachate, affecting its safety; (3) its composition and concentration are unstable, difficult Standardization of allergens; (4) spontaneous precipitation of the product; (5) long-term use in the treatment process may cause severe IgE-mediated allergic reactions in patients with high-sensitivity constitution, which may cause local or systemic adverse reactions, severe cases Can cause death; (6) the decline in production caused by the problem of raw material storage; (7) the validity period of the product cannot be clarified.

Summary of the invention

It is an object of the present invention to provide a recombinant vaccine for treating psyllium pollen allergic diseases.

Another object of the present invention is to provide a method for preparing the above vaccine.

In order to achieve the above object, the present invention constructs a yeast two-hybrid alfalfa pollen cDNA library by DSN homogenization library construction method, and identifies the amino acid sequences of 15 proteins of the valerian allergen by screening, and the sequences thereof are SEQ ID No. .5, 7, 9, 11, 13, 15, 17, 19, 21, 23, 25, 27, 29, 31, and 33.

Based on this, the present invention first provides a recombinant protein of valerian pollen, which comprises each protein of the amino acid sequence shown above, or an equivalent function of the protein obtained by inserting, deleting or replacing one or several amino acids. Derived protein.

Further, the present invention provides a valerian pollen allergen vaccine comprising any of the above proteins, or a recombinant mixed valerian pollen allergen vaccine comprising two or more of said proteins.

Further, the present invention provides a therapeutic or diagnostic preparation comprising the recombinant protein of valerian pollen. Each protein in the formulation is obtained by recombinant expression in vitro and is configured to obtain the formulation. Since the recombinant protein of the present invention is obtained by recombinant expression in vitro, the preparation can have a defined and constant ratio.

Further, the formulation is a vaccine. The formulation also includes a suitable adjuvant.

Further, the invention provides a gene encoding the protein. Preferably, the nucleotide sequence thereof is as shown in SEQ ID NO. 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30 and 32.

Further, the present invention provides a vector containing the gene. The vector may be a cloning vector or an expression vector such as pPIC9K-attR1-CmR-ccd-attR2.

Further, the invention provides a host cell comprising the vector. For example, yeast, E. coli.

Further, the present invention provides a method of producing the protein, which induces expression by culturing the host cell, and isolates the protein. By separately culturing the host cells, inducing expression, and isolating the protein, different proteins can be obtained, and then a formulation having a constant ratio can be obtained.

The invention provides a method for constructing a yarrow pollen cDNA yeast expression library, which comprises the following steps:

(1) The target fragment attR1-Cm ^R -ccd-attR2 was amplified from pDEST-22 and cloned into the destination vector pPIC9K by SnaBI and AvrII to obtain the final vector pPIC9K-attR1-Cm ^R- ccd-attR2;

(2) Extraction of total RNA from alfalfa pollen by Trizol method;

(3) isolating mRNA from the total RNA extracted by (2);

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

(5) Preparing the primary full-length homogenization library plasmid obtained in (4), and then recombining the primary homogenized library mixed plasmid into the yeast vector pPIC9K-attR1-CmR-ccd-attR2 by LR reaction, and then recombining The product was transformed into DH10B competent cells, and the library capacity was detected by using a resistant LB plate to obtain a full-length homogenous yeast expression library.

The cDNA library obtained by the above method is a full-length homogenization library, which is characterized by reducing the peak expression gene by more than 100 times, and effectively enriching the low-abundance expression gene, and the average insert size does not change significantly after homogenization.

The method for constructing the yeast expression library of the psyllium pollen cDNA is prepared by extracting the plasmid of pPIC9K-attR1-Cm ^R -ccd-attR2 yeast expression library, and the obtained plasmid is electrotransformed into methanol yeast Pichia Pastoris GS115, and is resistant. Positive screening was performed on the slabs; the inserts were randomly amplified by PCR, and the clones with inconsistent fragment size were selected by SDS-PAGE electrophoresis for sequencing, and 40 cDNA sequences of the following inserts were obtained and predicted. The amino acid sequence of the 15 proteins that potentially become valerian allergens.

On the basis of the above, a recombinant vaccine of valerian pollen is further prepared: according to the selected selected cDNA, a suitable expression strain is constructed and screened. The target protein is cultured and induced to be expressed, the purification tag (6His) is removed by protease (TEV) and the target protein is purified; and the unlabeled protein can be purified by other conventional purification methods. The 15 proteins obtained by purification were serologically confirmed to be the major allergenic proteins or minor allergens of valerian pollen. It is also determined that the main allergenic protein and the secondary allergen are present in a constant ratio, and a suitable adjuvant is used to obtain a standardized psyllium pollen recombinant allergen vaccine.

The present invention also provides the use of the recombinant vaccine of valerian pollen in a specific immunotherapy selected from the group consisting of allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.

The present invention also provides the use of 15 proteins obtained by purification or a combination thereof for diagnosing an allergic disease selected from the group consisting of allergic asthma, allergic rhinitis, atopic dermatitis and chronic urticaria.

