CN106916214B - Poplar allergen - Google Patents
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Abstract
The invention discloses a poplar pollen allergen sensitization component 1 and sensitization component 2, and expression and purification thereof. The purpose of deep research of the allergen is mainly to carry out allergen detection and use as specific treatment, and a certain test basis is provided for the identification of the sensitizing component 1 and the sensitizing component 2 as the sensitizing component 1 and the sensitizing component 2 in the aspects of clinical diagnosis application of poplar allergy and development of desensitization vaccines.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to expression and purification of a poplar pollen allergen sensitizing component 1 and a sensitizing component 2.
Background
With the rapid development of economy, the industrialization degree is higher and higher, and the incidence of pollinosis in China is also obviously increased due to the change of human immunity caused by environmental pollution and the change of modern life style. Pollinosis is a seasonal disease due to IgE-mediated hypersensitivity reaction [1] produced by atopic individuals after exposure to pollen, and is classified into spring and summer-autumn pollinosis according to the season in which pollen is spread [2,3] which is mainly tree pollen and is mainly caused by gramineous plants and weeds pollen [4,5 ]. In northern China, two peak periods of the disease of the pollinosis are mainly 4-5 months and 8-9 months (6) every year.
Poplar is one of the widest tree species planted in China, and is the main tree species of choice no matter whether protection forest, timber forest or urban afforestation is built [7 ]. Particularly, in recent ten years, the poplar forestation area in China is continuously enlarged, and the poplar forestation area is the largest country in the artificial poplar forest in the world. However, allergic and environmental problems caused by pollen produced by male poplar and flying floc produced by female poplar each year also put a great burden on sensitized patients and their families and society [8], and allergic diseases caused by the allergic problems include allergic asthma [9] and rhinitis. According to statistics, about 1000 million people exist in our country who have allergy to pollen, and the pollen becomes one of main non-infectious diseases which threaten the health of residents in our country and influence the quality of life of people, so the harm of the pollen is concerned more and more [10 ]. Therefore, the allergenicity research is carried out on poplar, hypoallergenic variety breeding is carried out, new allergenicity related protein is screened, the formation and the propagation of plant allergenicity sources can be effectively reduced and inhibited, the prevention and the control of plant allergic diseases are promoted, the incidence rate of pollen and catkin allergy is reduced, and the method has great practical significance for improving the health level of people [11 ].
Disclosure of Invention
The invention aims to provide a poplar pollen allergen sensitizing component 1.
The other purpose is to provide a poplar pollen allergen sensitizing component 2.
In order to achieve the purpose, the invention adopts the following technical scheme: the allergen-sensitized component 1 of poplar pollen is obtained by separating or purifying and recombining the allergen-sensitized component 1 from poplar pollen.
The amino acid sequence of the allergen sensitizing component 1 is shown as SEQ ID NO. 1.
The fusion protein pET28 a-sensitized component 1 of the sensitized component 1 has an apparent molecular weight of 22kDa as determined by SDS-PAGE.
The preparation method of the poplar pollen allergen sensitizing component 1 comprises the following steps:
(1) subcloning the target gene sensitizing component 1 into pET28a plasmid, transferring the obtained recombinant plasmid into escherichia coli BL21(DE3), and inducing and expressing target protein by IPTG;
(2) and ultrasonically crushing the obtained recombinant protein, centrifuging at low temperature, purifying the solution supernatant or precipitate by nickel column affinity chromatography, and eluting to obtain the purified recombinant protein.
The poplar pollen allergen sensitizing component 2 is obtained by separating or purifying and recombining the poplar pollen, wherein the allergen sensitizing component 2 is obtained by separating or purifying the poplar pollen.
The amino acid sequence of the allergen sensitizing component 2 is shown as SEQ ID NO. 2.
The fusion protein pET28 a-sensitized component 1 of the allergen-sensitized component 2 had an apparent molecular weight of 25kDa as determined by SDS-PAGE.
The preparation method of the poplar pollen allergen sensitizing component 2 comprises the following steps:
(1) subcloning the target gene sensitizing component 2 into pET28a plasmid, transferring the obtained recombinant plasmid into escherichia coli BL21(DE3), and inducing and expressing target protein by IPTG;
(2) and ultrasonically crushing the obtained recombinant protein, centrifuging at low temperature, purifying the solution supernatant or precipitate by nickel column affinity chromatography, and eluting to obtain the purified recombinant protein.
