CN113846115B - Recombinant protein of house dust mite I allergen pro-Der p1, and preparation method and application thereof - Google Patents
Recombinant protein of house dust mite I allergen pro-Der p1, and preparation method and application thereof Download PDFInfo
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Abstract
The invention discloses a house dust mite I allergen pro-Der p1 recombinant protein, a preparation method and application thereof, wherein the pro-Der p1 gene sequence has an optimized insect codon and a 3' -end is coupled with a His protein sequence, and the sequence is shown as SEQ ID NO: 1. Specifically, the pro-Der p1 gene sequence with optimized insect codons and His protein sequence linked thereto was constructed on the basis of the shuttle vector pFastBac TM In the Dual modified pFastBac-SCUVI, recombinant baculovirus plasmid Bacmid-Der p1 is obtained, and the recombinant Der p1 baculovirus is prepared and amplified by transfecting insect cells Sf9 to obtain the expressed pro-Der p1 recombinant protein. The pro-Der p1 recombinant protein has physical and chemical properties closer to those of natural allergen protein components, so that the detection sensitivity is high, the non-specificity is less, the correlation between ELISA and liquid chip detection results and the detection result of the clinical allergen specific antibody to detect the ImmunoCAP is high, and the method is particularly suitable for the rapid detection of house dust mite allergens.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a house dust mite I allergen pro-Der p1 recombinant protein, a preparation method and application thereof.
Background
Dust mites, pollen, animal hair, etc. are major allergens responsible for allergic diseases, especially dust mites, an important allergen in the environment where people live. Currently common dust mites include two types, one being house dust mites (Dermatophagoides pteronyssinus) and the other being dust mites (Dermatophagoides farinae). About 60% to 80% of allergic disease patients are reported to be allergic to house dust mites. Secretions, excretions and degradation products of house dust mites after death of the insects can all become allergens. Studies have shown that house dust mite crude extract contains several components that bind to human serum IgE antibodies, but that the cause of the disease in a patient may be associated with only one or some of these components, which are called "components". As for the constituent components of house dust mites, there are 30 or more components which have been named by the International Association of immunology, and among them, component 1 and component 2 are considered as the most main components in house dust mite allergens.
The allergen specificity detection method commonly used at present mainly comprises the following steps: in vivo tests (intradermal test or skin prick test), organ stimulation tests (via nasal mucosa, conjunctiva or airways), serum specific IgE (specific immunoglobulin E, sIgE) detection, patch test, etc. The serum-specific IgE detection is used as a safe, effective and convenient detection means and has been widely applied to diagnosis of allergic diseases. However, the currently commercialized serum-specific IgE detection reagents mostly use crude extracts, and the crude extracts may have the phenomena of partial component loss or more impurity proteins due to the differences of the extraction method, the post-treatment and the preservation method, so that the occurrence of false negative results is easy to occur. In addition, substances having similar epitopes to the true allergen may be present in the crude protein extract, and these similar epitopes may cause cross-reactions to give false positive results. Therefore, improving the accuracy of serum-specific IgE detection results is an urgent problem to be solved at present. Meanwhile, the crude extract contains a plurality of allergen components, so that the content of the same component in different batches of products is inconsistent, and the standardization of the products is not facilitated.
Therefore, the allergen components of the house dust mites are needed to be known, the allergen single-component recombinant protein, in particular the house dust mite class I allergen Der p1 recombinant protein is prepared, a technical means is provided for the rapid diagnosis of the house dust mite allergic diseases, and a basis is provided for the clinical establishment of an effective desensitization treatment scheme.
Disclosure of Invention
In order to overcome the defects of the prior art, one of the purposes of the invention is to provide a recombinant protein of a room dust mite I allergen pro-Der p1, which is particularly suitable for the rapid detection and desensitization treatment of room dust mite allergens.
Specifically, the recombinant protein of the room dust mite I allergen pro-Der p1, wherein the pro-Der p1 gene sequence has an optimized insect codon and is coupled with a His protein sequence at the 3' end, and the sequence is shown as SEQ ID NO: 1. The pro-Der p1 recombinant protein has higher expression quantity and similar biological activity with natural protein.
