Patent Application
20250109176
The instant application contains a Sequence Listing which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety. Said XML copy is named GBNJIP146-Sequence-Listing.xml, created on 07/01/2024, and is 20,762 bytes in size.
The present disclosure belongs to the technical field of biological medicines and relates to a recombinant type I allergen of Artemisia annua pollen, the characterization and activity of which are consistent with those of a native allergen, and a preparation method and a use thereof.
Pollen is one of the main triggers of seasonal allergy. Unlike food allergy, pollen allergy is transmitted through the air and is often difficult to avoid. Pollen allergy can induce a series of allergic reactions such as rhinitis, dermatitis and asthma, which seriously affect the quality of life of patients. Pollen allergy affects about 7% of adults and 9% of children in the United States (NIAID, National Institute of Allergy and Infectious Diseases), and prevalence is estimated to be as high as 40% in Europe (G.D 'Amato, 2007). In recent years, with the increasing area of green land and the “grain for green” program in China, the incidence of pollen allergy has been increasing year by year, and in the high incidence area can reach 5%.
The Artemisia pollen is one of the important allergens that cause hay fever in summer and autumn. The results of 215210 allergen specific IgE tests in China from 2008 to 2010 showed that Artemisia pollen had the highest positive rate among inhaled allergens. Qiongliang Yang et al. showed in 2015 that Artemisia pollen was the most important allergen in northern China.
Artemisia L. is one of the most abundant genera in the Asteraceae family. There are more than 300 Artemisia species in the world, which are widely distributed in temperate, temperate cold and subtropical regions of the Northern Hemisphere. Artemisia annua and Mugwort are common pollen-sensitized plants. Among them, Artemisia annua, which can be used to extract artemisinin, is one of the most common Artemisia plants in China, and it is also the earliest Artemisia allergic plant studied in China. Mugwort, also known as northern Artemisia vulgaris, is widely distributed in China, Mongolia, Russia, Europe, the United States, Canada and other places. It is one of the most deeply studied pollen allergens. Molecular Biological Analysis and Component Diagnosis of Artemisia Pollen Allergens in China (Zhejiang University, Fu Wanyi, 2018) showed that Artemisia annua was the most important allergen in Artemisia pollen. Other major sensitized Artemisia species include Artemisia sieversiana, Artemisia capillaris, Artemisia lavabdykufikua, and Artemisia desertorum, Artemisia argyi, etc. The main allergenic proteins of different Artemisia pollen are type I and type III allergens. Type I allergen belongs to the defensin-like protein family with molecular weight of about 12kD. Type III allergens belong to non-specific lipid transport protein (nsLTP), which has relatively high variability in different Artemisia pollen.
The World Health Organization (WHO) proposes a “four-in-one” approach to allergic diseases: allergen avoidance, symptomatic drug treatment, specific immunotherapy, and physician and patient education. Among them, avoiding exposure to allergens refers to doing a good job in environmental prevention and control on the basis of identifying allergens and avoiding exposure to allergens as far as possible. Avoiding allergens in the treatment of allergic diseases can not only reduce the incidence of allergy, but also improve the efficacy of drugs and help patients desensitize faster. Specific immunotherapy, namely desensitization therapy, is the only “cause-specific” therapy that may affect the natural course of allergic diseases and change the immune response mechanism. It uses gradually increasing doses of allergens to improve the patient's tolerance to the allergen, reduce the symptoms caused by exposure to the allergen, and eventually achieve tolerance or even immune tolerance.