The amino acid sequence of the sensitization component 1 is as follows: MANPRVYFDMTIGGQPAGRIVMELFADTTPRTAENFRALCTGEKGKGRSGKPLHYKGSTFHRVIPGFMCQGGDFTAGNGTGGESIYGSKFADENFIKKHTGPGILSMANAGPGTNGSQFFVCTAKTEWLDGKHVVFGRVVEGLDVVKAIEKFGSSNGRTSKPVVVADCGQLS are provided.
The amino acid sequence of the sensitizing component 2 is as follows: ATFEIRNSCPYTVWAAASPGGGRRLERGQTWNLNVPAGTSMARIWGRTNCNFDGGGKGRCQTGDCTGGLECKGWGVPPNTLAEYALNQFGNLDFYDISLVDGFNIPIEFSPTSGGGKCQALLCTADINGQCPNELRAPGGCNNPCSVFKTNEYCCTNGQGSCGPTKFSRFFKDRCPTSYSYPQDDPTSTFTCPGGTNYRVIFCPRGSPHFPLEMVEEKRAE
The purpose of intensive research on allergens is mainly allergen detection and specific immunotherapy (SAVT) [12], but most of pollen extracts used in vivo and in vitro allergen detection and specific immunotherapy in domestic clinical practice are crude extracts. The pollen crude extract contains various protein components including major and minor allergens and other miscellaneous proteins. When the pollen immersion liquid is used for in vivo experiments and specific immunotherapy, severe anaphylactic reaction can be caused to patients with high allergy constitution, while a plurality of impurities influence the specificity and sensitivity of the experiments, and the dose of the SAVT for obtaining good curative effect is difficult to master because of non-standardization or side effect and the like [13 ]. At present, the development of allergen preparations with high purity and low sensitization is a hotspot of research in the field at home and abroad in recent years [14], so that the identification of the sensitizing component 1 and the sensitizing component 2 into the sensitizing component 1 and the sensitizing component 2 provides a certain test basis in the aspects of clinical diagnosis application of poplar allergy and development of desensitization vaccines.
Drawings
FIG. 1 shows the SDS-PAGE results of the expression-induced expression identification of the fusion protein pET28 a-sensitizing component 1 of the example, wherein 1 is a western blot band of the non-induced sensitizing component 1, 2 is the induced protein, 3 is the supernatant of the protein after ultrasonic induction at a temperature of 11 ℃ by adding 0.5mM IPTG, 4 is the precipitate of the protein after ultrasonic induction at a temperature of 11 ℃ by adding 0.5mM IPTG, and M is a protein molecular weight standard of 180.0, 140.0, 100.0, 80.0, 60.0, 45.0, 35.0, 25.0, 15.0 and 10.0kDa from top to bottom, respectively.
FIG. 2 shows the SDS-PAGE results of separation and purification of pET28 a-sensitized fraction 1 of the fusion protein of the example, wherein 1 is the protein of sensitized fraction 1 which has not been purified, 2 is the eluate after purification of 30mM imidazole, 3 is the target protein after purification of 50mM imidazole, 4 is the target protein after purification of 100mM imidazole, 5 is the target protein after purification of 150mM imidazole, 6 is the target protein M after purification of 200mM imidazole as the protein molecular weight standard, 7 is the target protein after purification of 250mM imidazole as the protein molecular weight standard, and M is the protein molecular weight standard and is 180.0, 140.0, 100.0, 80.0, 60.0, 45.0, 35.0, 25.0, 15.0 and 10.0kDa from top to bottom, respectively.
FIG. 3 shows Western-Blot assay results for sensitizing component 1 protein, where 1 is an uninduced protein and serves as a control; 2 is IPTG induced protein, His-antibody as primary antibody for binding.
FIG. 4 shows the Western-Blot results of immunobinding of sensitizing component 1 to IgE, wherein 1 is primary antibody against serum from a healthy subject, and 2 is primary antibody against a mixed serum from a poplar pollen allergic patient.
FIG. 5 shows the SDS-PAGE results of the induced expression and purification of the fusion protein pET28 a-sensitized fraction 2 in the examples, wherein 1 is the Western blot band of the non-induced sensitized fraction 2, 2 is the induced protein, 3 is the supernatant of the protein after ultrasonic induction at 11 ℃ by adding 0.5mM IPTG, 4 is the supernatant of the protein after ultrasonic induction at 11 ℃ by adding 0.5mM IPTG, 5 is the inclusion body solubilized with urea, 6 is the target protein after purification with 250mM imidazole, and M is the protein molecular weight standard, from top to bottom, 180.0, 140.0, 100.0, 80.0, 60.0, 45.0, 35.0, 25.0, 15.0, 10.0 kDa.