The second object of the present invention is to provide a recombinant plasmid comprising the above-mentioned house dust mite group I allergen pro-Der p1 recombinant protein. The recombinant plasmid is prepared by inserting a pro-Der p1 recombinant gene sequence with optimized insect codon and 3' -end coupled His protein sequence into a shuttle vector pFastBac-based recombinant gene sequence by utilizing Xho I and KpnI enzyme cutting sites TM Obtained in Dual engineered pFastBac-SCUVI. Further, the pro-Der p1 recombinant plasmid is transformed into DH10Bac strain to obtain a pro-Der p1 recombinant baculovirus plasmid (i.e., recombinant baculovirus plasmid Bacmid-Der p 1).
The third object of the present invention is to provide an expression vector comprising the recombinant plasmid. Specifically, the recombinant baculovirus plasmid Bacmid-Der p1 is transfected into Sf9 cells to obtain a pro-Der p1 expression vector.
The fourth object of the present invention is to provide a process for the preparation of the recombinant protein of the allergen pro-Der p1 of house dust mite class I. Specifically, a pro-Der P1 gene sequence of the His protein gene sequence of SEQ ID No.1 is synthesized, and is connected to a shuttle vector pFastBac-SCUVI through Xho I and KpnI cleavage sites, a recombinant baculovirus plasmid Bacmid-Der P1 is obtained by transforming DH10Bac competent cells, then Sf9 insect cells are transfected to prepare recombinant baculoviruses, the Sf9 cells are infected by the P2 generation recombinant baculoviruses, and the supernatant is collected by centrifugation.
Specifically, the protein was purified as follows: the collected supernatant was filtered with a 0.45um filter; loading the filtrate His chromatographic column, eluting with 100mM imidazole and 200mM imidazole respectively; running the eluent on SDS-PAGE gel to obtain a pure pro-Der p1 band with the size of about 34 kD; concentrating the eluent by using an ultrafiltration membrane bag and replacing the eluent by using PBS to obtain the purified pro-Der p1 recombinant protein resuspended by using PBS.
The fifth object of the invention relates to the application of the recombinant protein of the allergen pro-Der p1 of the house dust mite I in preparing diagnostic reagents for detecting allergic diseases caused by house dust mites or biological products for desensitizing treatment and the like.
The sixth object of the present invention relates to the use of the recombinant protein of the allergen pro-Der p1 of house dust mites I as described above for the preparation of diagnostic reagents or biological products for the detection of house dust mites.
The seventh object of the invention relates to the application of the recombinant protein pro-Der p1 of the house dust mite I allergen in a method for detecting house dust mites. The method comprises in vitro diagnosis methods based on antigen and antibody, such as ELISA, liquid phase chip, solid phase chip, colloidal gold, and the like, and can be used for rapidly detecting house dust mite allergens.
The recombinant pro-Der p1 protein has physical and chemical properties closer to those of natural allergen protein components, and has higher cysteine protease activity, so that the detection sensitivity is high, the non-specificity is less, the correlation between ELISA and liquid chip detection results and the ImmunoCAP results is high, the trend is consistent, and the recombinant pro-Der p1 protein is particularly suitable for the rapid detection of house dust mite allergens.
The pro-Der p1 gene sequence of the room dust mite I allergen pro-Der p1 recombinant protein provided by the invention has an optimized insect codon, has stronger specificity to room dust mites, and is beneficial to the specificity detection of room dust mites. And the 3' end of the pro-Der p1 gene sequence is coupled with His protein, which is beneficial to the purification of the protein, the elution and separation of His chromatographic column can be facilitated, and the purity of the obtained recombinant protein is higher. The high-purity recombinant protein is beneficial to rapid etiology diagnosis and treatment of clinical house dust mite allergens, is beneficial to accurate diagnosis of house dust mite allergy components, shortens treatment period, saves medical cost and generates good social benefit.
Drawings
FIG. 1 is a diagram showing the alignment of the pro-Der p1 original sequence and the optimized sequence.
FIG. 2 shows a map of a pro-Der p1 recombinant plasmid.
FIG. 3 shows a comparison of the sequencing of the pro-Der p1 recombinant protein with the original pro-Der p1 protein.
FIG. 4 shows SDS-PAGE detection of pro-Der p1 recombinant proteins.
Lane 1: sf9 cell culture supernatant; lane 2: passing the protein sample through a nickel column and then flowing through the solution; lane 3:50mM imidazole eluent; lane4:200mM imidazole eluent; lane 5: the eluent was regenerated from 1M imidazole.
FIG. 5 shows the evaluation of the activity of pro-Der p1 recombinant protease.