In 2021, Zhejiang Wolwo pharma's desensitization drug Artemisia annua pollen allergen sublingual drops for the treatment of allergic rhinitis caused by Artemisia annua/Mugwort pollen was approved for marketing. Its patent CN101905022A states that “as raw material, the Artemisia pollen is defatted, extracted and concentrated to produce allergen extract from Artemisia pollen”. However, natural allergen extracts inevitably have quality problems due to the limitation of raw material sources and production methods, such as the presence of undefined non-allergic substances, contaminants, and high variability in allergen content and biological activity (Valenta R, P. et al.Allergen Extracts for in vivo diagnosis and treatment of allergy: is there a future[J].Journal of allergy & Clinical immunology in practice, 2018). The EAACI Guidelines on allergen immunotherapy: allergic rhinoconjunctivitis (2018) issued by the European Academy of Allergy and Clinical Immunology also states that: mixed allergens present a number of potential disadvantages, including dilution effects, potential allergen degradation due to the enzymatic activity of some allergens, and difficulties in adequately demonstrating the efficacy of allergen combinations. The standardized desensitizing drugs approved for marketing by EMA, HMA, and FDA are basically limited to the major allergenic proteins. For example, ODACTRA for house dust mite allergy contains major allergenic proteins Der p 1, Der p 2, Der f 1 and Der f 2. GRAZAX for timothy allergy contains Phl p 5. RAGWIZAX for ragweed allergy contains the major allergenic protein Amb a 1. On the other hand, the use of natural extracts for allergen diagnosis has the problems of low sensitivity and specificity, and it is not possible to determine reaction degree to allergen components, which may easyly lead to misdiagnosis.
The recombinant main allergens by genetic engineering can achieve high purity, high homogeneity, and the same immune activity as native allergens, which can effectively avoid the disadvantages of natural extracted mixed allergens. At present, recombinant allergens are widely used in the development of new diagnostic devices for allergic diseases. For example, the use of recombinant allergen proteins for molecular diagnosis of allergens can identify allergenic proteins, improve the accuracy of allergen diagnosis, and help to identify cross-allergy. At the same time, it has been confirmed that highly purified recombinant major allergens can be used for desensitization treatment of allergic diseases, and it is also important for the development of the second generation of desensitization drugs. At present, there is no recombinant Artemisia pollen allergen protein as drug on the market or in clinical trials.
The present disclosure provides a method for recombining and expressing the major allergens of Artemisia annua pollen. Compared with natural extraction methods, the allergens generated by recombining expression technology have more stable and controllable product quality, which is more suitable for the diagnosis and treatment of allergic diseases caused by Artemisia annua pollen.
An objective of the present disclosure is to provide a protein for treating Artemisia pollen allergy, which is a recombinant Art a 1 protein. Art a 1 is type I allergen protein of Artemisia annua and is major allergenic protein. It is a glycoprotein composed of the N-terminal defensin domain and the C-terminal hydroxyproline-rich part, belonging to the defensin-like protein family. Numerous researchs have shown that the type I allergen proteins of different Artemisia species are all defensin proteins with highly conserved sequences. The binding ability of each subtype to sIgE antibody is similar, and its immune activity is mainly determined by the N-terminal defensin domain. “Molecular biological analysis and component diagnosis of Artemisia pollen allergen in China” published by Fu Wanyi et al.in 2018 showed that, same as foreign patients, Chinese Artemisia pollen allergic patients had higher recognition degree of Artemisia annua pollen type I allergen (Art a 1) than other components, it is the main allergen in Artemisia annua pollen. The amino acid sequence, disulfide bond and molecular weight of the recombinant Art a 1 protein is completely consistent with that of the native protein, and it has similar biological activity to the native protein.
Preferably, the amino acid sequence of Art a 1 protein is set forth in SEQ ID NO. 4.
The amino acid sequence, molecular weight, amino acid coverage and disulfide bond of the recombinant Art a 1 protein in the present disclosure are completely consistent with the native Art a 1 protein and recombinant Art a 1 have similar immunological activity with native Art a 1 protein. Compared with the pollen extract of natural Artemisia annua, variation in content and activity between batches is avoided, the process and quality is more stable and controllable, the degradation of main allergens and other allergic reactions caused by the interaction of other components in natural pollen is avoided, so as to meet the requirements of safe, effective and controllable quality of modern biological products. It can be used in the treatment and diagnosis of Artemisia pollen allergy such as allergic rhinitis and asthma, and improve the accuracy of Artemisia pollen desensitization immunotherapy and the accuracy of Artemisia pollen allergy diagnosis. A further objective of the present disclosure is to provide DNA sequence encoding the Art a 1 protein with base sequence as set forth in SEQ ID NO: 14. This sequence is codon-optimized for the Pichia pastoris expression system, which is more conducive to the expression of Art a 1 in Pichia pastoris. Surprisingly, different codon optimization methods have significant impact on the yield of Art al. Compared to the wild-type Art al, the yield with optimized codon reach 200 mg/L. Compared with the native protein, the purified recombinant Art a 1 protein showed similar immunoreactivity with specific antibodies in the serum of allergic patients in vitro.