The invention also provides the application of the sorghum pollen recombinant vaccine for precise specific immunotherapy, according to the patient's allergic disease, using the recombinant vaccine for diagnosis, confirming the patient's sedge pollen allergen component, and producing the corresponding component The recombinant vaccine provides precise and specific immunotherapy for patients.

Compared with the crude leachate, the recombinant allergen has the following advantages: (1) It can be produced on a large scale and can be expressed in milligrams or micrograms, and can maintain extremely high purity with good batch consistency. Medical quality requirements for allergens; (2) production by fermentation technology greatly reduces production costs; (3) can be quantified, good stability, easy quality control, standardized production; (4) can be the product of the immunogen Is differentiated from its sensitization, thereby greatly improving the therapeutic index of recombinant allergens; (5) the bioavailability and route of administration of recombinant allergens are superior to those of crude leaching allergens; (6) specificity High, improved diagnostic sensitivity, and advanced the development of specific immunotherapy convenience reagents.

The Easy Select Pichia Expression System used in the present invention has the following advantages: (1) belongs to the eukaryotic expression system, has a certain protein post-translation processing, is favorable for the expression of eukaryotic proteins; (2) AOX potent promoter, outside The source gene product has a high expression level and can reach the level of several grams of expression product per liter; (3) yeast cultivation, transformation, high-density fermentation and the like are close to prokaryotes, far simpler than eukaryotic systems, and are very suitable for large-scale industrial production; 4) It can induce expression, and can also secrete expression for product purification; (5) It can replace IPTG with methanol as an inducer, and some methanol yeast can replace glucose as a carbon source with industrial products such as methanol, and the production cost is low.

The invention screens a series of potential allergens and performs expression by sequencing and bioinformatics analysis of the psyllium cDNA expression library, thereby obtaining a recombinant allergen vaccine, which not only covers all potential allergens, but also It is safe and efficient to carry out precise and specific immunotherapy of alfalfa pollen, and it can accurately and effectively diagnose allergic diseases induced by alfalfa pollen. Individualized treatments can be performed for different patient populations or individuals to maximize treatment outcomes and minimize side effects. This product can also be administered in combination with all recombinant allergens in a 1:1 ratio; or in proportion to the proportion of sensitizing proteins in each component of the natural leachate.

The bioavailability and route of administration of recombinant allergens are superior to those of crude leaching allergens, with high specificity, high diagnostic sensitivity, easy storage and transportation, easy to popularize and use, low cost, quick effect, short cycle, and large Reducing the medical burden of light patients is conducive to the development of specific immunotherapy convenience reagents.

DRAWINGS

Figure 1 is the identification of the reservoir capacity (dilution 1:1000, coating amount 50uL, number of clones 310);

Figure 2 is a PCR SDS-PAGE electrophoresis map of 24 cloned colonies;

Figure 3 is a plasmid map of pDEST-22Vector;

Figure 4 is a map of the pPIC9K Vector plasmid;

Figure 5 is a result of NC membrane recombinant protein activity;

Figure 6 is a result of an ELISA-binding inhibition test of a recombinant mixed alfalfa allergen vaccine against a natural valerian allergen infusion;

Figure 7: Verification of basophil activation test of recombinant mixed alfalfa allergen vaccine;

Figure 8a Single-component sorghum recombinant allergen When the immunotherapy was performed on rats, the specific sIgG 2a antibody was elevated, and the histogram in the figure was consistent from the top to the bottom in the order from top to bottom;

Figure 8b The total specific sIgG 2a antibody was elevated when the 15 components were mixed with valerian recombinant allergen for immunotherapy in rats.

Specific implementation method

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions of the methods, steps or conditions of the invention are intended to be included within the scope of the invention.

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

Example 1 Construction of DSN homogenized full-length cDNA library

The construction of the DSN homogenized full-length cDNA library was carried out in accordance with the Superscript Full length library construction kit II specification. Proceed as follows:

1. pPICK9K vector transformation:

The target fragment (attR1-Cm ^R -ccd-attR2) was amplified from pDEST-22 (Fig. 3) and cloned into the destination vector pPIC9K (Fig. 4) by SnaBI and AvrII to obtain the final vector pPIC9K-attR1-Cm ^R -ccd. -attR2.

2.total RNA extraction:

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

3. mRNA separation:

use

The MAG mRNA isolation Kit was used to separate mRNA from the second-stage extracted total RNA, and the quality of mRNA concentration and purity was detected by SDS-PAGE and Thermo nanodrop nucleic acid analyzer.