FIG. 6 shows the correlation analysis of the ELISA (TMB method) of the pro-Der p1 recombinant protein sIgE antibody with the detection result of ImmunoCAP.
FIG. 7 shows the correlation analysis of the result of the chemiluminescent enzyme immunoassay (CLEIA) with the detection of the pro-Der p1 recombinant protein sIgE antibody and the result of the ImmunoCAP.
FIG. 8 shows the correlation analysis of the liquid chip of the pro-Der p1 recombinant protein sIgE antibody and the detection result of the ImmunoCAP.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. It will be apparent to those skilled in the art that several simple deductions or substitutions may be made without departing from the spirit of the invention, and these should be considered to be within the scope of the invention.
Wherein pFastBacTM Dual is purchased from Thermo Fisher company, sf9 insect cells are purchased from American Type Culture Collection (ATCC), 2 XPCR reaction solution, KOD FX enzyme, dNTPs are purchased from Toyobo (Shanghai) Biotechnology Co., ltd, anti-human HRP-IgE, chemiluminescent substrates are purchased from Thermo Fisher company, and liquid chip microspheres and protein coupling kit are purchased from Bio-Rad company.
The specific implementation flow is as follows:
(1) Preparation of the house dust mite component I-pro-Der p1 recombinant protein: the gene sequence of Der p1 is found out from NCBI database, insect codon optimization is carried out, after His protein gene sequence is connected at 3' end, the gene sequence is inserted into a pFastBac-SCUVI vector modified based on a shuttle vector pFastBac Dual, DH10Bac competent cells are transformed and selected to obtain Bacmid rod particles, and Sf9 insect cells are transfected to prepare recombinant baculovirus, so that pro-Der p1 recombinant protein is obtained and purified.
Insect codon optimization references: horse garden, zhong Liang, wangguo, et al, foot and mouth disease virus VP1 gene codon preference analysis, chinese animal quarantine, 2021. 28 (4): 116-123.
(2) Evaluation of the activity of the pro-Der p1 recombinant protein: the protein activity was evaluated by comparing the fluorescence intensity after purification of the pro-Der p1 recombinant proteolytic cysteine protease substrate Z-phe-arg-MCA.
(3) Application of pro-Der p1 recombinant protein: the obtained pro-Der p1 recombinant protein is used for detecting Der p1 sIgE antibody, ELISA (TMB method), chemiluminescent enzyme immunoassay (CLEIA) and liquid chip method are adopted respectively, and the obtained pro-Der p1 recombinant protein shows good correlation by comparing with the detection result of the ImmunoCAP, and is particularly suitable for rapid detection of house dust mite allergens.
EXAMPLE 1 preparation of recombinant protein of the house dust mite component I pro-Der p1
Construction of a pro-Der p1 recombinant plasmid
The gene sequence of Der p1, which contains the pro peptide portion, was found from NCBI database, and after insect codon optimization of this sequence and addition of His protein gene sequence at its 3' end (FIG. 1), RT-PCR amplification or direct synthesis and insertion of the optimized pro-Der p1 into shuttle vector pFastBac SCUV using XhoI and KpnI cleavage sites, the pro-Der p1 recombinant plasmid pFastBac-SCUVI-Der p1 was obtained (FIG. 2).
2. Preparation and identification of baculovirus plasmid (Bacmid)
(1) Transformation and amplification
To 100ul DH10 MultiBac competent cells, 250ng of the recombinant plasmid pFastBac-SCUVI-Der p1 was added, and the mixture was subjected to ice bath for 30 minutes and heat shock at 42℃for 90 seconds, and after 3 minutes on ice, 900ul of non-resistant LB liquid medium was added for further culture for 5 hours. 200ul of bacterial liquid is coated on an LB resistant plate added with IPTG and X-gal (containing 10mg of kanamycin sulfate, 10mg of tetracycline hydrochloride and 1.4mg of gentamicin sulfate), after culturing for 48 hours at 37 ℃, target white bacterial colonies are screened out through blue white spots, and after re-streaking on the LB resistant plate added with IPTG and X-gal for 24 hours at 37 ℃,1 white bacterial colony is picked up in 5ml of LB liquid culture medium (containing 10mg of kanamycin sulfate, 10mg of tetracycline hydrochloride and 1.4mg of gentamicin sulfate) for continuous culturing for 16 hours.
