CN110343160A - Yellow meal worm YM47 albumen, its encoding gene and application - Google Patents

Abstract

The invention discloses the application of yellow meal worm YM47 albumen, the gene and yellow meal worm YM47 albumen and its encoding gene that encode the albumen, YM47 albumen is the yellow meal worm allergic protein that can cause human allergy.The amino acid sequence of yellow meal worm YM47 albumen of the present invention is as shown in SEQ ID NO:3；The gene of coding YM47 albumen of the invention, nucleotide sequence is as shown in SEQ ID NO:4.Present invention firstly discloses yellow meal worm specificity allergic protein and its encoding gene, it can be achieved that cultivating without sensitization system yellow meal worm.The allergic protein of other resource insects can be deduced by the amino acid sequence and corresponding gene order of the allergic protein, can reduce the risk of the development and utilization of the worm sources protein food by the potential sensitization initiation of insect protein to a certain extent.

Description

Yellow meal worm YM47 albumen, its encoding gene and application

Technical field

The invention belongs to field of biotechnology, and in particular to yellow meal worm YM47 albumen, its encoding gene and application.

Background technique

The yellow meal worm (Tenebrio molitor L.) of coleoptera, Tenebrionidae, powder genus is commonly called as bread worm, be after The third-largest economic insects after silkworm and honeybee.With vivo protein content is high, type is rich and varied, nutritional health function is good The advantages that, it is considered to be it is not fully developed the new protein sources utilized.But the albuminoid food and other foods containing protein Product equally have a potential risk (Sicherer, 2011) for causing a small number of eater's allergy, how predictive diagnosis and to solve this Confinement problems become alleviate protein resource shortage there is an urgent need to.It is existing about four mesh, ten kinds of common edible insects at present The study found that in the case where not discussing Cross-reactivity, tropomyosin and arginine kinase (tropomyosin And arginine kinase) it is almost anaphylactogen (the de Gier and that most of insect leads to human allergy Verhoeckx,2018；Liu et al.,2009)；Myosin (myosin) is then mealworm (yellow meal worm, barley worm (Zophobas atratus), Alphitobius (Alphitobius diaperinus)), cockroach (Periplaneta americana And/or Blatella germanica), allergic protein (the Broekman et of locust (Locusta migratoria) etc. al.,2015；Huss et al.,2001)；Pyruvate kinase (pyruvate kinase), protein in hemolymph (hemolymph Protein), glycoprotein (glycoprotein), chitinase (chitinase) are also detected to may be some edible insects Allergic protein (Jeong et al., 2016；Phiriyangkul et al.,2015)；In addition, vitellogenin (vitellogenin), hemocyanin (hemocyanin) may be cockroach and termite (Coptotermes formosanus) Specific allergic protein (Mattison et al., 2017；Jiing-Guang et al., 2011), cuticula albumen (cuticle protein), six aggressiveness 1B (hexamerin 1B) are yellow meal worm, cricket (Acheta domesticus) respectively Specific allergic protein (Srinroch et al., 2015；Verhoeckx et al.,2014).

In the prior art, the research about insect allergic protein is very few, and the correlative study temporarily without the nearly edge species of yellow meal worm Research is compared as reference.In addition, due to other worm sources albumen or containing in a certain insect food of daily consumption The cross influence of the food of quick original can also interfere with the prediction and diagnosis of allergy.Meanwhile existing most of researchs fail clearly to inhale Enter between type allergy, contact-type allergy and intake type allergy with the presence or absence of influencing each other or this kind of individual for having allergic symptom With the presence or absence of the possibility for having various Co-morbities.Above situation increases the difficulty of the positioning screening of insect specificity anaphylactogen, And resulting in the insect allergy original measured in existing most of correlative studys is examined in the case where there is cross allergy mostly It measures.

Summary of the invention

In view of the above-mentioned problems, the present invention provides yellow meal worm YM47 albumen and encoding the gene of the albumen, which is energy Cause the yellow meal worm allergic protein of human allergy.

The present invention also provides the applications of yellow meal worm YM47 albumen and its encoding gene.

The technical solution adopted by the invention is as follows:

Yellow meal worm YM47 albumen of the present invention, amino acid sequence is as shown in SEQ ID NO:3 or the amino acid sequence Column are through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition.

The gene of the above-mentioned albumen of coding of the present invention, nucleotide sequence is as shown in SEQ ID NO:4；Or the nucleosides Acid sequence is substituted, lacks or increases one or more nucleotide and expresses the nucleotide sequence of identical function albumen.

Carrier containing said gene, engineering bacteria or transgenic cell line of the present invention.

Application of the YM47 albumen of the present invention in yellow meal worm molecular breeding.

Application of the gene of the present invention in yellow meal worm molecular breeding.

Compared with prior art, the invention has the following advantages:

Present invention firstly discloses yellow meal worm specificity allergic protein and its encoding gene, it can be achieved that cultivating without sensitization system Huang Mealworm.The sensitization egg of other resource insects can be deduced by the amino acid sequence and corresponding gene order of the allergic protein It is white, the wind of the development and utilization of the worm sources protein food by the potential sensitization initiation of insect protein can be reduced to a certain extent Danger.

Detailed description of the invention

Attached drawing 1 is that Sephacryl S-200 chromatography of the invention purifies figure；Wherein, Peak 1~5 is respectively referred to 5 peaks of Sephacryl S-200 chromatography detection.