FIG. 6 shows Western-Blot assay results for sensitizing component 2 protein, and 1 is non-induced protein as a control; 2 is IPTG induced protein, His-antibody as primary antibody for binding.
FIG. 7 shows the Western-Blot results of immunobinding of sensitizing component 2 to IgE, wherein 1 is primary antibody against serum from a healthy subject, and 2 is primary antibody against a mixed serum from a poplar pollen allergic patient.
Detailed Description
The invention will be further described with reference to specific embodiments and the accompanying drawings.
Example 1
pET28 a-sensitized component 1 plasmid construction:
the target gene is subcloned on a pET28a vector, transformed into escherichia coli BL21(DE3), and induced expression is carried out by IPTG, and the result shows that the sensitized component 1 protein exists in an inclusion body, the inclusion body is purified by a nickel column, and dialysis renaturation is carried out to obtain the purified sensitized component 1 protein, and the following gene region of the sensitized component 1:
atggcaaaccctagagtctacttcgacatgacaatcggcggccaaccagccggccggatcgtgatggaactgttcgccgacacaactccacgaaccgcagagaacttcagggctctttgcactggagagaaaggaaaaggccgaagcggcaagcctttacactacaaaggctcgactttccatcgagtcatccctggattcatgtgccaaggaggagatttcactgcagggaatggaaccggaggggaatcgatctacggatcgaaatttgctgacgagaattttataaagaaacatactgggccagggattttgtccatggccaatgctgggcctgggactaacgggtcgcagttctttgtctgtacagccaagactgaatggctcgatggaaaacacgtggtgtttggaagagtagtggagggtctggatgttgtgaaggctatagagaagtttgggtcgtctaatggaaggacctctaagcctgttgttgttgctgactgtggacagctttct
the following is the amino acid sequence of the post-translational sensitizing component 1:
MANPRVYFDMTIGGQPAGRIVMELFADTTPRTAENFRALCTGEKGKGRSGKPLHYKGSTFHRVIPGFMCQGGDFTAGNGTGGESIYGSKFADENFIKKHTGPGILSMANAGPGTNGSQFFVCTAKTEWLDGKHVVFGRVVEGLDVVKAIEKFGSSNGRTSKPVVVADCGQLS
the theoretical molecular weight of pET28 a-sensitizing component 1 after fusion was approximately 22 kDa.
The experimental method comprises the following steps:
transforming the pET28 a-sensitized component 1 plasmid into escherichia coli BL21(DE3), and placing on ice for 20 min; thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 5 min; adding 600 μ L of LB culture solution preheated at 37 ℃; after centrifugation, the whole was spread on LB plates containing 50. mu.g/mL kanamycin antibiotic after shaking at 37 ℃ and 220rpm for 1 hour, and cultured overnight in an inverted state at 37 ℃.
IPTG induction of pET28 a-sensitizing component 1 fusion protein expression: picking up the single clone on the transformation plate and inoculating to a tube containing 50 ug/mL kanamycin antibiotic in 3mL LB culture solution, shaking at 37 ℃ and 220rpm overnight; the following day is as follows: 100 in 50 u g/mL kanamycin antibiotic 30mL LB culture solution, 37 degrees C220 rpm shake until the thallus OD600 is 0.4 (about 2 h); taking out 1mL of culture, centrifuging at 10000g of room temperature for 2min, discarding the supernatant, and resuspending the thallus precipitate with 100 μ L of 1 × loading buffer; IPTG was added to the remaining culture to a final concentration of 0.5mM and shaken at 220rpm for 8h, respectively, to induce expression of the fusion protein.