(2) Bacmid-Der p1 Bacmid extraction
1) Taking 1 sterile 2ml centrifuge tube, collecting 6ml bacterial liquid of the stem grains to be extracted three times, centrifuging at 12000 Xg for 1 min each time, and discarding the supernatant after centrifuging at room temperature. The following operations were performed separately for each centrifuge tube.
2) Mu.l Buffer S1 (RNase had been added) (Axygen plasmid miniDNA extraction kit AP-MN-P-250) was added and bacterial pellet was resuspended with a 1ml pipette.
3) Add 250. Mu.l Buffer S2 (from Axygen plasmid extraction kit), gently and thoroughly flip up and down 6 times to mix well to allow the cells to lyse well until a clear solution is formed, and leave it stand for 3 minutes. This step did not exceed 5 minutes.
4) Add 350. Mu.l Buffer S3 (from Axygen plasmid extraction kit), gently and thoroughly mix up and down 8 times, place on ice for 5 minutes and centrifuge 12000 Xg for 10 minutes, collect supernatant into a new centrifuge tube, about 800. Mu.l.
5) An equal volume of DNA extract (phenol/chloroform/isoamyl alcohol=25:24:1) was added to the fume hood, gently mixed upside down, and after 12000 Xg, centrifuged for 5 minutes, and the supernatant (about 800 ul) was collected.
6) Adding 0.6 times volume (480 μl) of isopropanol, mixing well, and standing at-20deg.C for 1 hr; centrifuging at 12000 Xg for 20 min, and discarding supernatant;
7) Mu.l of 70% ethanol was added to each tube to rinse the precipitate for desalting, and the supernatant was discarded after centrifugation at 12000 Xg for 5 minutes.
8) Centrifuge 12000 Xg for 5 minutes, pipette the remaining supernatant off in a clean bench, leave for several minutes, and air dry the pellet.
9) 30 μl of sterilized water is added into an ultra-clean bench for dissolution, and then the mixture is temporarily placed at 4 ℃ for preservation, thus obtaining the pro-Der p1 recombinant baculovirus plasmid (Bacmid-Der p 1).
(3) Identification of Bacmid-Der p1
PCR was performed on the extracted bacmid using specific primer P10 (F: ATACGGACCTTTAATTCAACCCAAC; R: ACCCGTGCGTTTTATTCTGTCTTTT) and STAP3 (F: CCGGCTCGTATGTTGTGTGGAATT; R: ATGTGGACAAAATACCTGGTTACCC). The total volume of the PCR reaction system was 50. Mu.l as shown in the following Table.
Reaction system for identifying baculovirus plasmid by PCR:
PCR reaction procedure:
STAP3 at 94℃for 2 min; (98 ℃,10 seconds; 68 ℃,5 minutes), 26 cycles; 68 ℃ for 20 minutes; 4 ℃, and preserving.
P10 is 94 ℃ for 2 minutes; (98 ℃,10 seconds; 57 ℃,30 seconds; 68 ℃,2 minutes), 26 cycles; 68 ℃ for 20 minutes; 4 ℃, and preserving.
As shown in the results of the PCR product gene sequencing analysis and PCR product measurement of FIG. 3, the recombinant protein is pro-Der p1 protein.
3. Preparation of recombinant baculoviruses and expression of recombinant pro-Der p1
(1) Culturing insect cells Sf9 in a serum-free Grace's culture medium, taking cells in a logarithmic growth phase, transfecting the obtained recombinant baculovirus plasmid bacmid-Der P1 into the Sf9 cells, continuously culturing for 4-5 days after Grace's complete culture medium (5% FBS) is replaced, and collecting supernatant to obtain the P1 generation recombinant baculovirus.
(2) Taking Sf9 cells in logarithmic growth phase, and adjusting cell density to 3.5X10 6 And (3) infecting Sf9 cells with P1 generation recombinant baculovirus liquid according to the volume ratio of 1:50 per ml for virus amplification, culturing for 4 th day, and collecting supernatant, namely the P2 generation recombinant baculovirus.
(3) Taking Sf9 cells in logarithmic growth phase, and adjusting cell density to 3.5X10 6 And (3) infecting Sf9 cells with P2 generation recombinant baculovirus liquid according to the volume ratio of 1:50 per ml for virus amplification, culturing for 4 th day, and collecting supernatant, namely the P3 generation recombinant baculovirus.
(4) Taking Sf9 cells in logarithmic growth phase, and adjusting cell density to 3.5X10 6 And (3) infecting Sf9 cells with P3 generation recombinant baculovirus liquid according to the volume ratio of 1:50 for virus amplification, culturing the cells at the 4 th day, collecting cell culture liquid, and centrifugally collecting supernatant for protein purification.