Attached drawing 2 is that AKTA explorer fast protein liquid chromatography system of the invention isolates and purifies figure；Wherein, Peak1 Q1~Q7 respectively refers to 7 peaks that AKTA explorer is detected in PANEL A；Peak2 Q1~Q10 is respectively referred in PANEL B 10 peaks of AKTA explorer detection；Peak3 Q1~Q10 respectively refers to AKTA explorer is detected in PANEL C 10 Peak；Peak4 Q1~Q5 respectively refers to 5 peaks that AKTA explorer is detected in PANELd；Peak5 Q1~Q5 respectively refers to PANEL 5 peaks that AKTA explorer is detected in E.

Attached drawing 3 is that yellow meal worm purifying protein SDS-PAGE electrophoresis of the invention is composed；Wherein in attached drawing 3aRespectively For total protein, peak1, peak1Q5, peak1Q6, peak2Q7, peak2Q8, peak2Q9, peak3, peak3Q4, peak3Q7, Peak4, peak4Q3, peak4Q4, peak5,It is protein labeling；In attached drawing 3bRespectively total protein, peak1, peak1Q5、peak4、peak2Q7、peak2Q8、peak3Q4、peak3、peak1Q7、peak2Q4、peak2Q5、peak3Q6、 Peak4Q2, peak5Q2,It is protein labeling.

Attached drawing 4 is that 16 arrays of the yellow meal worm protein component isolated and purified of the invention and specific human body serum sample are swept Tracing；Wherein note: the left figure of a figure and b figure is 16 array of figure, each array corresponds to a kind of blood serum sample of right figure, from upper It is down respectively blank control, positive control, test group；Each point corresponds to corresponding a kind of albumen in right figure.

Attached drawing 5 is the immunoblotting of the yellow meal worm protein component isolated and purified and specific human body serum sample of the invention Figure；Wherein,Respectively total protein, peak1, peak1Q5, peak4, peak2Q7, peak2Q8, peak3Q4, Peak3, peak1Q7, peak2Q4, peak2Q5, peak3Q6, peak4Q2, peak5Q2,It is protein labeling.

Attached drawing 6 is peak3-20 group sub-sequence of the invention and derives from yellow meal worm serine protease (ABC88745.1) Matching figure.

Attached drawing 7 is peak3-30 group sub-sequence of the invention and derives from yellow meal worm serine protease (ABC88747.1) Matching figure.

Specific embodiment

Thinking of the invention is, successively using liquid chromatography (Sephacryl S-200 liquid phase column chromatography and AKTA Explorer system) total protein extracting solution that isolates and purifies yellow meal worm, 32 kinds of the effective protein component isolated and purified.So Afterwards, using the blood serum sample for having allergies and the patient without allergies to tenebrio molitor protein, in conjunction in immunology Microarray and Western Western blot finds there are 2 protein component energy after carrying out 32 protein components of successive analysis detection And have allergies to tenebrio molitor protein specific human serum generate Immunofluorescence Reactions, corresponding purifying protein it is opposite Molecular mass respectively may be about 20kDa and 30kDa.Finally, by the sequencing result of MALDI-MS it is found that the two cause specificity The yellow meal worm protein component that human serum sample generates immune response corresponds to serine protease.

Embodiment 1

It is specific as follows present embodiment discloses yellow meal worm total protein extraction, purifying:

A, yellow meal worm total protein extraction

Choose the dry simultaneously grind into powder of fresh and alive yellow meal worm mature larva rapid freezing in liquid nitrogen.Then, in 10g worm 150mL lysis buffer (the phosphate buffer solution and 3 containing 0.5%TritonX-100 that 150mL pH is 7.4 is added in powder The protease without EDTA that piece Thermo Fisher Scientific company produces inhibits tablet), use MSE Soniprep 150 ultrasound for homogenization instrument ultrasonications.When using ultrasound for homogenization instrument, by the mixed liquor of worm powder and the lysis buffer of 150mL It is divided in the centrifuge tube of 4 50mL, the process centrifuge tube of entire ultrasonication is both placed in the box of ice and keeps low temperature.Together When, pause instrument is 30 seconds cooling after every processing 15 seconds, so recycles until worm powder is thoroughly dissolved in lysis buffer.

After mixed liquor is centrifuged 15 minutes (1000rpm, 4 DEG C) in Allegratm 64R low temperature desk centrifuge, supernatant is taken Liquid, and supernatant is filtered by 0.45 μm of Nalgene injection filter.Obtained yellow meal worm total protein solution is placed on- It is stand-by in 20 DEG C of refrigerator.

B, yellow meal worm total protein isolates and purifies

The protein solution of acquisition uses Sephacryl liquid phase column using fast protein liquid chromatography (FPLC) (Sephacryl S-200) isolate and purify for the first time, and the yellow meal worm total protein sample for reserving sufficient amount is used for follow-up test. Whole process phosphate solution (pH 7.4) is buffer solution, flow velocity 120mL/h.Yellow meal worm total protein solution is passed through After Sephacryl S-200 chromatography, it can be seen that detector detects by the obtained scanning figure (attached drawing 1) for isolating and purifying map 5 peaks (Peak 1~5).According to chromatographic theory, 5 peaks respectively correspond 5 class macromolecular crude protein.It is corresponding to collect this 5 peaks Sample, respectively dialysis 72h (centre replacement 3 times be used for dialysis ultrapure waters) after be divided in 50mL centrifuge tube.