Determination of allergen-sensitized component 1 (by immunoblotting method): 1mL of the culture was removed, centrifuged at 12000g at room temperature for 2min, the supernatant was discarded, and the pellet was resuspended in 100. mu.L of 1 Xloading buffer. The samples were separated using 12% separation gel, 5% concentration gel. Starting voltage at 70V, raising the voltage to 120V after the front edge of the bromophenol blue enters the separation gel, performing constant-voltage electrophoresis until the front edge of the bromophenol blue reaches the lower boundary of the gel, and stopping electrophoresis. And taking down the gel for film transfer. Transferring the protein to the PVDF membrane by constant current for 60 min. And (3) putting the PVDF membrane into a plate containing a sealing solution, and sealing for 3 hours. The blocked PVDF membrane was placed in a primary antibody at 4 degrees overnight. The following day the strips were washed with TBST for 3 x 10 min. And then placing the mixture into a mouse antibody (secondary antibody), placing the mixture on a shaker at room temperature for 2 hours, washing the mixture for 3 x 5min by using TBST after the reaction is finished, carrying out substrate color development, and stopping the reaction when protein bands are clearly developed. Separation and purification of allergen-sensitized component 1:
resuspending the culture thallus sediment for induced expression by using 20mL of extract Ni-IDA Binding-Buffer, ultrasonically crushing (the power is 400W, the working time is 4sec, the intermittence time is 8sec, and the total time is 20min), centrifuging for 20min at the temperature of 4 ℃ and 10000g, taking supernatant, and loading the supernatant to a Ni-IDA Binding-Buffer pre-balanced Ni-IDA-Sepharose CL-6B affinity chromatographic column at the flow rate of 0.5 mL/min; flushing with Ni-IDA Binding-Buffer at a flow rate of 0.5mL/min until the effluent OD280 value reaches the baseline; washing with Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1mL/min until the effluent OD280 value reaches baseline; eluting the target protein with Ni-IDA Elution-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0) at the flow rate of 1mL/min, and collecting the effluent; dialyzing the eluted recombinant protein with dialysis buffer PBS (pH7.4), changing the dialysate every 12 hr for 3 times, taking out the dialyzed protein solution, storing at-20 deg.C for use, and performing 12% SDS-PAGE analysis, the results are shown in the figure.
FIG. 1 shows the SDS-PAGE results of the expression-induced expression identification of the fusion protein pET28 a-sensitizing component 1 of the example, wherein 1 is a western blot band of the non-induced sensitizing component 1, 2 is the induced protein, 3 is the supernatant of the protein after ultrasonic induction at a temperature of 11 ℃ by adding 0.5mM IPTG, 4 is the precipitate of the protein after ultrasonic induction at a temperature of 11 ℃ by adding 0.5mM IPTG, and M is a protein molecular weight standard of 180.0, 140.0, 100.0, 80.0, 60.0, 45.0, 35.0, 25.0, 15.0 and 10.0kDa from top to bottom, respectively.
FIG. 2 shows the SDS-PAGE results of separation and purification of pET28 a-sensitized fraction 1 of the fusion protein of the example, wherein 1 is the protein of sensitized fraction 1 which has not been purified, 2 is the eluate after purification of 30mM imidazole, 3 is the target protein after purification of 50mM imidazole, 4 is the target protein after purification of 100mM imidazole, 5 is the target protein after purification of 150mM imidazole, 6 is the target protein after purification of 200mM imidazole, 7 is the target protein after purification of 250mM imidazole, and M is the protein molecular weight standard and is 180.0, 140.0, 100.0, 80.0, 60.0, 45.0, 35.0, 25.0, 15.0, 10.0kDa from top to bottom, respectively.
FIG. 3 shows Western-Blot assay results for sensitizing component 1 protein, where 1 is an uninduced protein and serves as a control; 2 is IPTG induced protein, His-antibody as primary antibody for binding.
FIG. 4 shows the Western-Blot results of immunobinding of sensitizing component 1 to IgE, wherein 1 is primary antibody against serum from a healthy subject, and 2 is primary antibody against a mixed serum from a poplar pollen allergic patient.
Example 2
pET28 a-sensitized component 2 plasmid construction:
the target gene is subcloned on a pET28a vector, transformed into escherichia coli BL21(DE3), and induced expression is carried out by IPTG, and the result shows that the protein of the sensitizing component 2 exists in an inclusion body, the inclusion body is purified by a nickel column, and dialysis renaturation is carried out to obtain purified Pop4 protein, wherein the following gene region of the sensitizing component 2:
gccacctttgaaatccgaaatagttgtccttacactgtgtgggccgcagcctcacctggtggtggacgccgtctagaacgtggccaaacttggaatcttaatgtgcctgctggcacctccatggctcgtatttggggcaggacaaattgtaacttcgatggtggtggtaagggtcgttgccaaactggggattgtactggtggcctcgagtgcaaaggctggggtgtccctcccaacactctagcagaatatgcattaaatcagtttggtaacttggatttttatgatatatcgcttgttgatggatttaatatccctatagaatttagtccaacatcaggcggtgggaagtgtcaagcgcttctttgcacagcagatattaatgggcaatgtcctaatgaattgagggctcctggggggtgtaataacccatgttccgtgttcaaaactaacgaatattgctgcactaatgggcaggggagctgtggccctaccaaattttcaaggttttttaaggataggtgccctacttcttatagctatccccaggatgaccctacaagcacatttacatgccctggcgggaccaactatagggttatcttttgccctcgggggtctcctcatttccccttggagatggttgaagaaaagcgtgcagag
the amino acid sequence of the sensitizing component 2 after translation is shown below, and the theoretical molecular weight of pET28 a-sensitizing component 2 after fusion is about 25 kDa.