Purification and identification of pro-Der p1 recombinant proteins
(1) Filtering the collected cell culture supernatant with a 0.45um filter membrane; and purifying the sample by a nickel ion affinity chromatographic column, eluting with 50mM, 200mM and 1M imidazole respectively, and collecting the flow-through liquid obtained in each stage.
(2) Purifying and identifying: and (3) performing SDS-PAGE gel electrophoresis on the eluent, and then performing coomassie brilliant blue staining, wherein as shown in figure 4, the target protein is mainly concentrated in 200mM imidazole eluent, so as to obtain purer pro-Der p1 protein with the size of about 34 kD.
(3) Concentrating and desalting: concentrating 200mM imidazole eluent by using an ultrafiltration membrane bag and replacing the imidazole eluent by using PBS to obtain the pro-Der p1 recombinant protein resuspended by PBS, wherein the concentration is 3.5mg/ml, and the protein yield is 25mg per 1L of insect cell culture supernatant.
EXAMPLE 2 evaluation of the Activity of the pro-Der p1 recombinant protein
The protease activity of the expressed recombinant pro-Der p1 was detected using the specific fluorogenic substrate Z-amphetaminoyl-arginine-7-amino-4-methylcoumarin hydrochloride (Z-phe-arg-MCA) of cysteine protease.
1. Adding a proper amount of pro-Der p1 into phosphoric acid-sodium citrate buffer with pH of 5.5, wherein the total reaction volume is 3ml, the final concentration of pro-Der p1 is 25ug/ml, and the water bath is carried out at 37 ℃ for 30 minutes; if inhibition of the enzymatic activity of pro-Der p1 is desired, pro-Der p1 is reacted beforehand with E64 for 15 minutes at 37 ℃.
2. After 30 minutes in the water bath, 2ul of Z-phe-arg-MCA substrate buffer (final concentration 20 umol/L) was added to the solution and the reaction was continued for 30 minutes at 37 ℃.
3. After the reaction, 100ul of each reaction solution was placed in a 96-well plate, and the fluorescence intensity was measured at 370nm for excitation light and 440nm for emission light. The protease activity of Pro-Der p1 is proportional to the fluorescence intensity in this result.
As a result, as shown in FIG. 5, the fluorescence intensity was highest after the pro-Der p1 hydrolyzed the cysteine protease substrate Z-phe-arg-MCA, compared with the other groups, demonstrating that the recombinant Der p1 did have cysteine protease activity.
EXAMPLE 3 use of pro-Der p1 recombinant protein for ELISA (TMB method) for detection of sIgE antibodies
(1) Coating: the recombinant pro-Der p1 protein was diluted to 5ug/ml with carbonate buffer, 100 ul/Kong Baobei in 96-well ELISA plates, covered with sealing film overnight at 4 ℃.
(2) Washing the plate: the plate coated overnight was removed, the existing liquid was removed by shaking, PBST 300. Mu.l/well was added, shaking was performed for 20 seconds, washing solution was removed by shaking, washing was repeated 3 times, and the liquid in the plate was dried by shaking.
(3) Closing: 200 μl of blocking solution was added to the wells and the plates were allowed to stand in an incubator at 37deg.C for 2 hours.
(4) Washing the plate: washing the plate 3 times, and the same step (2).
(5) Sample adding: diluting a clinical serum sample with a sample diluent according to a ratio of 1:20, adding 100 μl/well into the wells, and two pairs of wells; simultaneously setting a PBS negative control group; incubate at 37℃for 2 hours. At present, there is no standard for Der p1 sIgE in the market of China, so that high-level sIgE serum-fold dilution detected by ImmunoCAP is selected as the standard.
(6) Washing the plate: washing the plate 3 times, and the same step (2).
(7) Detection of antibodies: the diluted HRP-labeled anti-human IgE antibody was added and incubated at 37℃for 1 hour at 100. Mu.l/well.
(8) Washing the plate: washing the plate 5 times, and drying the plate for the last time in the same step (2).
(9) Adding a substrate: the substrate TMB was added at 100. Mu.l/well and reacted at 37℃for about 5 minutes in the absence of light.
(10) Adding a stop solution: observing color change in the hole, adding stop solution H 2 SO 4 (1 mol/L) 50. Mu.l/well.