5 obtained the protein component sample isolated and purified for the first time is used into AKTA explorer fast protein liquid phase Chromatographic system (55 liquid phase column of Mono Q) is isolated and purified again.During isolating and purifying for the second time, temporary not used egg White component sample is placed in -20 DEG C of refrigerator and saves for use.Two kinds of buffers of A, B, buffer solution A are used during isolating and purifying For the Tris buffer of pH8, buffer solution B is the Tris solution for having 20mM NaCl in every 1000mL, flow velocity 60mL/h.AKTA It after the operation of explorer system, is first washed using buffer solution A and pumps 5min, carry out separation chromatography using buffer solution B after sample introduction.Add every time Buffer solution A will be used to wash pump 5min before yellow meal worm total protein sample, once be loaded 5mL, sample-adding number obtains as needed The amount of yellow meal worm protein component sample determines.The protein component sample isolated and purified for the second time is packed into after dialysis In the flat centrifuge tube of 25mL, it is placed in freeze overnight in -80 DEG C of refrigerator, next day freezes dry using CRYODOS freeze drier It is dry to obtain yellow meal worm protein component powder.Protein component sample is stored in -20 DEG C with after the dissolution of 200 μ L ultrapure waters.

The 5 yellow meal worm macromolecular crude protein sample AKTA that will be purified by Sephacryl S-200 chromatography After explorer fast protein liquid phase systems isolate and purify, figure (attached drawing 2) is isolated and purified it is found that Peak1 according to what is obtained (PANEL A) has been detected 7 peaks (Peak1 Q1~Q7)；Peak2 (PANEL B) has been detected 10 peak (Peak2 Q1~Q10)；Peak3 (PANEL C) has been detected 10 peaks (Peak3 Q1~Q10)；Peak4 (PANELd) is detected 5 peaks (Peak4 Q1~Q5)；Peak5 (PANEL E) has been detected 5 peaks (Peak5 Q1~Q5).It is former according to chromatography Reason, each peak respectively correspond a kind of protein component, therefore, separate by AKTA explorer fast protein liquid chromatography system pure The sample of 37 yellow meal worm protein components is obtained after change.The protein component that wherein 5 concentration are negative value is rejected, 32 Huangs are obtained Mealworm protein component sample.

Embodiment 2

Present embodiment discloses the SDS-PAGE of protein sample detections.

Referring to the experimental method and Optimal improvements of the Laboratory Manual that Thermo company provides, yellow meal worm total protein is chosen Sample, the protein component sample isolated and purified for the first time (4 samples) and the concentration that obtains after isolating and purifying for the second time are higher Protein component sample (9 samples), totally 14 samples.This 14 samples be respectively as follows: total protein, peak1, peak1Q5, peak1Q6, peak2Q7、peak2Q8、peak2Q9、peak3、peak3Q4、peak3Q7、peak4、peak4Q3、peak4Q4、peak5。

Each sample takes 6.5 μ L that 3 μ L sample buffer (Pierce are added^TMSDS-PAGE Sample Prep Kit) and Mixed liquor is placed in 70 DEG C of heating 12min in PCR instrument by 0.5 μ L dithiothreitol (DTT) (DTT).Meanwhile preparing 1L electrophoretic buffer (50mL NuPAGE SDS solution and 950mL ultrapure water), with prepared buffer solution for cleaning gel (NuPAGE 4-12%Bis- Tris), gel is then put into electrophoresis apparatus and fills prepared buffer.With liquid-transfering gun by heat 14 mixing samples (each 10 μ L of sample) squeezes into gel pore respectively, and 10 μ L NOVEX sharp protein standard samples are squeezed into the last one hole, has been loaded Bi Hou, 200V constant pressure terminate electrophoresis when until indicator is apart from glue bottom about 1cm.

Gel is taken out, is put into coomassie brilliant blue R250 stained over night, next day decolourizes and scans, as a result as depicted in figure 3 a.

In attached drawing 3aRespectively total protein, peak1, peak1Q5, peak1Q6, peak2Q7, peak2Q8, Peak2Q9, peak3, peak3Q4, peak3Q7, peak4, peak4Q3, peak4Q4, peak5,It is protein labeling.

The shallower lower result one of concentration with spectrophotometric determination of the electrophorogram color of number protein sample It causes, therefore, recombines two results and pick out 6 protein samples and be replaced, obtain result as shown in fig. 3b.Attached drawing 3b In Respectively total protein, peak1, peak1Q5, peak4, peak2Q7, peak2Q8, peak3Q4, peak3, peak1Q7,peak2Q4,peak2Q5,peak3Q6,peak4Q2,peak5Q2；It is protein labeling.

It wherein, in 1,2,3,5,6,8,9,11 and Fig. 3 b 1,2,3,5,6,8,4 is respectively identical egg two-by-two in Fig. 3 a White sample can be seen that protein band is consistent twice by electrophorogram Fig. 3 a, Fig. 3 b, illustrate test repeatability preferably.

Meanwhile by 3 yellow meal worm purifying protein SDS-PAGE electrophoresis of attached drawing spectrum it can be seen from stripe size be evenly distributed, Clearly, no disperse illustrates the yellow meal worm protein sample for having obtained meeting follow-up test requirement twice after isolating and purifying.