4. Construction of a DSN homogenized full-length cDNA library using Superscript Full length library construction kit II:

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

(2) subjecting the cDNA obtained in (1) to RNase I treatment, and enriching the one-strand cDNA having a 5' cap structure with a biotin-labeled cap antibody needle;

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

(3) PCR synthesis of the second strand of cDNA using the one-strand cDNA with a 5' cap structure in (2);

5' Connector Adapter Sequences: (SEQ ID NO. 2, 3)

(4) hybridizing the cDNA obtained in (3), and performing DSN homogenization treatment of the cDNA double strand;

(5) Perform linearized PCR amplification on the cDNA after homogenization of DSN in (4), determine the best DSN processing sample according to the results of SDS-PAGE electrophoresis; and then use correspondingly according to the previously determined processing sample and its number of cycles. The appropriate internal reference gene is subjected to PCR amplification under the same conditions to ensure that the electrophoresis results are consistent with the previous preliminary experiments;

(6) fractionating and purifying the cDNA double strand;

(7) performing the Gateway recombination reaction on the fractionated and purified cDNA obtained in (6) and the pDONR222 vector (primer see SEQ ID NO. 34, 35);

(8) electroporating the recombinant product into DH10B competent cells to form a cDNA library;

(9) The quality of cDNA library was determined by various methods: positive bacteria were screened by kanamycin plate, library capacity (number of recombinant clones) was detected by colony counting method; 32 clones were randomly picked from each library for colony PCR, electrophoresis detection of recombinant clone ratio (recombination rate), and average insert size; each library randomly picked 32 clones extracted plasmid for bidirectional sequencing, sequence analysis to determine whether the insert contains the full-length ORF.

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

The primary full-length homogenization library plasmid obtained in the previous stage was extracted and the primary homogenized library mixed plasmid was recombined into the yeast vector pPIC9K-attR1-Cm ^R- ccd-attR2 by LR reaction (see SEQ ID NO. for the sequence). 38), then the recombinant product was transformed into DH10B competent cells (see Example 2), and the library capacity (number of recombinant clones) was detected by using a resistant LB plate, and 24 clones were randomly picked from each library. Colony PCR, electrophoresis detection of recombinant clone ratio (recombination rate), and average insert size (see Example 3).

Example 2: Construction of a ppic9k yeast two-hybrid library

2.1 Primary library plasmid extraction

The library containing 5×10 ⁶ to 1×10 ⁷ positive clones was inoculated into 100 mL of broth culture containing kanamycin (working concentration 50 ug/mL), and cultured at 30 ° C and 250 rpm until the OD600 was 1.0. . use

The library plasmid was extracted using the HiPure Plasmid Filter Midiprep Kit.

2.2 Library Plasmid Recombination

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

Add the ingredients according to the table below:

Primary library plasmid (300ng/μL)	1μL
PPIC9K (300ng/μL)	1μL
LR Clonase II Mix	4μL
ddH 2 O	14μL
total capacity	20μL

Mix and place at 25 ° C for 16-20 hours

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

2.2.3 Add 180 μL dd-water, 1 μL Glycogen (20 ug/μL), 200 μL 5MNH4OAc, 1000 μL 100% ethanol, -80 ° C, let stand for 15 min; 14,000 rpm, 4 ° C, centrifuge for 25 min, remove the supernatant;

2.2.4 Add 1 ml of 70% ethanol to wash the precipitate; 14,000 rpm, 4 ° C, centrifugation for 3 min, remove the supernatant; repeat this step;

2.2.5 Drying the recombinant product at room temperature for 5-10 minutes;

2.2.6 repeated soaking with 8 μL of TE Buffer for 30-40 times to dissolve the recombinant product;

2.2.7 Firstly, pre-cool the 1mm electric rotor-80°C, add the recombinant product and 50μL electro-transformed competent cell DH10B to the electric rotor on ice, and set it on ice for 45min;

2.2.8 The electric shock condition on the TX ECM 630 is shown as:

Voltage	2.0kV
resistance	200 ohms
capacity	25uF

Immediately after the electric shock, 1 mL of SOC medium was added to the electric rotor;

2.2.9 Take the electroporate into a new 15mL centrifuge tube, and place it at 37 ° C, 225-250 rpm shaker for 1 hour;

2.2.10 After the end of the culture, 10 μL of the bacterial solution was diluted to 1:1000 to take 50 μL of the coated plate for reservoir capacity identification; the remaining culture was added with glycerol to a final concentration of 20% at -80 ° C, which is the yeast library solution. .

Example 3 Quality Identification of Yeast Library

3.1 Identification of the library capacity

After the transformed bacterial stock solution was diluted 1000-fold by 10 μL, 50 μL of the coated LB plate (containing the corresponding resistance) was taken out therefrom, and cultured at 37 ° C overnight to count the next day.

CFU/mL=Number of clones on the plate/50 μL×1000××1×10 ³ μL=Number of clones on the plate 310/50 uL×1000×1000 uL=6.2×10 ⁶ cfu/mL

Total CFU of the library=CFU/mL× Total volume of library solution (mL)=6.2×10 ⁶ cfu/mL×3mL=1.86×10 ⁷ cfu

3.2 Recombination rate and insert length identification

24 clones were randomly picked for colony PCR identification (5' AOX and 3' AOX sequences are shown in SEQ ID NO. 36, 37), and the following reaction solutions were prepared:

ddH 2 O	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 2720 PCR machine under the following conditions:

Table 1, 24 library insert length and recombination rate

clone	Insert size (kb)	clone	Insert size (kb)	clone	Insert 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	twenty one	1.1
6	1.4	14	1.0	twenty two	1.2
7	1.5	15	1.1	twenty three	1.1
8	1.2	16	1.0	twenty four	1.5