(11) Reading a plate: the plate was placed in an microplate reader and the OD was recorded by reading the plate at a wavelength of 450 nm.
(12) And (3) calculating results: and (3) establishing a standard curve according to the concentration of the standard substance and the corresponding OD value to obtain a calculation formula, and calculating the corresponding sIgE concentration according to the average OD value of the sample compound holes.
The detection results of ImmunoCAP were used as a control by detecting Der p1 sIgE results (grade 0-6, 3 samples per grade) from 21 sera. The results are shown in Table 1 and FIG. 6, the OD value of Der p1 sIgE detected by ELISA and the converted concentration value thereof are obviously related to the house dust mite sIgE concentration detected by the ImmunoCAP method, and the correlation coefficients are 0.938, P <0.001 and 0.927, and P <0.001 respectively.
TABLE 1 pro-Der p1 detection of human serum Der p1 sIgE results by ELISA (TMB method)
EXAMPLE 4 use of pro-Der p1 recombinant proteins for chemiluminescent enzyme immunoassay (CLEIA) detection of sIgE antibodies
(1) The Derp 1 stock was diluted with coating solution to a concentration of 5. Mu.g/ml, 100. Mu.l/well was added to a light-resistant microplate and coated overnight at 4 ℃.
(2) Washing the plate: the overnight coated plates were removed, washed 3 times with 200. Mu.l/well PBST and the plates were slapped last.
(3) Closing: blocking solution was added at 200. Mu.l/well and incubated at 37℃for 2 hours.
(4) Washing the plate 3 times, and the same step (2).
(5) Sample adding: serum samples were diluted with sample dilutions at 1: dilution was performed at a ratio of 20, 100. Mu.l/well, and two wells were set for each sample, and the sample dilutions were used as negative controls for incubation at 37℃for 2 hours.
(6) Washing the plate 3 times, and the same step (2).
(7) Detection of antibodies: HRP-labeled anti-human IgE antibody was prepared as follows: 2000 dilutions, 100. Mu.l/well, 37℃for 1 hour, 200rpm shaking incubate.
(8) Washing the plate 5 times, and the same step (2).
(9) Adding a substrate: superSignal chemiluminescent substrate was prepared in advance, added at 50 μl/well, and incubated for 1 min with shaking in the absence of light.
(10) Reading a plate: the plate was placed in a microplate reader BIOTEK rotation 3 at 425nm wavelength light to detect relative light units within 1-5 minutes after substrate addition.
(11) And (3) calculating results: and establishing a standard curve from the concentration of the standard substance and the corresponding RLU value to obtain a calculation formula, and calculating the corresponding sIgE concentration from the average RLU value of the sample compound well.
The Der p1 sIgE results of 14 sera were tested, and the results of the test of the imunocap were used as a control, and the CLEIA chemiluminescence values and converted sIgE concentrations thereof were significantly correlated with the imunocap results, with correlation coefficients of 0.921, p <0.001 and 0.929, p <0.001, respectively, as shown in table 2 and fig. 7.
TABLE 2pro-Der p1 detection of human serum Der p1 sIgE results Using chemiluminescent enzyme immunoassay (CLEIA)
EXAMPLE 5 use of pro-Der p1 recombinant protein in liquid phase chip detection of sIgE antibodies
1. Coupling pro-Der p1 with magnetic beads:
(1) Resuspension of the dispersed magnetic beads: the beads were vortexed on a vortexing apparatus at 1400rpm for about 30 seconds and then sonicated with a water bath for 15 seconds.
(2) Cleaning magnetic beads: adding 100 μl of resuspended magnetic beads into a sterile 1.5ml centrifuge tube, standing on a magnetic reaction rack for 1 min to make the magnetic beads completely adsorbed, and carefully removing supernatant; 100 μl of the bead washing liquid was added, vortexed for 30 seconds, and then placed on a magnetic rack for standing for 1 minute, and the supernatant was carefully aspirated.
(3) Activating magnetic beads: the beads were resuspended with 100 μl of bead activation solution and vortexed for 30 seconds; 10. Mu.l of 50mg/ml S-NHS was added, and immediately after gentle mixing 10. Mu.l of 50mg/ml EDC was added, and again gentle mixing was performed; high-speed oscillation and resuspension for at least 30 seconds; incubate at 25℃for 20 min with shaking at 1100rpm in the absence of light.