Embodiment 3

Present embodiment discloses the method for extracting of test human serum, specifically:

According to the biomedical research scheme drafted, yellow meal worm allergies (test group) have been filtered out and without yellow meal worm allergy The subject of history (positive controls) each 5.Method particularly includes: edible yellow meal worm has been provided by voluntary principle screening and has been undergone 60 volunteers, inquiry understand they once eat yellow meal worm after whether have adverse reaction relevant to allergy, and body Health, No respiratory disease, disease in the blood system etc. are unsatisfactory for the medical history of experiment sieving condition, and volunteer is not thermophilic cigarette, excessive drinking Person.Meanwhile any other clinical test is not participated in the surrounding of this on-test.

The whole blood 40mL (4 pipes, 10mL/ pipe) of every subject is acquired using the vacuum glass bloodletting tube (red cap) of 10mL, The serum sample that everyone obtained after standing 2 hours is no less than 16mL dispenses label with the serum tube of 2mL.All serum sample marks - 80 DEG C of refrigerator number is placed on to save.The acquisition and follow-up study of human serum need to obtain the examination & approval and supervision of Ethics Committee, Experiment should sign subject's informed consent form with subject according to the rules before carrying out.

Embodiment 4

Proteomic studies

20 μ L protein component samples are placed in 16 hole nitrocellulose layer glass slides in Biomek Automation workstation In after, by the glass slide containing protein component be put into Glass carrier box and by the phosphate containing 3% bovine serum albumin(BSA) (BSA) it is slow Solution (PBS) is rushed as confining liquid and fills Glass carrier box, has avoided bubble.Glass carrier box is placed in close in 37 DEG C of insulating boxs and is carried The protein component sample 3h of surface of glass slide.After 3h, 37 DEG C at a temperature of with 0.05%PBST (the Tween 20:PBS=that contains 0.05%m/V, 250mg/500ml) washing glass slide 2min, it is repeated 3 times.With milli-Q water 5min, then glass slide is taken Out, centrifuge dripping 10min.

Glass slide is caught in sample-adding box, moistens 16 pulls containing protein component on glass slide with 150 μ L PBST respectively Hole outwells the 3, hole 6-14 after PBST and is separately added into 100 μ L with diluted 10 volunteers' of the 0.2%PBST containing 2%BSA Human serum (30% serum), 4, No. 5 holes are added placebo solution (30%PBS), use rubber belt sealing.In humidification temperature controller In (Thermo Hybaid HyPro-20) after 37 DEG C of concussion heating 3h, with 0.05% in board-washing machine (BioTek ELx50) PBST is cleaned glass slide 3 times.Dilution secondary antibody (the Anti-Human that 100 μ L concentration are 2mg/mL is added in each hole again IgE is purchased from Vector Laboratories, the U.S.), 37 DEG C of concussion heating 30min in humidification temperature controller are put into, are taken out.First use 0.05%PBST is cleaned 3 times, then is cleaned 3 times, centrifuge dripping 10min with ultrapure water, with GenePix 4000B biochip scanning Data are read in instrument scanning.

The blood serum sample of ten subjects (37 blooms have been screened out into conjunction with 32 yellow meal worm protein component samples respectively 5 protein concentrations are the sample of negative value in worm protein component), wherein two subjects and egg with yellow meal worm allergies White sample produces immune response.The specific yellow meal worm protein component isolated and purified such as attached drawing 4 and specific human body serum sample 16 array scanning figures shown in.It can be seen from attached drawing 4a compared with blank control, positive control, number be HFC-05 by Examination person's blood serum sample and protein sample peak2Q4 produce Immunofluorescence Reactions；Number is HFC-07 it can be seen from attached drawing 4b Experimenter's serum sample and protein sample peak3Q6 produce Immunofluorescence Reactions.According to the immune of arrays of immobilized protein experiment Fluorescence principle, for the serum of subject when with it can be caused to generate anaphylactoid protein binding, contact site will generate can quilt Scan the immunofluorescence read.Illustrate, protein sample peak2Q4 and peak3Q6 can cause subject HFC-05 and HFC- respectively 07 generates allergic reaction.

Embodiment 5

Western western blot test

SDS-PAGE: by the blood serum sample of ten subjects respectively and including being produced in proteomic studies with human serum 14 kinds of yellow meal worm protein component samples including two kinds of protein samples of raw specific immunofluorescence reaction carry out immunoblotting examination Test, binding protein Microarray assays as a result, twice testing sieve select have more significantly for certain protein components produce The serum of raw Immunofluorescence Reactions is consistent.Specific experiment result only present generate Immunofluorescence Reactions experimental group (HFC-05, HFC-07), 1 positive control (HFC-10) without any Immunofluorescence Reactions is arbitrarily selected, human serum is added without and is only added The blank control (negative control group) of dilution.Electrophoresis takes around 40min.

Protein screening standard: in the protein component sample SDS-PAGE detection isolated and purified, second used when detecting Protein sample (including generating two kinds of protein samples that specific immunofluorescence reacts with human serum in proteomic studies)；

Protein screening result: total protein, peak1, peak1Q5, peak4, peak2Q7, peak2Q8, peak3Q4, peak3、peak1Q7、peak2Q4、peak2Q5、peak3Q6、peak4Q2、peak5Q2。

Serum screening standard: the human serum sample of Immunofluorescence Reactions is generated with yellow meal worm protein sample.