ATFEIRNSCPYTVWAAASPGGGRRLERGQTWNLNVPAGTSMARIWGRTNCNFDGGGKGRCQTGDCTGGLECKGWGVPPNTLAEYALNQFGNLDFYDISLVDGFNIPIEFSPTSGGGKCQALLCTADINGQCPNELRAPGGCNNPCSVFKTNEYCCTNGQGSCGPTKFSRFFKDRCPTSYSYPQDDPTSTFTCPGGTNYRVIFCPRGSPHFPLEMVEEKRAE
The experimental method comprises the following steps:
respectively transforming pET28 a-sensitized component 2 into Escherichia coli BL21(DE3), and placing on ice for 20 min; thermally shocking at 42 deg.C for 90sec, and rapidly placing in ice for 5 min; adding 600 μ L of LB culture solution preheated at 37 ℃; after centrifugation, the whole was spread on LB plates containing 50. mu.g/mL kanamycin antibiotic after shaking at 37 ℃ and 220rpm for 1 hour, and cultured overnight in an inverted state at 37 ℃.
IPTG induction of pET28 a-sensitizing component 2 fusion protein expression: picking up the single clone on the transformation plate and inoculating to a tube containing 50 ug/mL kanamycin antibiotic in 3mL LB culture solution, shaking at 37 ℃ and 220rpm overnight; the following day is as follows: 100 in 50 u g/mL kanamycin antibiotic 30mL LB culture solution, 37 degrees C220 rpm shake until the thallus OD600 is 0.4 (about 2 h); taking out 1mL of culture, centrifuging at 10000g of room temperature for 2min, discarding the supernatant, and resuspending the thallus precipitate with 100 μ L of 1 × loading buffer; IPTG was added to the remaining culture to a final concentration of 0.5mM and shaken at 220rpm for 8h, respectively, to induce expression of the fusion protein.
Determination of allergen-sensitized component 2 (by immunoblotting method): 1mL of the culture was removed, centrifuged at 12000g at room temperature for 2min, the supernatant was discarded, and the pellet was resuspended in 100. mu.L of 1 Xloading buffer. The samples were separated using 12% separation gel, 5% concentration gel. Starting voltage at 70V, raising the voltage to 120V after the front edge of the bromophenol blue enters the separation gel, performing constant-voltage electrophoresis until the front edge of the bromophenol blue reaches the lower boundary of the gel, and stopping electrophoresis. And taking down the gel for film transfer. Transferring the protein to the PVDF membrane by constant current for 60 min. And (3) putting the PVDF membrane into a plate containing a sealing solution, and sealing for 3 hours. The blocked PVDF membrane was placed in a primary antibody at 4 degrees overnight. The following day the strips were washed with TBST for 3 x 10 min. And then placing the mixture into a mouse antibody (secondary antibody), placing the mixture on a shaker at room temperature for 2 hours, washing the mixture for 3 x 5min by using TBST after the reaction is finished, carrying out substrate color development, and stopping the reaction when protein bands are clearly developed. Separation and purification of allergen-sensitized component 2:
resuspending the culture thallus sediment for induced expression by using 20mL of extract Ni-IDA Binding-Buffer, ultrasonically crushing (the power is 400W, the working time is 4sec, the intermittence time is 8sec, and the total time is 20min), centrifuging for 20min at the temperature of 4 ℃ and 10000g, taking supernatant, and loading the supernatant to a Ni-IDA Binding-Buffer pre-balanced Ni-IDA-Sepharose CL-6B affinity chromatographic column at the flow rate of 0.5 mL/min; flushing with Ni-IDA Binding-Buffer at a flow rate of 0.5mL/min until the effluent OD280 value reaches the baseline; washing with Ni-IDA Washing-Buffer (20mM Tris-HCl, 20mM imidazole, 0.15M NaCl, pH8.0) at a flow rate of 1mL/min until the effluent OD280 value reaches baseline; eluting the target protein with Ni-IDA Elution-Buffer (20mM Tris-HCl, 250mM imidazole, 0.15M NaCl, pH8.0) at the flow rate of 1mL/min, and collecting the effluent; dialyzing the eluted recombinant protein with dialysis buffer PBS (pH7.4), changing the dialysate every 12 hr for 3 times, taking out the dialyzed protein solution, storing at-20 deg.C for use, and performing 12% SDS-PAGE analysis, the results are shown in the figure.