Another objective of the present disclosure is to provide secretory signal peptide design that is beneficial to the expression of Art a 1 protein in Pichia pastoris expression system, which not only improves the expression of Art a 1 protein, but also the molecular characterization of the obtained recombinant Art a 1 protein is completely consistent with that of the native protein. Such signal peptides are yeast α-factor signal peptide, melanomycin signal peptide, acid phosphatase signal peptide (PHO), Saccharomyces cerevisiae signal peptide (SUC2) or Art al protein wild type signal peptide, more preferably α-factor signal peptide (SEQ ID NO: 11) and wild type signal peptide (SEQ ID NO: 12). The inventors found that different secretory signal peptides had significant effect on the uniformity and expression of the recombinant Art a 1 protein, and the preferred signal peptide was more conducive to the correct and efficient expression, and the recombinant Art a 1 obtained was not only highly expressed, but also was completely consistent with native Art a 1 in the primary structure, molecular weight, amino acid coverage and disulfide bond.
A further objective of the present disclosure is to provide a vector containing the gene encoding Art a 1 as described above. Preferably, the vector is pAO815, pPIC9, pPIC9K, pPIC3.5, pIC3.5K, pPICZαA, B, C or pGAPZαA, B, C, and more preferably, the vector is pPICZαA or pGAPZαA.
A further objective of the present disclosure is to provide a Pichia pastoris strain comprising the vector described above. Preferably, the Pichia pastoris strain is SMD1168, GS 115, KM71, X33 or KM71H, more preferably is the KM71 or X33 strain.
The recombinant protein coding gene of the present disclosure is more conducive to Pichia pastoris expression. The inventors found that different combinations of signal peptides, expression vectors, and host bacterial have a significant impact on the yield of recombinant Art a 1. The best combination has a yield of 220 mg/L. The purified recombinant Art a 1 protein has the same amino acid sequence, disulfide bond and molecular weight as the native protein, and showed similar activity of immune response in vitro with specific antibodies in the serum of allergic patients as the native protein.
A further objective of the present disclosure is to provide expression method of Art a 1 protein, comprising the steps of:
A. Construction of a vector containing the gene encoding Art a 1 described above:
The commonly used pPICZ series or pGAPZ series vectors do not contain signal peptides in the exogenous gene expression cassette, so they can be used to construct Art a 1 expression cassette containing wild type signal peptides. Specifically, the artificial sequence of Art a 1 containing wild type signal peptide and start codon and a stop codon is cloned into the polyclonal sites (such as EcoRI and NotI) of the corresponding vector, so that the open reading frame is located downstream of the promoter and upstream of the terminator to construct a recombinant expression vector.
The common pPICZα series or pGAPZα series vectors contain the α-factor signal peptide and its signal peptide cleavage site: Kex2(amino acid sequence is KR), Ste 13(amino acid sequence is EAEA, SEQ ID NO: 15) in the exogenous gene expression cassette, and Kex2 is located upstream of Ste 13. The gene coding Art a 1 is cloned into downstream of Kex2 site (the α-factor signal peptide is excised by Kex2 protease) or downstream of the Ste 13 site (the α-factor signal peptide could be excised by Kex2 and/or Ste 13 protease at the same time). Taking the pPICZαA vector as an example, if the gene coding Art a 1 is located downstream of Kex2, the target gene could be cloned between XhoI and NotI sites. If the gene coding Art a 1 is located downstream of Ste13, the target gene could be cloned between EcoRI and NotI sites. The recombinant expression vector expressing Art a 1 with α-factor signal peptide is constructed by example.
B. The vector of step A is linearized and transferred into Pichia pastoris strain, and the clone with high expression is screened under the pressure of antibiotic, and the expression level is further verified by cultivation and purification in suitable conditions.
C. Recovery of purified proteins.
Such vectors are preferably pPICZα A or pGAPZα A.
The Pichia pastoris strains described above are preferably KM71 or X33 strains.
A further objective of the present disclosure is to provide purification method of recombinant Art a 1 protein as follows:
A. Art a 1 fermentation broth is centrifugated at low temperature and high-speed, the supernatant is collected and concentrated by ultrafiltration with 3KD membrane package, replaced by pH7.0 25 mM PB buffer, and filtered by 0.45 μm filter membrane.