Example 4 Identification of recombinant protein activity by double membrane method

40 recombinant expression libraries were inoculated onto the MM plate with a sterile toothpick while GS115 blank control bacteria transfected with the pPIC9K plasmid containing no peptide of interest was inoculated as a control clone. After growing into a clone with a diameter of about 2 mm, the NC membrane was sterilized by moist heat, and the clone attached to the MM plate was labeled, inverted, and cultured at 29 ° C until the cloned position was wetted (about 3 to 4 days). ), the NC film was peeled off and dried at 37 ° C to form solid phase antigen spots. After 1 hour of mass fraction 1% BSA, 15 patients were mixed with serum pool as primary antibody, washed at 37 °C for 2 hours, 3 times, added with 1:1000 enzyme-labeled mouse anti-human IgE, 37 °C for 1 h, washed 3 times, added ECL The substrate was developed with color, and the color of the spot was observed to determine the activity of the recombinant protein. The corresponding transformants were subjected to single colony sequencing. There were 17 colonies showing positive results, see Figure 5. The results of single colony sequencing are shown in Table 2. It is found that Hum s3 is identical to the Hum s3:ADB97919.1 cDNA sequence in the NCBI database previously discovered by the applicant; Hum SX2 is identical to the registered EU088286.1 cDNA sequence in the NCBI database. Not listed in Table 2.

Table 2 positive single colony sequencing results

Remarks: NCBI registration number has not been disclosed yet

Example 4 Rapid screening of methanol yeast high expression strain by double membrane method

The transformants were picked from the MD plate and inoculated into 96-well culture plates. The wells were added with G418 YPD solution (1% yeast extract, 2% peptone, 2% glucose, 2.5 mg/mL G418). Incubate at 29 °C for 5-6 days, inoculate the grown colonies into another new plate with the same concentration of G418, and culture at 29 °C for 5-6 days; inoculate the grown colonies into a new plate with 4.0 mg/mL G418. The medium was cultured at 29 ° C for 5 to 6 days, and the grown colonies were inoculated into another new plate of the same mass concentration of G418, and cultured at 29 ° C for 5 to 6 days. The bacterial liquids to be screened in 2.5 mg/mL G418 and 4.0 mg/mL G418 plates were respectively streaked onto YPD-G418 plates of corresponding mass concentration, and cultured at 29 ° C until single colonies appeared. 60 transformants on different mass concentrations of G418 plates were randomly picked up with sterile toothpicks and inoculated onto MM plates (mass fraction 1.34% YNB, 4×10 ^-5 % biotin, 0.5% methanol and 1.5% agar). GS115 control isolates which had been transfected with the pPIC9K plasmid containing no peptide of interest were used as control clones. After growing into a clone with a diameter of about 2 mm, the NC membrane was sterilized by moist heat, and the clone attached to the MM plate was labeled, inverted, and cultured at 29 ° C until the cloned position was wetted (about 3 to 4 days). ), the NC film was peeled off and dried at 37 ° C to form solid phase antigen spots. After blocking for 1 h with mass fraction of 1% BSA, rabbit anti-porcine ZP3 antibody was added, washed at 37 °C for 2 h, 3 times, adding 1:500 enzyme goat anti-rabbit IgG, 37 ° C for 1 h, washing 3 times, adding DAB-H ₂ O ₂ The substrate was developed with color and the spot color was observed. The darkest (strongly positive) and positive clones on the NC membrane were picked and induced by conventional shake flask culture according to the Invitrogen manual. The supernatant was taken for routine SDS-PAGE and Western Blotting.

Example 5 Expanding culture, inducing expression and purification

The corresponding yeast strain was inoculated into the BMG medium, cultured at 30 ° C overnight, and transferred to a fermentor for cultivation. The ammonia water was maintained at a pH of 6.0, the dissolved oxygen degree was maintained at 60%, and high-density culture was carried out. Thereafter, the cells were cultured in BMM medium, maintaining a methanol concentration of 1%, and inducing expression for 3-4 days. The supernatant was collected by centrifugation, and PMSF was appropriately added as a protease inhibitor. The Ni column and the waste collection vessel were fixed, 20% ethanol was discharged from the column, and the Ni column was washed by adding PBS 1 CV. The supernatant solution is applied to the column, and the flow rate is controlled at 150 cm/h. The syringe needle and the pipette tip can be appropriately used to control the flow rate, and the flow-through sample is taken, and the sample is used as a flow-through sample. The Ni column was prepared by taking a PBS containing 25 mM imidazole solution at 1-1.5 CV, and taking a sample of the washing liquid. The target protein was eluted by using PBS to prepare a 200 mM imidazole solution at about 1 CV, and collected into a centrifuge tube. This sample was a purified protein of interest. TEV Protease was added to the freshly purified protein solution containing high concentration of imidazole, and the imidazole was removed by dialysis at 4 ° C to obtain the target allergen protein.