Serum screening result: HFC-07, HFC-05.

Electrotransfer: during carrying out electrophoresis, 1L transfering buffering liquid (50mL NuPAGE Tansfer Buffer is prepared With 950mL ultrapure water), and electrotransfer mold is got out, after nitrocellulose filter is moistened with methanol, with electrotransfer plate, filter paper It is immersed in transfering buffering liquid together stand-by.

After electrophoresis, by gel according to from bottom to top be transfer blade+filter paper+gel+nitrocellulose filter+filter paper+turn It moves after plate is put into electrotransfer mold and is placed in electrophoresis apparatus and fills prepared transfering buffering liquid.30V constant pressure, about 1h.

Enzyme immunolocalization: the nitrocellulose filter for being printed on protein band is taken out from electrophoresis apparatus and is dried, is then placed in The centrifuge tube of 50mL pours into PBS cleaning 2 times after outwelling solution and the PBS that 5mL contains 3%BSA is added, rotates under 37 DEG C of constant temperature Heat 3h.It outwells the solution in centrifuge tube and is cleaned 1 time with PBS, 5mL dilution human serum is added and is placed on refrigerator low temperature mistake Night.

Next day pours out human serum, and nitrocellulose filter is cleaned 3 times at 37 DEG C with 0.2%PBST, each 10min.So Afterwards, the diluted secondary antibody of 5mL is added, is put into rotary heating 3h in 37 DEG C of insulating boxs.

After 3h, after cleaning 3 times (37 DEG C, each 10min) with 0.2%PBST, 5mL streptavidin is added The dilution of (Streptavidin HRP, Bio-Rad) (is added in the 0.2%PBST containing 1%BSA with the ratio of 400:1 Streptavidin-solution), it is placed in 1h at 37 DEG C.

After 1h, streptavidin dilution is poured out, cleans nitrocellulose filter 3 at 37 DEG C with 0.2%PBST It is secondary, each 10min.With Bio-Rad ECL chemiluminescent substrate (Clarity Western ECL Substrate) dyeing after Scanning analysis in gel imaging system.

All experimenter's serums are subjected to western blot test with the 14 kinds of albumen component samples picked out respectively, In, two subjects with yellow meal worm allergies produce corresponding immune response with protein sample.Pass through gel imaging system The map (attached drawing 5) obtained after scanning in system can be seen that experimental group HFC-05 and positive controls and feminine gender in attached drawing 5a Control group is compared, and 8. produces immune response in the position that relative molecular mass is about 20kDa in number (peak3) band；Attached drawing Experimental group HFC-07 in 5b is similarly generated in 8. number (peak3) band compared with positive controls and negative control group Immune response, the relative molecular mass for generating the protein component of immune response is about 30kDa.

10. number in addition, being carried out after Western blotting test with the blood serum sample of the subject of experimental group (peak2Q4) immunization sites are not generated in band, illustrates no immunoblotting reaction.

Embodiment 6

Matrix Assisted Laser Desorption/ionization mass spectrometry sequencing

Mass spectrum inspection will be carried out after digesting (trypsase) from the protein sample concentrate extracted in PAGE gel It surveys, parsing then is carried out to the mass spectrometric data of acquisition and Mascot is retrieved.

It is searched for by Mascot, and is carried out in Non-redundant protein sequences database (NCBI) Blast compares analysis.The result shows that peak3-20 group sub-sequence in immunoblotting measurement with from yellow meal worm serine egg The matching degree of white enzyme (ABC88745.1) is 12%, is detailed in wire part in attached drawing 6；Peak3-30 group sub-sequence with from Huang The matching degree of mealworm serine protease (ABC88747.1) is 21%, is detailed in wire part in attached drawing 7.Speculate two and causes spy The yellow meal worm protein component sample that anisotropic human serum sample generates immune response is serine protease.

Yellow meal worm allergic protein of the present invention: the amino acid sequence of YM45 albumen is as shown in SEQ ID NO:1.By utilizing In line analysis platform ExPASy ProtScale program (https: //web.expasy.org/protscale/) analysis it is found that The relative molecular mass of YM45 is 27968.97, totally 265 amino acid residues.Molecular formula (Formula) is C₁₂₅₁H₁₉₈₃N₃₂₁O₃₈₅S₉, molecule total atom number is 3949.Each amino acid content is as shown in table 1.Theoretical isoelectric point (Theoretical pI) is 4.39；Charge residue residue totally 43, wherein negatively charged amino acid residue has 28 (Asp+Glu), positively charged amino acid residue totally 15 (Arg+Lys).

1 ABC88745.1 amino acid of table composition

The amino acid sequence ABC88745.1 of YM45 albumen is imported into yellow meal worm proteomic data library, uses GO, COG (http://www.ncbi.nlm.nih.gov/COG/), KEGG and IPR (Interproscan software) database carry out respectively Protein function, structure, access annotation.It knows that YM45 albumen participates in the proteolysis in bioprocess, and there is molecule function Can --- serine-type endopeptidase activity (GO annotation, i.e. GeneOntology gene ontology).In addition, the albumen belongs to COG note Release the posttranslational modification in (i.e. Cluster of Orthologous Groups prokaryotes ortholog) 26 major class, albumen Matter is had enough to meet the need, in chaperone (Posttranslational modification, protein turnover, chaperones) Secretion trypsin-like serine protease (serine proteinase).