FIG. 5 shows the SDS-PAGE results of the induced expression and purification of the fusion protein pET28 a-sensitized fraction 2 in the examples, wherein 1 is the Western blot band of the non-induced sensitized fraction 2, 2 is the induced protein, 3 is the supernatant of the protein after ultrasonic induction at 11 ℃ by adding 0.5mM IPTG, 4 is the supernatant of the protein after ultrasonic induction at 11 ℃ by adding 0.5mM IPTG, 5 is the inclusion body solubilized with urea, 6 is the target protein after purification with 250mM imidazole, and M is the protein molecular weight standard, from top to bottom, 180.0, 140.0, 100.0, 80.0, 60.0, 45.0, 35.0, 25.0, 15.0, 10.0 kDa.
FIG. 6 shows Western-Blot assay results for sensitizing component 2 protein, and 1 is non-induced protein as a control; 2 is IPTG induced protein, His-antibody as primary antibody for binding.
FIG. 7 shows the Western-Blot results of immunobinding of sensitizing component 2 to IgE, wherein 1 is primary antibody against serum from a healthy subject, and 2 is primary antibody against a mixed serum from a poplar pollen allergic patient.
As noted above, while the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limited thereto. Various changes in form and detail may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.
Reference documents:
1. crowing, Zhongbei, Weiqingyu, research overview of humulus pollinosis, Liaoning traditional Chinese medicine J2004: 601-602.
2. Yi Jia, Yue Feng Min, Wang Liang Lu, summer and autumn pollinosis patients allergic rhinitis progresses to the clinical research of the allergic asthma process, Chinese medicine journal 2006, 1628-.
3.Goldblum RM,Ning B,Judy BM et al.A single mouse monoclonal antibody,E58modulates multiple IgE epitopes on group 1cedar pollen allergens.Molecular immunology.2016；74:106-112.
4. Yang Qio Liang, Europe Ting, face Red, pollen allergy research progress, agricultural Notification of China 2015 163-167.
5.Sikora M,Valek M,Susic Z et al.Tree pollen spectra and pollen allergy risk in the Osijek-Baranja County.Arhiv za higijenu rada i toksikologiju.2013；64:115-122.
6. Baili, Jinzhongying, clinical analysis of acupuncture treatment for pollen diseases, journal of traditional Chinese medicine and pharmacology, 2006:751.
7. The technical research progress of the artificial poplar forest cultivation in China, the application of the ecology newspaper 2008:2308 and 2316.
8.Celik G,Mungan D,Pinar M et al.Poplar pollen-related allergy in Ankara,Turkey:how important for patients living in a city with high pollen loadAllergy and asthma proceedings.2005；26:113-119.
9.Panzner P,Malkusova I,Vachova M et al.Bronchial inflammation in seasonal allergic rhinitis with or without asthma in relation to natural exposure to pollen allergens.Allergologia et immunopathologia.2015；43:3-9.
10. Research on flowering habit and allergenicity of poplar [ Master ]. national institute of forestry science; 2014:
11. cause, prevention and treatment of Liu Shi Hua, pollen allergy, biological teaching, 2013:52-53.
12.Abramson MJ,Puy RM,Weiner JM.Allergen immunotherapy for asthma.The Cochrane database of systematic reviews.2003:Cd001186.
13.Jutel M,Kosowska A,Smolinska S.Allergen Immunotherapy:Past,Present,and Future.Allergy,asthma&immunology research.2016；8:191-197.
14.Letran A,Espinazo M,Moreno F.Measurement of IgE to pollen allergen components is helpful in selecting patients for immunotherapy.Annals of allergy,asthma&immunology:official publication of the American College of Allergy,Asthma,&Immunology.2013；111:295-297.