Example 6 ELISA binding inhibition test

The IgE binding inhibition assay evaluates two antigen or antigen mixtures containing similar epitopes. This example evaluates the similarity in the IgE binding epitope between the recombinant mixed alfalfa pollen allergen vaccine (in this case, mixing different recombinant allergens in equal proportions) and the natural allergen infusion. Reactivity) to assess the feasibility of using a recombinant recombinant psyllium pollen allergen vaccine to replace valerian pollen natural allergen infusion for immunotherapy.

1 coating: The natural valerian allergen infusion was diluted with a coating buffer (Na ₂ CO ₃ 0.16 g, NaHCO ₃ 0.293 g, 100 ml, pH 9.6), and coated in a 96-well microtiter plate at 5 μg/well ( In the AD row, columns 1-12), the sealing plate was coated overnight at 4 °C.

2 Wash the plate: discard the liquid, wash the plate 3 times with PBST, each time for 3 minutes, absorb the liquid in the dry hole.

3 Blocking: Add blocking solution 1% BSA-PBST, 200 μl per well, and let stand for 2 hours at room temperature.

4 Wash the plate: discard the liquid, wash the plate 3 times, each time for 3 minutes, the absorbent paper absorbs the liquid in the dry hole.

5 inhibitor sample preparation:

Diluting the natural valerian allergen infusion to 5 μg / ml as the first dilution, placed in A3-12, B3-12 (multiple pores), diluted in 3 × ratio, 50 μl / well;

The recombinant mixed valerian allergen vaccine was diluted to 0.05 μg/ml as a first dilution, placed in C3-12, D3-12 (multiple well), diluted 3× times, 50 μl/well.

6 Yin, positive serum pool dilution preparation:

The dilutions were diluted 1:5 in a positive serum pool (15 aliquots of valerian allergic patients). 50 μl/well was added to A2-A12, B2-12, C2-12, D2-12.

The dilutions were diluted 1:5 in a negative serum pool (5 healthy controls, mixed in equal proportions). 50 μl/well was added to A1-D1.

Then, 50 μl/well of 1% BSA-PBST dilution was added to A1-D1, A2-D2 to make the negative and positive control serum titers the same as A3-A12, B3-12, C3-12, D3-12 serum titers.

7 primary antibody incubation: After sealing at 37 ° C for 2 h, incubate overnight at 4 ° C.

8 Wash the plate: discard the liquid, wash the plate 3 times, each time for 3 minutes, the water paper absorbs the liquid in the hole.

9 Incubation of biotinylated goat anti-human IgE antibody (secondary antibody): dilution 1:1000 dilution antibody, 100 μl per well, 1 h at room temperature.

10 Wash the plate: discard the liquid, wash the plate 3 times, each time for 3 minutes, the water paper absorbs the liquid in the hole.

11 Incubation of horseradish enzyme labeled streptavidin: dilute 1:500 diluted secondary antibody, 100 μl per well, room temperature 0.5 h.

12 Wash the plate: discard the liquid, wash the plate 3 times, each time for 3 minutes, the water paper absorbs the liquid in the hole.

13 color development: Add coloring solution, 100ul per hole, and avoid light for 5min at room temperature.

14 Termination: Add stop solution at 50 ul per well and read at 450 nm in 30 minutes.

For data analysis, the log dilution is plotted on the abscissa and the inhibition rate is plotted on the ordinate. OD _{negative control} = (A1 + B1 + C1 + D1) / 4; OD _{positive control} = (A2+B2+C2 + D2) / 4; OD value of each competitive inhibition dilution takes the mean value of the re-pore; each competitive inhibition The inhibition rate of dilution % = (OD _{positive control -} OD _{dilution mean} ) / (OD _{positive control -} OD _{negative control} ) * 100%. A3-A12, B3-12 to investigate the competitive inhibition of gradient dilution of natural alfalfa pollen allergen infusion on serum IgE antibody in patients with natural alfalfa pollen allergen and alfalfa pollen allergy Effect of C3-12, D3-12 on the dilution of recombinant valerian pollen allergen vaccine against the reaction of serum IgE antibody in natural sorghum pollen allergen and valerian pollen allergic patients Inhibition effect.

The results are shown in Fig. 6. It can be seen that in the present embodiment, at the first dilution, the natural valerian pollen allergen (5 μg/ml) inhibited the coated natural alfalfa pollen allergen by 97.5%, and the recombinant mixture was recombined. The valerian vaccine (0.05 μg/ml) achieved 69% inhibition of the coated natural alfalfa pollen allergen. The results showed that the recombinant mixed alfalfa vaccine and the natural alfalfa pollen allergen had a similarity of 69%/97.5%=70.8%, indicating that the similarity between the two was high, and the cross-reaction was large, in the aspect of human IgE antigen recognition. The recombinant mixed alfalfa vaccine can basically replace the natural alfalfa pollen infusion; it also shows that the recombinant mixed alfalfa vaccine only needs the natural alfalfa pollen infusion when it reaches the titer of 70.8% of the natural alfalfa pollen infusion. 1% (0.05μg/ml/5μg/ml), indicating that under the same concentration conditions, the recombinant mixed valerian vaccine has a higher biological activity due to the removal of non-allergen components in psyllium pollen. Because the composition of the recombinant mixed valerian vaccine is clear, the content of each component is easy to control, and it is easy to purify and combine with one component, it represents the development direction of the accurate molecular diagnosis and treatment of the allergen.