(4) Washing: immediately after the incubation was completed, 150. Mu.l MES (0.05M pH 5.0) was added and washed with high speed shaking for 10 seconds; placing the centrifuge tube in a magnetic rack for 1 minute, and sucking and discarding supernatant after the magnetic beads are precipitated; add 100. Mu.l MES to resuspend the beads and shake at high speed for 30 seconds.
(5) Coupling protein: to the beads, 4. Mu.g rpro-Der p1 was added and the final volume was adjusted to 500. Mu.l with MES and the beads were dispersed by high-speed shaking for 1 minute; incubate at 25℃for 2 hours with shaking at 1200rpm in the absence of light.
(6) Washing: standing the product in a magnetic rack for 1 minute, and carefully sucking and discarding the supernatant; mu.l MES was added, washed with vortexing for 30 seconds and then placed on a magnetic rack for 1 minute, and the supernatant was aspirated off.
(7) Closing: adding 125 μl of magnetic bead sealing solution, vortex oscillating for 30 seconds, placing in a magnetic rack for 1 minute, and removing supernatant; adding 250 μl of magnetic bead sealing solution, shaking by vortex to re-suspend the magnetic beads, and incubating at 25deg.C under shaking at 1200rpm in dark place for 30 min; the product was placed on a magnetic rack for 1 min and the supernatant carefully aspirated.
(8) Storing and counting: adding 500 μl of magnetic bead storage solution, vortex oscillating for 30 seconds, placing in a magnetic rack for 1 minute, and removing supernatant; 150 μl of the magnetic bead stock was added, the beads were resuspended by vortexing, and after completion by a cell counter plate, placed at 4deg.C in the dark.
2. Detection of Der p1 sIgE using Luminex platform
(1) Magnetic bead count: taking out 150 μl of magnetic Beads coupled with rpro-Der p1 (rpro-Der p 1-Beads), shaking for 30 seconds, mixing, adding 1 μl of PBS solution, mixing, and counting to obtain rpro-Der p 1-Beads with density of 10.35X106/ml; the coupled beads were diluted with low cross-reaction solution to a concentration of 6X 104/ml.
(2) After the coupling by shaking at 1400rpm, the beads were subjected to shaking for 30 seconds (light shielding), and 50. Mu.l/well was added to the 96-well reaction microplate.
(3) Washing: the plate was placed on a magnetic plate washing station and washing was automatically performed three times.
(4) Sample dilution: according to the pre-experiment result, the dilution factor of the sample is set to be 20 times by using a low cross reaction liquid, and meanwhile, the sample with the highest Der p1 sIgE concentration detected by the ImmunoCAP in the sample is used as a standard substance for concentration gradient dilution: 1:10, 1:40, 1:160, 1:640, 1:2560, 1:10240, 1:40960.
(5) Sample adding: 50 μl of diluted sample and standard were added to wells of the existing magnetic beads, respectively, and a low cross-reaction solution was added to the blank wells as a control.
(6) The mixture was shaken at 1100rpm at room temperature for 30 seconds under light-shielding conditions, and incubated at 700rpm for 1 hour.
(7) Washing: the same as in the step (3).
(8) The biotin-labeled anti-human IgE was diluted to 1. Mu.g/ml with sample dilution (1:750 dilution).
(9) 50 μl of diluted detection antibody was added to each well.
(10) The cells were incubated at room temperature for 30 minutes at 1100rpm under shaking at 700rpm for 30 seconds in the absence of light.
(11) Washing: the same as in the step (3).
(12) SA-PE (100X) was diluted to 1X concentration with sample dilution, 50. Mu.l of diluted SA-PE was added to each well, and incubated at room temperature for 30 seconds with shaking at 1100rpm in the absence of light, and at 700rpm for 15 minutes.
(13) Washing: the same as in the step (3).
(14) The beads were resuspended by adding 125 μl of sample dilution per well and were run on a plate shaker at 1100rpm for 1 minute.
By detecting Der p1 sIgE results of 19 serum, and taking the detection result of the ImmunoCAP as a control, the results are shown in Table 3 and FIG. 8, the fluorescence value of the liquid phase chip and the converted sIgE concentration value thereof are obviously related to the ImmunoCAP result, and the correlation coefficients are respectively 0.939, P <0.001 and 0.800, and P <0.005.