B. In the first step of cation chromatography, the column is equilibrated with equilibration buffer, and then the fermentation liquid obtained in step A is passed through the separation filler by the purification system, then the elution buffer is used for gradient elution, and the elution peak is collected; the equilibration buffer is 25 mM PB, pH7.0, and elution buffer is 25 mM PB, 1.0M NaCl, pH7.0.
C. In the second step, the Art a 1 protein peak collected in B is diluted with equilibration buffer, and the chromatographic column is equilibrated by equilibration buffer. The diluted Art a 1 protein solution is loaded onto the hydrophobic chromatographic packing and collect the elution peak. Equilibration buffer is 1.0M(NH4)2SO4, 25 mM PB, pH7.0, and elution buffer is 25 mM PB, pH7.0.
D. In the third step, the target protein peak collected in C is ultrafiltered and replaced with pH7.0 25 mM PB buffer. Art a 1 protein stock solution is obtained after filtration and remove bacteria.
After optimizing the cultivation process and purification method, the recombinant Art a 1 prepared by the present disclosure meets the requirements of recombinant DNA products for human use in terms of purity, process impurity residue, molecular characterization, etc. The SEC-HPLC purity is >99%, and the expression level reaches 220 mg/L. It has the same amino acid sequence, disulfide bond and molecular weight as the native protein, and its in vitro activity of immune response with specific antibodies in the serum of allergic patients is equivalent to the native protein. It has good potential for medicinal use. Compared with naturally extracted allergen products, recombinant allergen molecules have many advantages, such as variation in content and activity between batches is avoided; the process and quality is more stable and controllable; avoid the degradation of main allergens and avoid other allergic reactions caused by the interaction of other components in native pollen; meet the needs of safe, effective and controllable quality of modern biological products; in addition, allergy diagnostic kits using recombinant allergen protein can accurately identify the allergen protein that triggers the body reaction.
The present disclosure was further described below in connection with specific examples, and it was understood that the examples were cited only to illustrate the present disclosure and not to limit the scope of the present disclosure.
According to the DNA sequence of Art a 1 published in NCBI (Genbank accession number: KC700033.1, containing wild type signal peptide), as set forth in SEQ ID NO: 1, two gene sequences containing wild type signal peptide, Art a 1-01 and Art a 1-02, were obtained after codon optimization, the nucleotide sequences were set forth in SEQ ID NO: 2 and SEQ ID NO: 3, respectively, and the amino acid sequence was set forth in SEQ ID NO: 4. Comparison of base sequences before and after codon optimization was shown in
The GC content can affect the expression level of genes. The ideal GC content is 30%-70%, and GC content beyond this range will affect transcription and translation efficiency. The average GC base content of Art a 1 gene in
1. pPIC Expression Plasmid was Constructed
Enzyme cutting site sequence EcoR I and XhoI were introduced to the 5′ end and the 3′ end of the codon-optimized Art a 1-01 and 02 genes in Example 1, and synthesize the whole gene. The synthesized gene fragment was constructed into pPICZ plasmid (provided by GenScript Biotech Corporation) to obtain long-term preservation plasmids, which were denoted as pPICZ-Art a 1-01 and pPICZ-Art a 1-02 according to different optimization methods.
2. pGAP Expression Plasmid was Constructed
pPICZ-Art a 1-01 and pPICZ-Art a 1-02 plasmids were used as templates for PCR amplification with the following primer sequences:
5′AOX primer was used as forward primer, with a sequence as set forth in SEQ ID NO: 5; 3′AOX primer was used as reverse primer, with a sequence as set forth in SEQ ID NO: 6.
The total volume of reaction system was 50 μL, including 2.5 μL of each primer at a concentration of 10 μmol/L, 1 μL of dNTP at a concentration of 10 mmol/L, and 0.5 μL of 2U/μL DNA polymerase Q5(purchased from New England Biolabs). The reaction condition was 98° C. for 5 seconds, 55° C. for 45 seconds, and 72° C. for 30 seconds, and after 25 cycles, the products were analyzed by 1.0% agarose gel electrophoresis, which showed that the product had the expected size (400 bp) (the results were shown in
Using pPICZ-Art a 1-01 plasmid as template, the Art a 1-01 gene without signal peptide was obtained by PCR amplification, as set forth in SEQ ID NO: 13. Primer sequences was used as follows: the forward primer was SEQ ID NO: 7; the reverse primer was SEQ ID NO: 8.