Example 7 Basophilocyte activation test

The recombinant valerian pollen allergen vaccine prepared by the invention carries out basophil activation test on whole blood of patients with allergic to valerian pollen, thereby verifying that it has allergic reaction with sIgE antibody and activated basophil degranulation Learning function. It is also theoretically confirmed that it has the characteristics of a specific immunotherapy vaccine. Therefore, it has a function as a recombinant allergen vaccine.

Test principle: Allergen and patient whole blood cell reaction can mimic the allergic process in human body: that is, specific IgE antibody binds to the cell surface by bridging with the corresponding allergen, and activates intracellular signal cascade to cause basophils (CCR3 is continuously expressed in basophils, which is a specific marker for its active degranulation). In this process of degranulation, the intracellular complex affects the transmembrane protein CD63 (gp53), which is expressed on the cell surface and exposed to the extracellular matrix, thus relying on the principle of flow cytometry The basophils are labeled with the factor receptor CCR3-phycoerythrin (anti-CCR3-PE) using an anti-human CD63 monoclonal antibody-fluorescein isothiocyanate (anti-CD63-FITC) for the activated state. The basophils were labeled, the non-specific cell activator fMLP was used as a positive property, and the percent change in basophil degranulation was used to determine whether the control was allergic to a particular allergen. METHODS: Patients with healthy controls and valerian allergy were selected and their EDTA anticoagulated whole blood samples were taken to stimulate the buffer (negative control) and the recombinant valerian pollen allergen vaccine mixture (mix the components in equal proportions, dilute to 2×10-7mg/ml), fMLP stimulating solution (positive nature control) as an activator of basophils, added to whole blood, then added anti-CD63-FITC, anti-CCR3-PE staining, within 48h Upstream cytometry for detection. Result: See Figure 7. When the healthy control is a negative control, a recombinant psyllium pollen allergen vaccine, and a positive control is a basophil activation, the basophil activation rate should be <15%, <15%, ≥15%, respectively. When the grass pollen allergy patients were negative control, recombinant valerian pollen allergen vaccine, and the positive control was basophil activated matter, the basophil activation rate should be <15%, ≥15%, ≥15%, respectively. . Conclusion: The recombinant psyllium pollen allergen vaccine can effectively bind sIgE antibody and activate basophil degranulation. Therefore, it possesses antigenicity and corresponding biological functions, and can be effectively applied to the specific diagnosis of patients with valerian pollen allergy. It can also be used as a potential therapeutic vaccine with development and application prospects.

Example 8 Evaluation of Immunotherapy for Mouse Allergic Asthma Model by Recombinant Mixed Valerian Pollen Allergen Vaccine

Objective: (1) Establish a mouse model of asthma that is allergic to mixed psyllium pollen allergen; (2) Use a psyllium pollen allergen recombinant vaccine (including 15 valerian-sensitizing proteins described in Table 2, and The allergic asthma mouse-specific immunotherapy according to the Hum s 3 and Hum SX2 sensitizing protein molecules obtained in Examples 4 and 5 of the present invention. Methods: (1) Establishing a mouse model of asthma mixed with recombinant valerian pollen allergen using a method of subcutaneous subcutaneous sensitization plus nebulization; (2) Applying successful modeling mice, using a subcutaneous subcutaneous injection method To give specific time immunotherapy to asthmatic mice that are allergic to mixed valerian pollen allergens. RESULTS: (1) The sRAW value of the model group was significantly higher than that of the control group, especially when the acetylcholine concentrations were 25 mg/ml and 50 mg/ml (p<0.01), which proved that the mouse asthma model was established successfully. After specific immunotherapy, the sRAW values of the treatment group were lower than those of the model group, especially at the acetylcholine concentrations of 25 mg/ml and 50 mg/ml (p<0.01), demonstrating the recombinant mixed valerian pollen allergen vaccine to mice. Immunotherapy in the asthma model reduced and improved airway hyperresponsiveness in mice. (2) The total number of inflammatory cells, neutrophils, lymphocytes, mononuclear cells, and number of eosinophils in the model group were significantly higher than those in the control group (P<0.01); The airway developed a serious inflammatory reaction, and the infiltration of inflammatory cells was mainly EOS, and the EOS% was also significantly increased (P<0.01), indicating successful modeling. After specific immunotherapy, the total number of inflammatory cells, neutrophils, lymphocytes and eosinophils in the treatment group were significantly lower than those in the model group (P<0.01), and EOS% was also significantly decreased (P<0.05). <0.01), indicating that immunotherapy effectively inhibits airway inflammation in mice and reduces cell infiltration. (3) The results showed that the total IgE and mixed recombinant psyllium pollen allergen sIgE in the model group were significantly higher than those in the control group (P<0.01), which proved that the model was successful and could induce mice to produce. A lot of IgE. After specific treatment, the expression level of total IgE and mixed recombinant alfalfa pollen allergen sIgE in the treatment group was significantly lower than that in the model group (P<0.01), which proved that immunotherapy successfully inhibited the production of IgE. In addition, the expression level of sIgG in the treatment group mixed recombinant valerian pollen allergen vaccine was significantly higher than that in the model group (P<0.01), which proved that immunotherapy successfully induced the production of sIgG antibody. (4) HE staining of lung tissue showed that a large number of inflammatory cells infiltrated in the tracheal and peripheral blood vessels of the model group, forming a multi-layered cell ring accompanied by airway thickening and edema. No abnormalities were observed in the control group. After specific immunotherapy, the infiltration and edema of inflammatory cells in the lungs and peripheral blood vessels of the mice were significantly relieved; AB-PAS staining of lung tissue revealed that the airway wall of the model group was widely stained blue-violet, and the airway could also be seen to diffuse. The blue-violet color indicates that a large amount of mucus material is produced; the goblet cells proliferating in the airway epithelium account for >75% of the total number of airway epithelial cells. No obvious abnormalities were observed in the control group. The proliferation of goblet cells in the airway epithelium accounted for <25% of the total number of airway epithelial cells. The airway wall of the treatment group was stained blue-violet, and the cup-shaped cells proliferating in the airway epithelium accounted for <50% of the total number of airway epithelial cells. Conclusion: (1) The mouse model of asthma with mixed reconstituted valerian pollen allergen vaccine was established successfully; (2) The specific immunotherapy of asthma mice with mixed recombinant valerian pollen allergen vaccine allergy was effective.