Sequence listing
<110> first subsidiary hospital of Nanjing medical university
<120> Populus allergen
<160>2
<210>SEQ ID N0.1
<211>516
<212>PRT
<221>CDS
<400>
Atg gca aac cct aga gtc tac ttc gac atg aca atc ggc ggc caa 45
Met Ala Asn Pro Arg Val Tyr Phe Asp Met Thr Ile Gly Gly Gln
1 5 10 15
cca gcc ggc cgg atc gtg atg gaa ctg ttc gcc gac aca act cca 90
Pro Ala Gly Arg Ile Val Met Glu Leu Phe Ala Asp Thr Thr Pro
20 25 30
cga acc gca gag aac ttc agg gct ctt tgc act gga gag aaa gga 135
Arg Thr Ala Glu Asn Phe Arg Ala Leu Cys Thr Gly Glu Lys Gly
35 40 45
aaa ggc cga agc ggc aag cct tta cac tac aaa ggc tcg act ttc 180
Lys Gly Arg Ser Gly Lys Pro Leu His Tyr Lys Gly Ser Thr Phe
50 55 60
cat cga gtc atc cct gga ttc atg tgc caa gga gga gat ttc act 225
His Arg Val Ile Pro Gly Phe Met Cys Gln Gly Gly Asp Phe Thr
65 70 75
gca ggg aat gga acc gga ggg gaa tcg atc tac gga tcg aaa ttt 270
Ala Gly Asn Gly Thr Gly Gly Glu Ser Ile Tyr Gly Ser Lys Phe
80 85 90
gct gac gag aat ttt ata aag aaa cat act ggg cca ggg att ttg 315
Ala Asp Glu Asn Phe Ile Lys Lys His Thr Gly Pro Gly Ile Leu
95 100 105
tcc atg gcc aat gct ggg cct ggg act aac ggg tcg cag ttc ttt 360
Ser Met Ala Asn Ala Gly Pro Gly Thr Asn Gly Ser Gln Phe Phe
110 115 120
gtc tgt aca gcc aag act gaa tgg ctc gat gga aaa cac gtg gtg 405
Val Cys Thr Ala Lys Thr Glu Trp Leu Asp Gly Lys His Val Val
125 130 135
ttt gga aga gta gtg gag ggt ctg gat gtt gtg aag gct ata gag 450
Phe Gly Arg Val Val Glu Gly Leu Asp Val Val Lys Ala Ile Glu
140 145 150
aag ttt ggg tcg tct aat gga agg acc tct aag cct gtt gtt gtt 495
Lys Phe Gly Ser Ser Asn Gly Arg Thr Ser Lys Pro Val Val Val
155 160 165
gct gac tgt gga cag ctt tct 516
Ala Asp Cys Gly Gln Leu Ser
170 172
<210>SEQ ID N0.2
<211>663
<212>PRT
<221>CDS
<400>
gcc acc ttt gaa atc cga aat agt tgt cct tac act gtg tgg gcc 45
Ala Thr Phe Glu Ile Arg Asn Ser Cys Pro Tyr Thr Val Trp Ala
5 10 15
gca gcc tca cct ggt ggt gga cgc cgt cta gaa cgt ggc caa act 90
Ala Ala Ser Pro Gly Gly Gly Arg Arg Leu Glu Arg Gly Gln Thr
20 25 30
tgg aat ctt aat gtg cct gct ggc acc tcc atg gct cgt att tgg 135
Trp Asn Leu Asn Val Pro Ala Gly Thr Ser Met Ala Arg Ile Trp
35 40 45
ggc agg aca aat tgt aac ttc gat ggt ggt ggt aag ggt cgt tgc 180
Gly Arg Thr Asn Cys Asn Phe Asp Gly Gly Gly Lys Gly Arg Cys
50 55 60
caa act ggg gat tgt act ggt ggc ctc gag tgc aaa ggc tgg ggt 225
Gln Thr Gly Asp Cys Thr Gly Gly Leu Glu Cys Lys Gly Trp Gly
65 70 75
gtc cct ccc aac act cta gca gaa tat gca tta aat cag ttt ggt 270
Val Pro Pro Asn Thr Leu Ala Glu Tyr Ala Leu Asn Gln Phe Gly
80 85 90
aac ttg gat ttt tat gat ata tcg ctt gtt gat gga ttt aat atc 315
Asn Leu Asp Phe Tyr Asp Ile Ser Leu Val Asp Gly Phe Asn Ile
95 100 105
cct ata gaa ttt agt cca aca tca ggc ggt ggg aag tgt caa gcg 360
Pro Ile Glu Phe Ser Pro Thr Ser Gly Gly Gly Lys Cys Gln Ala
110 115 120
ctt ctt tgc aca gca gat att aat ggg caa tgt cct aat gaa ttg 405
Leu Leu Cys Thr Ala Asp Ile Asn Gly Gln Cys Pro Asn Glu Leu
125 130 135
agg gct cct ggg ggg tgt aat aac cca tgt tcc gtg ttc aaa act 450
Arg Ala Pro Gly Gly Cys Asn Asn Pro Cys Ser Val Phe Lys Thr
140 145 150
aac gaa tat tgc tgc act aat ggg cag ggg agc tgt ggc cct acc 495
Asn Glu Tyr Cys Cys Thr Asn Gly Gln Gly Ser Cys Gly Pro Thr
155 160 165
aaa ttt tca agg ttt ttt aag gat agg tgc cct act tct tat agc 540
Lys Phe Ser Arg Phe Phe Lys Asp Arg Cys Pro Thr Ser Tyr Ser
170 175 180
tat ccc cag gat gac cct aca agc aca ttt aca tgc cct ggc ggg 585
Tyr Pro Gln Asp Asp Pro Thr Ser Thr Phe Thr Cys Pro Gly Gly
185 190 195
acc aac tat agg gtt atc ttt tgc cct cgg ggg tct cct cat ttc 630
Thr Asn Tyr Arg Val Ile Phe Cys Pro Arg Gly Ser Pro His Phe
200 205 210
ccc ttg gag atg gtt gaa gaa aag cgt gca gag 663
Pro Leu Glu Met Val Glu Glu Lys Arg Ala Glu
215 210 211
Claims (4)
1. The poplar pollen allergen sensitizing component 1 is characterized in that: the allergen sensitization component 1 is obtained by separating or purifying and recombining poplar pollen;
the amino acid sequence of the allergen sensitizing component 1 is shown as SEQ ID NO. 1.