Using pPICZ-Art a 1-02 plasmid as template, the Art a 1-02 gene without signal peptide was obtained by PCR amplification, as set forth in SEQ ID NO: 14. Primer sequences was used as follows: the forward primer was SEQ ID NO: 9; the reverse primer was SEQ ID NO: 10.
The total volume of reaction system was 50 μL, including 2.5 μL of each primer at a concentration of 10 μmol/L, 1 μL of dNTP at a concentration of 10 mmol/L, and 0.5 μL of 2U/μL DNA polymerase Q5 (purchased from New England Biolabs). The reaction condition was 98° C. for 5 seconds, 55° C. for 45 seconds, and 72° C. for 30 seconds, and after 25 cycles, the products were analyzed by 1.0% agarose gel electrophoresis, which showed that the product had the expected size (400 bp) (the results were shown in
1. pPICZα Expression Plasmid was Constructed
The purified product was ligated into pPICZαA plasmid (purchased from Invitrogen) with T4 ligase (M0202S, purchased from New England Biolabs) and transformed into DH5u competent cells (CB101, purchased from Beijing Tianggen Biochemical Technology Co., LTD.). Culture overnight at 37° C. in LB solid medium containing bleomycin (purchased from Invitrogen). The next day, the positive clones were selected for sequencing, which were completely consistent with the expected sequence. The Art a 1 codon-optimized expression plasmids were obtained, which were recorded as pPICZα-Art a 1-01 and pPICZα-Art a 1-02.
2. pGAPZα Expression Plasmid was Constructed
The purified product was ligated into pGAPZαA plasmid (purchased from Invitrogen) with T4 ligase (M0202S, purchased from New England Biolabs) and transformed into DH5u competent cells (CB101, purchased from Beijing Tianggen Biochemical Technology Co., LTD.). Culture overnight at 37° C. in LB solid medium containing bleomycin (purchased from Invitrogen). The next day, the positive clones were selected for sequencing, which were completely consistent with the expected sequence. The Art a 1 codon-optimized expression plasmids were obtained, which were denoted as pGAPZα-Art a 1-01 and pGAPZα-Art a 1-02.
Preparation of YPDS+Zeocin solid medium: According to the description of Pichia expression vectors for constitutive expression and purification of recombinant proteins of Invitrogen, yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, AGAR 15 g/L, sorbitol 18 g/L, and Zeocin at a final concentration of 0.1 mg/ml was included.
1, pPIC Expression Plasmid Transformation and Engineering Strain Screening
Electrocompetent cells were prepared according to the description of Easy Select Pichia Expression Kit of Invitrogen. Plasmids pPICZ-Art a 1-01, pPICZ-Art a 1-02, pPICZα-Art a 1-01 and pPICZα-Art a 1-02 obtained from Example 2 step 1 and Example 3 step 1 were digested and linearized with Sac I restriction enzyme (purchased from New England Biolabs), respectively. After ethanol precipitation, the linearized vectors were electrotransformed into Pichia pastoris X33 competent cells, coated in YPDS solid medium, and cultured at 30° C. until the transformants grew.
2, pGAP Expression Plasmid Transformation and Engineering Strain Screening
The electroconversion competent cells were prepared according to the description of Pichia expression vectors for constitutive expression and purification of recombinant proteins. Plasmids pGAPZ-Art a 1-01, pGAPZ-Art a 1-02, pGAPZα-Art a 1-01 and pGAPZα-Art a 1-02 obtained from Example 2 step 2 and Example 3 step 2 were digested and linearized with Avr II restriction enzyme (R0174S, purchased from New England Biolabs), respectively. After ethanol precipitation, the linearized vectors were electrotransformed into Pichia pastoris X33 competent cells, coated in YPDS solid medium, and cultured at 30° C. until the transformants grew.