TABLE 3 detection of human serum Der p1 sIgE results by ProDer p1 using Luminex liquid chip
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Sequence listing
<110> university of medical science in Guangzhou
<120> recombinant protein of house dust mite I allergen pro-Der p1, and preparation method and application thereof
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cgcccttcct ccatcaagac cttcgaggag tacaagaagg ctttcaacaa gtcctacgct 120
accttcgagg acgaggaggc tgcccgtaaa aacttcctgg agtctgtgaa gtacgtgcaa 180
tccaacggtg gtgccatcaa ccacctgtcc gacctgtccc tggacgagtt caagaaccgc 240
ttcttgatgt ccgctgaggc tttcgagcac ctgaagacac agttcgacct gaacgctgag 300
accaacgctt gttccatcaa cggtaacgct cctgccgaaa tcgacctgag acagatgcgc 360
accgtgaccc ctattcgtat gcaaggcggc tgcggctcct gctgggcttt ctctggtgtg 420
gctgctactg agtccgcata cctggcttac cgtaaccagt ccttggacct ggccgagcag 480
gagttggtgg actgcgcgtc ccagcacggt tgtcacggtg ataccatccc tcgtggtatc 540
gagtacattc agcacaacgg tgtggtgcag gagagctact accgctacgt tgcacgcgag 600
cagagttgcc gtaggccaaa cgctcagagg ttcggtattt ccaactactg ccaaatctac 660
cctcccaacg tcaacaagat cagggaggca ctggcccaga cccactccgc tatcgcagtc 720
atcattggta tcaaggacct ggacgcattc cgccactacg acggtcgcac catcatccaa 780
cgcgacaacg gctaccagcc caactaccac gctgtgaaca tcgtgggtta cagtaacgcc 840
cagggtgtgg actactggat cgtgcgcaac tcctgggaca ccaactgggg tgacaacggt 900
tacggctact tcgccgctaa catcgacctg atgatgattg aggagtaccc atacgtggtg 960
attctgggtt ctcatcatca ccaccaccac taaggtacc 999
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<210> 4
<211> 24
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
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ccggctcgta tgttgtgtgg aatt 24
<210> 5
<211> 25
<212> DNA/RNA
<213> Artificial sequence (Artificial Sequence)
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atgtggacaa aatacctggt taccc 25
Claims (6)
1. A gene encoding a recombinant protein of a room dust mite group I allergen pro-Der p1, which is characterized in that the pro-Der p1 gene sequence has an optimized insect codon and is coupled with a His protein sequence at the 3' end, and the sequence is shown as SEQ ID NO: 1.
2. Recombinant plasmid comprising the house dust mite group I allergen pro-Der p1 gene, characterized in that the pro-Der p1 recombinant gene sequence with optimized insect codon and 3' -end coupled His protein sequence is inserted into shuttle vector pFastBac based by utilizing Xho I and KpnI cleavage sites TM In the Dual modified pFastBac-SCUVI, a pro-Der p1 recombinant plasmid is obtained, and the pro-Der p1 recombinant gene sequence is shown as SEQ ID NO: 1.
3. Recombinant baculovirus plasmid comprising the recombinant plasmid of claim 2, wherein said pro-Der p1 recombinant plasmid is transformed into a DH10Bac strain to obtain a recombinant baculovirus plasmid Bacmid-Der p 1.
4. A host cell comprising the recombinant baculovirus plasmid of claim 3, wherein said recombinant baculovirus plasmid Bacmid-Der p1 is transfected into Sf9 cells.
5. A preparation method of room dust mite I allergen pro-Der P1 recombinant protein is characterized by synthesizing a pro-Der P1 gene sequence with optimized insect codon and connected with His protein gene sequence, connecting the pro-Der P1 gene sequence with a shuttle vector pFastBac-SCUVI through Xho I and KpnI enzyme cleavage sites, obtaining Bacmid rod particles through transforming DH10Bac competent cells, then transfecting Sf9 insect cells to prepare recombinant baculovirus, infecting Sf9 cells with P4 generation recombinant baculovirus, and centrifuging to collect supernatant.
6. The method for preparing a recombinant protein of the allergen pro-Der p1 of house dust mite group I according to claim 5, wherein the collected supernatant is filtered by a 0.45um filter; loading the filtrate His chromatographic column, eluting with 50mM, 100mM and 200mM imidazole respectively; running the eluent on SDS-PAGE gel to obtain a pure pro-Der p1 band with the size of about 34 kD; concentrating the eluent by using an ultrafiltration membrane bag and replacing the eluent by using PBS to obtain the purified pro-Der p1 recombinant protein resuspended by using PBS.
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