The amino acid sequence of the YM45 albumen measured is imported into yellow meal worm transcript profile database, is obtained with assembling The sequence of Unigenes has carried out blast comparison, occurs selecting the carry out subsequent Pfam of highest scoring when multiple corresponding genes (http://pfam.sanger.ac.uk/)、Swiss-Prot(http://www.ebi.ac.uk/uniprot/)、NT(NCBI nucleotide sequences)、NR(NCBI non-redundant protein sequences)、KOG(http:// www.ncbi.nlm.nih.gov/COG/)、GO(www.geneontology.org)、KEGG(http:// Www.genome.jp/kegg/) annotation analyzes the gene it is found that most like with the amino acid sequence (shown in SEQ ID NO:1) It is Cluster-24946.22118, nucleotide sequence is as shown in SEQ ID NO:2, homology 100%.From the results of view, It is considered that the encoding gene of YM45 albumen is exactly Cluster-24946.22118, sequence codified Ah method's peptide domain, cell are glutinous Attached element, tryptose, short-tail urea, chymotrypsin-like protease participate in passing through plasma membrane cell adhesion molecule in bioprocess Thermophilic specific cell-cell adherence and proteolysis, have protein domain specific binding and serine-type endopeptidase living The molecular function of property, is the component part of film in cellular component.

Yellow meal worm allergic protein of the present invention: the amino acid sequence of YM47 albumen is as shown in SEQ ID NO:3.By utilizing In line analysis platform ExPASy ProtScale program (https: //web.expasy.org/protscale/) analysis it is found that YM47 albumen relative molecular weight is 28182.32, there is 266 amino acid residues.Molecular formula (Formula) is C₁₂₄₁H₁₈₉₇N₃₃₅O₃₉₇S₁₀, molecule total atom number is 3880., each amino acid content it is as shown in table 2.Theoretical isoelectric point (Theoretical pI) is 4.57；Charge residue residue totally 51, wherein negatively charged amino acid residue has 35 (Asp+Glu), positively charged amino acid residue totally 16 (Arg+Lys).

2 ABC88747.1 amino acid of table composition

The amino acid sequence ABC88747.1 of YM47 albumen is imported into yellow meal worm proteomic data library, uses GO, COG (http://www.ncbi.nlm.nih.gov/COG/), KEGG and IPR (Interproscan software) database carry out respectively Protein function, structure, access annotation.It knows that YM47 albumen participates in the proteolysis in bioprocess, and there is molecule function Can --- serine-type endopeptidase activity (GO annotation, i.e. GeneOntology gene ontology).In addition, the albumen belongs to COG (i.e. Cluster of Orthologous Groups prokaryotes ortholog) posttranslational modification in 26 major class, protein week Turn, point in chaperone (Posttranslational modification, protein turnover, chaperones) Secrete trypsin-like serine protease (serine proteinase).

The amino acid sequence of the YM47 albumen measured is imported into yellow meal worm transcript profile database, is obtained with assembling The sequence of Unigenes has carried out blast comparison, occurs selecting the carry out subsequent Pfam of highest scoring when multiple corresponding genes (http://pfam.sanger.ac.uk/)、Swiss-Prot(http://www.ebi.ac.uk/uniprot/)、NT(NCBI nucleotide sequences)、NR(NCBI non-redundant protein sequences)、KOG(http:// www.ncbi.nlm.nih.gov/COG/)、GO(www.geneontology.org)、KEGG(http:// Www.genome.jp/kegg/) annotation analyzes the gene it is found that most like with the amino acid sequence (shown in SEQ ID NO:3) It is Cluster-24946.17391, nucleotide sequence is as shown in SEQ ID NO:4, homology 87.76%.The nucleotide Sequence codified trypsase and chymotrypsin participate in the proteolysis in bioprocess, have serine-type endopeptidase Bioactive molecule function.

Above-described embodiment is only one of the preferred embodiment of the present invention, should not be taken to limit protection model of the invention It encloses, as long as that in body design thought of the invention and mentally makes has no the change of essential meaning or polishing, is solved The technical issues of it is still consistent with the present invention, should all be included within protection scope of the present invention.