1, pPIC Clone Screening and Identification
The monoclonal engineering bacteria obtained in Example 4 step 1 was selected and cultured in 5 mL BMGY medium in a 50 mL sterile centrifuge tube at 30° C. When OD600=1.0-2.0, the bacterial solution was centrifuged at 4000 rpm for 10 minutes, resuspended in BMMY medium, and induced for expression, and methanol was added every 24 hours to a final concentration of 1%. After being cultured at 220 rpm for 72 hours, centrifugated the bacterial solution, collected the supernatant, and the supernatant was analyzed by SDS-PAGE gel electrophoresis to observe the brightness of the expressed product.
BMGY+zeocin medium preparation: according to description of Easy SelectPichia Expression Kit from Invitrogen, yeast extract 10 g/L, peptone 20 g/L, K2HPO4 3 g/L, KH2PO4 11.8 g/L, YNB 13.4 g/L, Biotin 4×10−4 g/L, glycerol 10 g/L, and Zeocin at a final concentration of 0.1 mg/ml was included.
BMMY+Zeocin medium preparation: according to description of Easy SelectPichia Expression Kit from Invitrogen, yeast extract 10 g/L, peptone 20 g/L, K2HPO4 3 g/L, KH2PO4 11.8 g/L, YNB13.4 g/L, Biotin 4×10−4 g/L, methanol 5 mL/L, and Zeocin at a final concentration of 0.1 mg/ml was included.
2, pGAP Clone Screening and Identification
The monoclonal engineering bacteria obtained in Example 4 step 2 were selected and cultured in 5 mL YPD medium in 50 mL sterile centrifuge tube at 30° C. and 220 rpm for 48 hours. Centrifugated the bacterial solution, collected the supernatant, and the supernatant was analyzed by SDS-PAGE gel electrophoresis to observe the brightness of the expressed product.
Preparation of YPD+Zeocin medium: according to the description of Pichia expression vectors for constitutive expression and purification of recombinant proteins from Invitrogen, yeast extract 10 g/L, peptone 20 g/L, glucose 20 g/L, and Zeocin at a final concentration of 0.1 mg/ml was included.
The clone selected in Example 5 was cultured at 1 liter using the method in Example 5, the fermentation broth was prepared, and the sample was purified by ion exchange and hydrophobic chromatography. The chromatographic packing was Hitrap SP HP, Hitrap Phenyl HP, and the specific steps were as follows:
1. Pretreatment of fermentation broth: centrifugated the fermentation broth at low temperature and high-speed, collected the supernatant, concentrated by 3KD membrane ultrafiltration, replaced to pH7.0 25 mM PB buffer, and filtered by 0.45 μm filter membrane.
2. Cation chromatography: the SP HP chromatography column was equilibrated with equilibration buffer, and then the ultrafiltered fermentation liquid in the previous step was passed through the separation filler with purification system. Then the elution buffer was used for gradient elution, and the elution peak was collected; The equilibration buffer was 25 mM PB, pH7.0, and the elution buffer was 25 mM PB, 1.0M NaCl, pH7.0. As shown in
3. Hydrophobic chromatography: The Art a 1 protein peak collected in the previous step was diluted with equilibration buffer, and Art a 1 protein solution was loaded on phenyl HP hydrophobic chromatography packing, the equilibration buffer was 1.0M(NH4)2SO4, 25 mM PB, pH7.0, and the elution buffer was 25 mM PB, pH7.0. Collect the elution peak. As shown in
4. Ultrafiltration replacement: the protein peaks of hydrophobic chromatography were collected and replaced to 25 mM PB pH7.0.
After the above purification steps, the expression level of the recombinant Art a 1 by pGAPZα-Art a 1-02(with a Kex 2 enzyme cutting site between the signal peptide and the target protein without Ste 13 site) was 100 mg/L, and the yield was 45%.
1. Pollen degreasing: Weigh the pollen of Artemisia annua, add ether at w/v ratio of 1:5, degrease for 24-48 hours at low temperature; Ether was removed and residual solvent was removed by rotary steaming.
2. Crude extract preparation: prepare pH7.0, 50 mM PB solution, add PB solution at w/v ratio of 1:10, and extract at low temperature for 48-72 hours; centrifugate at 4000 rpm, and the crude extract was obtained by collecting the supernatant.
3. Chromatographic purification: The crude extract collected in step 2 was loaded to SP FF cation chromatography packing, the equilibration buffer was 25 mM PB, pH7.0, and the elution buffer was 25 mM PB, 1.0M NaCl, pH7.0. The elution peak was collected and identified by electrophoresis. As shown in
4. Ultrafiltration replacement: Merge elution peak 5 in step 3, concentrate the sample, replace to pH7.4 PBS solution, and freeze it below −20° C. until use.