2. The method for producing a poplar pollen allergen sensitizing component 1 according to claim 1, comprising the steps of:
(1) subcloning a target gene encoding the poplar pollen allergen sensitizing component 1 of claim 1 into a pET28a plasmid, transferring the obtained recombinant plasmid into escherichia coli BL21(DE3), and inducing and expressing a target protein by IPTG;
(2) carrying out ultrasonic crushing and low-temperature centrifugation after resuspending the escherichia coli thalli by using an extraction solution, taking supernatant, purifying by nickel column affinity chromatography, and eluting to obtain the purified poplar pollen allergen sensitized component 1 of claim 1.
3. The poplar pollen allergen sensitizing component 2 is characterized in that: the allergen sensitization component 2 is obtained by separating or purifying and recombining poplar pollen;
the amino acid sequence of the allergen sensitizing component 2 is shown as SEQ ID NO. 2.
4. The method for producing a poplar pollen allergen sensitizing component 2 according to claim 3, comprising the steps of:
(1) subcloning a target gene encoding the poplar pollen allergen sensitizing component 2 of claim 3 into a pET28a plasmid, transferring the obtained recombinant plasmid into Escherichia coli BL21(DE3), and inducing and expressing a target protein by IPTG;
(2) carrying out ultrasonic crushing and low-temperature centrifugation after resuspending the escherichia coli thalli by using an extraction solution, taking supernatant, purifying by nickel column affinity chromatography, and eluting to obtain the purified poplar pollen allergen sensitized component 2 of claim 3.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101972472A (en) * | 2010-10-26 | 2011-02-16 | 北京新华联协和药业有限责任公司 | Method for purifying and preparing allergen vaccine |
| CN105481957A (en) * | 2016-01-08 | 2016-04-13 | 南京医科大学第一附属医院 | Platanus pollen allergen Pla a 3 and monoclonal antibody thereof |
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2017
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101972472A (en) * | 2010-10-26 | 2011-02-16 | 北京新华联协和药业有限责任公司 | Method for purifying and preparing allergen vaccine |
| CN105481957A (en) * | 2016-01-08 | 2016-04-13 | 南京医科大学第一附属医院 | Platanus pollen allergen Pla a 3 and monoclonal antibody thereof |
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| Title |
|---|
| Comparative Proteomic Analysis of Mature Pollen in Triploid and Diploid Populus deltoides;Zhang XL 等;《Intenational journal of molecular sciences》;20160903;第17卷(第9期);第1-17页 * |
| PREDICTED: osmotin-like protein OSM34 [Populus euphratica];无;《GenBank Database》;20150106;region和origin * |
| unknown [Populus trichocarpa x Populus deltoides];Ralph,S.G.等;《GenBank Database》;20090524;region和origin * |
| 日本柳杉花粉变应原CJP-6的重组表达和免疫反应性鉴定;蒋建国 等;《中国免疫学杂志》;20120530;第28卷(第5期);第444-448页 * |
| 李志良 等.杨树花粉变应原的提取及纯化.《新疆医学院学报》.1985,第8卷(第3期),第231-236页. * |
| 杨树花粉变应原的提取及纯化;李志良 等;《新疆医学院学报》;19851001;第8卷(第3期);摘要 * |
| 花粉变应原的分类和相关的进化分析;陈瑧 等;《基础免疫学》;20100115;第30卷(第1期);第56页摘要和57页表1 * |
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