SEQUENCE LISTING

<110>Sichuan Agricultural University

<120>yellow meal worm YM47 albumen, its encoding gene and application

<130> 2019

<160> 4

<170> PatentIn version 3.3

<210> 1

<211> 265

<212> PRT

<213>artificial sequence

<400> 1

Met Lys Arg Ile Ala Ile Phe Cys Leu Cys Phe Val Leu Val Trp Ala

1 5 10 15

Thr Pro Leu Gln Lys Ser Leu Leu Lys Glu Val Ser Val Lys Asp Ile

20 25 30

Asp Ser Arg Ile Leu Asn Gly Ala Gln Ala Ala Leu Gly Gln Phe Pro

35 40 45

Trp Glu Ala Ala Leu Tyr Val Asn Ile Gly Thr Thr Thr Tyr Phe Cys

50 55 60

Ser Gly Asn Ile Ile Ser Glu Glu Trp Ile Leu Thr Val Ala Gln Cys

65 70 75 80

Ile Ile Gly Ala Asp Ser Ile Asp Val Leu Ala Gly Leu Ile Asp Leu

85 90 95

Asn Gly Ser Gly Thr Val Ala Arg Gly Thr Glu Ile Val Leu His Gly

100 105 110

Asp Tyr Asp Pro Asp Ala Phe Asn Asn Asp Ile Gly Leu Ile Lys Leu

115 120 125

Ser Thr Pro Ile Thr Phe Asn Val Asn Val Ala Pro Ile Ala Leu Ala

130 135 140

Glu Thr Leu Leu Glu Asp Gly Ile Asp Val Arg Val Ser Gly Trp Gly

145 150 155 160

Ala Thr Ser Asp Val Gly Gly Val Ser Glu Phe Leu Ser Tyr Val Asp

165 170 175

Leu Val Thr Ile Arg Asn Ser Glu Cys Ile Ala Val Tyr Gly Asn Thr

180 185 190

Ile Val Asp Ser Ile Val Cys Ala Gln Ser Ala Thr Ala Leu Leu Lys

195 200 205

Ser Val Cys Lys Gly Asp Gly Gly Ser Pro Leu Val Ile Asp Ala Gly

210 215 220

Ile Ser Pro Val Leu Val Gly Leu Val Ser Phe Ile Ser Thr Asp Gly

225 230 235 240

Cys Glu Ser Gly His Pro Thr Gly Phe Thr Arg Thr Ala Ala Tyr Arg

245 250 255

Asp Trp Ile Arg Thr Asn Ser Gly Val

260 265

<210> 2

<211> 1532

<212> DNA

<213>artificial sequence

<400> 2

tccaaaatga aacgtatcgc cattttttgt ctatgctttg tgctagtgtg ggctacgccc 60

ttgcagaaaa gcctattgaa agaggtatct gtaaaggaca tagattccag aattctcaat 120

ggtgctcaag cggccttagg tcaatttcct tgggaagcag ctctatacgt aaatataggg 180

accactacgt atttttgtag tggtaacata ataagcgaag aatggatcct taccgttgcc 240

caatgtatta ttggagccga ttctattgat gttctggcag gattgataga tttgaatggc 300

tctggtacgg tggcacgggg tactgaaatc gtccttcacg gggattatga tcccgatgct 360

tttaataacg atattggtct tattaaacta tcgacaccaa ttacatttaa tgtcaacgta 420

gccccaatag ctcttgcgga aacccttctt gaagacggaa tcgatgttag agtaagtgga 480

tggggcgcaa caagcgacgt cggtggtgta agcgaatttt tgagctatgt ggatctagta 540

acgattcgca acagtgaatg tatagcagtt tacggaaata ccattgtgga ttcaatcgtt 600

tgcgcacaat cagcaacggc tctcttgaag agcgtttgca agggtgacgg tggttcgcca 660

ttagtcattg atgctggcat cagtcctgta ctcgttggtc ttgttagttt tatcagcacc 720

gacggttgtg aatctgggca tcctactgga tttacaagaa ctgctgctta cagagactgg 780

atacgaacta attccggtgt gtaaattaca tgagaaccaa acgcaattaa ttgtaatgaa 840

aaactatatg taataaaatt ggattattgt gaattgtata ttttgacagt tattatttaa 900

tcattgatat ggaaaaaata tctttaagta gagaaattgt atcaacggat tgggaaaggt 960

atcacgtcat tacagccgtg aatagccaaa tcggcgtatc tgcatgttac aacatttatt 1020

gtaaagtgag aaaaattctg aacgtctagc cgtaatcttg gttaataagc ataattacca 1080

ttttaactgg cgattttttg cgtttctctt aatgaaatat gtaaataata acttattgaa 1140

ctatgtatat aactcaaaaa atgtaattta actttttttc caaatatgga ggttttgctg 1200

attactgata ttatttttga taactaatct tgaaagacgg tatttattgt ctgtagatgc 1260

tctaacgatt ctcgttttgg gtgatgtttg agtattgttc tcttcctttc cccgaatggt 1320

gtttttgcaa aaaatacgaa gcaaaaactt attggaaaag aatccccaat ttacgtactc 1380

acagagcacg tgatgccatt ccgtgttact tatttttatt taaataagat tctaagagag 1440

ttctaggata aataggtttt tgtaagagtt ttaaatgaaa cgattaagca aacttcactt 1500

atttttgatt ttacaaagaa tcgtgttttg at 1532

<210> 3

<211> 266

<212> PRT

<213>artificial sequence

<400> 3

Met Lys Ser Ile Val Leu Ile Cys Leu Cys Val Val Ser Ala Trp Ala

1 5 10 15

Gly Leu Ile Thr Glu Arg Lys Pro Val Pro Val Lys Lys His Phe Gly

20 25 30

Gly Arg Ile Val Gly Gly Asp Glu Ala Ala Glu Asn Gln Phe Pro Trp

35 40 45

Gln Val Ala Val Tyr Phe Asp Thr Ser Asp Gly Thr Tyr Phe Cys Gly

50 55 60

Gly Ala Leu Val Ala Glu Asn Trp Val Leu Thr Ala Gly His Cys Val

65 70 75 80