LC-MS molecular weight can accurately reflect whether the primary sequence of biological macromolecules is correct, including the N and C terminal sequences missing, and post-translational modifications such as glycosylation, oxidation and deamidation. It is one of the most important analytical methods for biological macromolecules. The molecular weight of purified recombinant Art a 1 protein with different construction methods was analyzed by LC-MS, and the results were shown in Table 2. When the α-factor signal peptide was used for secretion expression and there was Ste 13 protease enzyme cutting site between the signal peptide and the target gene, the corresponding amino acid sequence could not be completely removed. The resulting target protein had amino acid residues at the N-terminus. The other construction forms produced Art a 1 protein with the N-terminal sequence completely consistent with the theory, with no residue.
{circle around (1)}The signal peptide was separated from the target protein by Ste 13 signal cleavage sequence (amino acid sequence EAEA).
{circle around (2)}There was a Kex 2 enzyme cutting site between the signal peptide and the target protein, with no Ste 13 site and no other sequence.
Peptide mass figerprinting is one of the most important identification methods in protein research. In theory, every protein has different peptides after digestion, and the mass of these peptides is the peptide map of this protein. Then the measured amino acid sequence is compared with the known sequence, and the correctness of the primary structure of the protein was determined.
Peptide analysis of pure Art a 1 expressed by strains with different construction methods in Example 8 and the native protein in Example 7 showed that, both two wide type signal peptides and α-factor signal peptide (signal peptide should be directly linked to the target protein and cannot be separated by Ste 13 signal cleavage in the vector) can achieve the recombinant expression of Art al protein with amino acid sequence consistent with the native protein, and all the coverage rates were 100% when compared with the theoretical sequence. The results indicated that the recombinant Art al protein had primary structure completely consistent with that of the native protein.
Whether disulfide bonds can be correctly paired is crucial for the maintenance of higher structure and activity of biological macromolecules. The disulfide bond of recombinant Art al protein obtained by pGAPZα-Art a1-02 construction and native Art al protein was determined by Thermo Scientific Q Exactive LC-MS system, and the results were shown in
The purity of the purified samples was identified by electrophoresis: Agilient 1260 HPLC, column Sepax Zenix SEC-80, mobile phase 20 mM PB+300 mM NaCl(pH7.0) buffer, flow rate 0.5 ml/min, equal elution, column temperature 25.0° C., 280 nm to detect the purity of the samples. The results of
1. The recombinant Art a 1 protein obtained by pGAPZ-Art a 1-02 construction method and the native Art a 1 protein purified in Example 7 were diluted to 10 μg/ml with 1×CB carbonate buffer, 100 μl per well, and coated at 4° C. overnight; CB buffer was added to the negative control, no protein was added.
2. The next day, the ELISA plate was washed 3 times with PBST, and 200 μl of 1% BSA/PBST solution was added to each well and blocked for 2 hours at 37° C.
3. After blocking, the blocking solution (2% BSA/PBST) was discarded, 100 μl of positive serum was added to each well (the serum was diluted 10 times with 1% BSA/PBST solution), slightly shake, and incubated at 37° C. for 1h.
4. After washing three times with PBST, secondary antibody of mouse anti-human IgE-HRP diluted 1:1500 was added, 100p per well, and incubated for 1h at 37° C.
5. Washed 3 times with PBST, and 100 μl TMB I color development solution was added to each well. After reaction at 37° C. for 10 min, 50p termination solution (2M H2SO4) was added to each well, and OD450 nm was immediately detected.
6. Result analysis: As shown in Table 2, the detection value of recombinant Art a 1 protein was slightly higher than that of native Art a 1 protein, indicating that in vitro immune response activity of recombinant protein with specific antibodies in serum of allergic patients was equivalent to that of native protein.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111654407.2 | Dec 2021 | CN | national |
This application is the national phase entry of International Application No. PCT/CN2022/144026, filed on Dec. 30, 2022, which is based upon and claims priority to Chinese Patent Application No. 202111654407.2, filed on Dec. 30, 2021, the entire contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/144026 | 12/30/2022 | WO |