Example 9 Individualized treatment of psyllium pollen recombinant vaccine

OBJECTIVE: To evaluate the immunoprotective response and toxicity of single and its mixed sorghum pollen recombinant molecular vaccine administered to SD rats by repeated subcutaneous injections, and to evaluate the humoral immune response induced by a single and mixed psyllium pollen recombinant vaccine in animals. Provide animal test data on pharmacology and safety for clinical trials and clinical applications of individualized molecular therapy for psyllium pollen recombinant vaccine.

Methods: 204 healthy SPF SD rats were selected, male and female. They were randomly divided into 17 groups according to body weight and gender, with 12 in each group. The #1-15 group is a valerian single molecule recombinant vaccine administration treatment group, respectively corresponding to the recombinant protein of the present invention Hum SC 1-15 (corresponding to the above Table 2, yeast induction, expression, purification, lyophilization, respectively); The 16 groups were treated with the sorghum mixed recombinant vaccine administration group, and the recombinant protein of the present invention Hum SC1-15 was mixed in equal proportions; the #17 group was the vehicle control group (0.2%-0.4% phenol, 4.5-5.5 g/ L sodium chloride, 0.025%-0.035% human serum albumin). Route of administration: subcutaneous injection. Dosing concentration and volume: #1-15 groups, each injection 0.05ug/ml*1.0ml. #16 groups, each injection of 0.06ug/ml*1.0ml (corresponding to the sum of Hum SC1-15 each valerian recombinant sensitizing protein 0.0015 ug/ml*1.0ml). Dosing frequency: 2 times a week, 2-3 days apart. Continuous administration for 6 months, recovery period: withdrawal of drug recovery for 1 month. The following examinations were performed on the experimental animals throughout the experiment: general symptom observation, injection site irritation, body weight, and food intake. Animals were euthanized (3/sex/group) at 6 months after continuous administration and at the end of one month after recovery. Animal gross anatomy and histopathological examination were performed to determine serum IgG2a content to evaluate antigen immunity. . Rat serum IgG2a content was measured using a Rat IgG2a ELISA Kit ((Jianglai Bio)).

RESULTS: (1) General symptoms of animals: no abnormalities in respiratory, exercise, eyelid indications, feces, urethra, skin, hair, salivation, etc., and related to the test articles were observed during the whole experiment. Toxic effects of sudden death and death. (2) Irritating part of the injection site: visual observation: During the 6-month period of administration and the recovery period, local injections of the rats in each group showed no local reactions such as congestion, edema, induration and necrosis. (3) Body weight and food intake: After 6 months of administration and 1 month of withdrawal, the body weight of male rats and female rats in each group increased. Compared with the CN control group, the difference was not statistically significant (P>0.05). ). (4) histopathological examination results: the gross anatomy of each animal was normal and the color was normal, the texture was uniform, and there was no lump. The mucosa of the hollow organ is intact and the color is normal. Under the microscope observation, at the end of the administration and at the end of the recovery period, the organs examined in each group (except for the subcutaneous tissue at the administration site) showed no pathological changes related to the test article. (5) After administration for 6 months and withdrawal for 1 month, there was no significant change in total IgG2a (total IgG2a, tIgG2a) in male rats and female rats, but the IgG2a of each component of valerian allergen ( Specific IgG2a, ie sIgG2a, such as Hum SC1s IgG2a, Hum SC2s IgG2a, Hum SC3s IgG2a..., Hum SC15s IgG2a) There was a statistically significant difference between the administration group and the control group (P<0.05), and the valerian recombinant vaccine (Hum SC1- There was a statistically significant difference in the sIgG2a content between the drug-administered group and the control group (P<0.05). See Figures 8a and 8b for details.