Tyr His Ala Lys Val Phe Thr Leu His Leu Gly Ser Asn Ser Leu Val

85 90 95

Asp Asp Asp Asp Asn Arg Val Thr Leu Gly Ala Ser Tyr Ser Val Pro

100 105 110

His Pro Asp Tyr Asp Pro Ser Asp Leu Glu Asn Asp Ile Gly Leu Ile

115 120 125

Arg Ile Asp Thr Ala Tyr Lys Thr Asn Asp His Ile Lys Val Ile Pro

130 135 140

Leu Ala Ser Ser Glu Leu Gly Ala Asp Val Asp Val Ile Val Ser Gly

145 150 155 160

Trp Gly Ala Ser Gly Asp Trp Asp Gly Val Glu Asn His Leu Arg Phe

165 170 175

Val Gly Leu Lys Thr Leu Ser Asn Asp Asp Cys Lys Ala Ile Tyr Gly

180 185 190

Glu Ala Val Ile Thr Asp Gly Met Val Cys Ala Val Gly Pro Asn Ser

195 200 205

Glu Gly Thr Cys Asn Gly Asp Ser Gly Gly Pro Leu Val Thr Asp Asp

210 215 220

Gly Ser Gly Asn Ser Val His Val Gly Val Val Ser Trp Ala Ser Ala

225 230 235 240

Ser Gly Cys Glu Thr Asn His Pro Ser Gly Tyr Thr Arg Thr Ala Ala

245 250 255

Tyr Arg Asp Trp Val Glu Ser Val Ile Gly

260 265

<210> 4

<211> 1731

<212> DNA

<213>artificial sequence

<400> 4

cagaagaaac cagttccctt caagaaacat ttcggtggac gaattgtagg aggtgatgta 60

gctgcagata accagttccc ttggcaagta gcagtctact ttgataccag cgacggtaca 120

tttttctgtg ggggtgctct gattgcagag aactgggtct tgactgctgg ccattgtgtc 180

taccatgctg aagtatttac tctccatttg ggatcaaatt cacttgtaga tgatgatgac 240

aatagagtga ctttgggcgc cagttactct gtaccgcatc ctgactacga tccttcgtct 300

ctggagaacg acattggtct tatcaggata gatgctgctt ataaaaccaa cgatcacatc 360

aaggtcattc ctttggccag cagcacgctt ggtgctgatc tagatgttac agttagtgga 420

tggggagcaa gcggtgattg ggatggtgtt gtcaacgatc ttaactttgt tggcttgaag 480

acaatttcca acgatgactg caaagccatt tatggtgacg caacgattct cgatggtatg 540

gtgtgtgctg ttggcccgag cacagaagga acttgcagcg gcgacagtgg tggtcctttg 600

gtaacagatg atggcagtgg taactctgtt catgttggtg ttgttagctg ggtgagctcc 660

agtggctgcg aaactgatca tccatcagga tatactagaa ctgccgctta tcgcgactgg 720

atagacagtc ttgttattag ttcaatgcac aaaactaagc aacagaagat atctctgatt 780

aatgaatgag gagttaatgg taacgaaaca atagaaatta ataaatcagt aacagaaagc 840

aactaaaaac aaaaagaaat ttatacaggg tgtattaaaa atggcgcata atatttatgt 900

aaggcgaatt tacgttgaat aacaaacaaa aatttttatt gtgaatttgg tcttagacga 960

ataatttttt aattattatt attattatca aaacttacat taatgacaat ccgtgttact 1020

ttctcacttt actcaatttc gaaagataaa gggtgaataa gagtcgtttt aataacttaa 1080

tttaaatcac aaaaaggtgc ttaggcaata taatttttga agagaaaaac aaaatttaaa 1140

acacaaaaat gaaaattgtg tggcaaagtt aaattaatat acatattcgt acaataagat 1200

tgacattaat attttttatt aattttatca ataagttttg gcaattttat ctctatatgt 1260

atcatgcgtt caaataaaaa cctgcgctga gtaggtgttg aaaaaagtaa accatttgga 1320

acagggtaaa gtttgaattt cccgcccttt gacatgaatg acttttgttt atgtttaatc 1380

ggcacatgat tgaacatgat tgttttcaga atggggtgcc tttagatatt tgcttggaga 1440

atagaattcg ctagttattc tatttttaag gtaggtaaaa cattacagag aaagaaaaaa 1500

caggaagatt ttttgtttat aaacttgagg atgtgattta cctatttttt ttagcacgtc 1560

atgaaaaatt taacatcaga acattttcaa aacttcaaca tgaatttgag aatcagctct 1620

gctattacat atttaaagaa taagtcgtga ccattttgaa cattcttttt gattaatatt 1680

gaaaaatgta ttacctaatt atttgtaaat attatattta acttaagtac a 1731

Claims (5)

1. yellow meal worm YM47 albumen, which is characterized in that its amino acid sequence is as shown in SEQ ID NO:3 or the amino acid sequence Through replacement, missing or one or several amino acids formed amino acid sequences with same function of addition.

2. encoding the gene of albumen as described in claim 1, which is characterized in that its nucleotide sequence is as shown in SEQ ID NO:4； Or the nucleotide sequence is substituted, lacks or increases one or more nucleotide and expresses the nucleotides sequence of identical function albumen Column.

3. carrier, engineering bacteria or transgenic cell line containing gene described in claim 2.

4. YM47 albumen described in claim 1 is in the application of yellow meal worm molecular breeding.

5. gene as claimed in claim 2 is in the application of yellow meal worm molecular breeding.