Conclusion: The present invention relates to a sorghum monomolecular allergen recombinant vaccine Hum SC 1-15 and a valerian allergen mixed recombinant vaccine (Hum SC 1-15 content of each component is uniform) under the protocol of the present embodiment, the rat The general symptom observation, body weight, food intake, organ weight, organ coefficient, gross anatomy and other results showed no toxicological changes related to the related single molecule vaccine, mixed vaccine and vehicle control, indicating that the recombinant single molecule of the present invention Vaccines and mixed vaccines have better safety. After receiving immunotherapy, both single-molecule vaccines and mixed vaccines produced higher titers of immunoprotective IgG2a antibodies (in human immunotherapy, high titers of IgG4 antibodies were produced, competing with IgE Binding, alleviating allergic inflammatory response), and the protective IgG2a antibody persisted after one month of withdrawal during the recovery period, confirming that the single and mixed valerian recombinant allergen vaccine immunotherapy according to the present invention has long-term efficacy and protection Function and safety.

Example 10 Individualized treatment plan for psyllium pollen recombinant vaccine

10.1 Accurate diagnosis

The pricking test was performed on all the potential allergens of valerian pollen, and saline was used as a negative control and histamine was used as a positive control. All patients discontinued anti-allergic drugs and hormones for 1 week before the skin test. The skin pricking site is selected from the curved side of the forearm. After disinfection with alcohol, an allergen prick is performed at intervals of 3 cm on the skin. After 8 min of pricking, the histamine reaction was observed, and the allergen reaction was observed for 15-20 min. According to the range of the allergen wind block and the standard histamine wheal range, the patient's sedge pollen allergen component was confirmed.

10.2 personalized treatment

According to the patient's diagnosis of the psyllium pollen allergen prick test, the composition and ratio of the recombinant vaccine were determined. The purified allergen is prepared according to the patient's needs and added with adjuvant to prepare various concentrations of the recombinant vaccine, and repeated doses or sublingual or repeated routes of administration to the patient through repeated administration, the dose is from small to large. The concentration is from low to high. After the maintenance dose is reached, sufficient course of treatment is maintained to improve the patient's tolerance to the allergen. When the allergen is contacted again, the allergic phenomenon is alleviated or no longer allergic. phenomenon.

The above description is only a preferred embodiment of the present invention, and it should be noted that those skilled in the art can make several improvements and retouchings without departing from the technical principles of the present invention. It should also be considered as the scope of protection of the present invention.

Claims (27)

1. A recombinant mixed psyllium pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 5.
2. A recombinant mixed psyllium pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 6.
3. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 9.
4. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 11.
5. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 13.
6. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 15.
7. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 17.
8. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 19.
9. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 21.
10. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 23.
11. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 25.
12. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 27.
13. A recombinant mixed psyllium pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 29.
14. A recombinant mixed alfalfa pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 31.
15. A recombinant mixed valerian pollen allergen vaccine comprising the amino acid sequence set forth in SEQ ID No. 33.
16. A recombinant mixed psyllium pollen allergen vaccine comprising two or more proteins selected from the group consisting of SEQ ID NO. 5, 7, 9, 11, 13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 and 33 are shown.
17. Valerian pollen allergen protein, which is a protein 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 shows
18. A therapeutic or diagnostic preparation comprising the valerian pollen recombination protein of claim 17.
19. The preparation according to claim 18, wherein each protein in the preparation is obtained by recombinant expression in vitro, and the preparation is obtained by formulation.
20. The preparation according to claim 18, wherein the preparation is a vaccine.
21. The formulation of claim 18, wherein the formulation further comprises an adjuvant.
22. A gene encoding the protein of claim 17.
23. A vector comprising the gene of claim 22.
24. A host cell comprising the vector of claim 23.
25. A method of producing the protein of claim 17, which comprises inducing expression by culturing the host cell of claim 24, and isolating the protein.
26. The method according to claim 23, wherein said host cells are cultured separately, expression is induced, and said protein is isolated.
27. A method for constructing a yeast expression library of valerian pollen cDNA, the method comprising the following steps:

    (1) The target fragment attR1-Cm ^R -ccd-attR2 was amplified from pDEST-22 and cloned into the destination vector pPIC9K by SnaBI and AvrII to obtain the final vector pPIC9K-attR1-Cm ^R -ccd-attR2;

    (2) Extraction of total RNA from alfalfa pollen by Trizol method;

    (3) isolating mRNA from the total RNA extracted by (2);

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

    (5) Preparing the primary full-length homogenization library plasmid obtained in (4), and then recombining the primary homogenized library mixed plasmid into the yeast vector pPIC9K-attR1-CmR-ccd-attR2 by LR reaction, and then recombining The product was transformed into DH10B competent cells, and the library capacity was detected by using a resistant LB plate to obtain a full-length homogenous yeast expression library.

Images