CN117004650A - Cat dander allergen component feld1 double-chain dimer recombinant protein, preparation method and application - Google Patents

Cat dander allergen component feld1 double-chain dimer recombinant protein, preparation method and application Download PDF

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CN117004650A
CN117004650A CN202310745735.6A CN202310745735A CN117004650A CN 117004650 A CN117004650 A CN 117004650A CN 202310745735 A CN202310745735 A CN 202310745735A CN 117004650 A CN117004650 A CN 117004650A
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CN117004650B (en
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黄宁
于清华
李滨雁
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Shandong Lifei Biological Industry Co ltd
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Abstract

The application relates to the technical field of recombinant proteins, and particularly discloses a cat dander allergen component feld1 double-chain dimer recombinant protein, a preparation method and application thereof. Firstly, preparing and synthesizing feld1 ch1, ch2 and Linker gene sequences, then fusing and connecting PCR, then constructing an expression vector by taking a plasmid pcDNA3.1 (+) as a vector, adding a connecting product into TOP10 competent cells for culture, collecting thalli, extracting plasmids, transfecting HEK293 cells with genes, expressing, purifying and obtaining ELISA titers. The method changes complex natural extraction into rapid, simple and stable recombinant expression, obtains target protein very similar to natural conformation, and improves the detection accuracy of allergen.

Description

Cat dander allergen component feld1 double-chain dimer recombinant protein, preparation method and application
Technical Field
The application relates to the technical field of recombinant proteins, in particular to a cat dander allergen component feld1 double-chain dimer recombinant protein, a preparation method and application.
Background
Cat dander allergen feld1 is 34kd dimer protein, which is formed by first connecting two protein chains of ch1 and ch2 in series and then polymerizing, and the protein conformation of the cat dander allergen feld1 is a double-chain dimer like a butterfly shape. Proteins exist mostly in single-or double-stranded monomeric form during recombination, however, proteins with a natural state can only stimulate allergic reactions in humans.
The current medical allergen detection raw materials are mostly extracted naturally, and target allergenic proteins are purified and separated in multiple ways and times from cat dander of multiple genera. Currently recombinant feld1 is a single-or double-stranded monomer, and is very different from the natural state, and a butterfly-shaped double-stranded dimer with biological activity is difficult to form.
Because of the species limitation of cats, the content and purity of the natural extraction target sensitized protein are extremely low, and the purification process is complicated, so that the protein is degraded and inactivated in the purification process, and the allergen detection difference is extremely large. Meanwhile, recombinant feld1 is in a single-chain or double-chain monomer form and has extremely low reaction to IgE of allergic people.
Disclosure of Invention
In order to improve the accuracy of cat dander allergen detection and reduce the difficulty of natural allergen extraction, the application provides a cat dander allergen component feld1 double-chain dimer recombinant protein, a preparation method and application.
In a first aspect, the application provides a preparation method of a double-stranded dimer recombinant protein of a cat dander allergen component feld1, which adopts the following technical scheme:
a method for preparing a double-chain dimer recombinant protein of a cat dander allergen component feld1, which comprises the following steps:
(1) Preparation of concatemer target fragments
1.1, the sequences of the feld1 ch1, ch2 and Linker genes are respectively SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3;
1.2 fusion and connection PCR to obtain a ch2-C4bp alpha-ch 1 concatemer target fragment, the sequence of which is SEQ ID NO.4;
(2) Construction of expression vectors
1.1 amplifying a target fragment of an enzyme cleavage site;
1.2pcDNA3.1 (+) plasmid double enzyme digestion, connecting target fragments;
(3) Protein expression
Adding the connection product into TOP10 competent cells for culturing, collecting thalli, and extracting plasmids;
(4) HEK293 cells are transfected by ch2-C4bp alpha-ch 1 gene, expressed and purified
(5) ELISA titers.
Preferably, the step (2) constructs an expression vector by using the plasmid pcDNA3.1 (+) as a vector.
Preferably, step (3) is added to TOP10 competent cells in an ice bath.
Preferably, the culture in the step (3) adopts LB-AG liquid medium.
Preferably, the purification in step (4) is performed by affinity chromatography using a nickel column.
In a second aspect, the application provides a double-stranded dimer recombinant protein of a cat dander allergen component feld1, which adopts the following technical scheme:
the cat dander allergen component feld1 double-chain dimer recombinant protein has the amino acid sequence as follows:
RGAVAVTAGVKMATCYDVAVANGNDSTKVNATRTAMKKDCYVNGSRVDGVMTTSSSKDCMGAVNTVDKNTGRMLGSQSISCSESGTWYPEVPRCEQEASEDLKPALTGMDAACTVAATADCCAVKRDVDTGTDYVVAYKAVVNARKNCVDAKMTDKNASDKYTSC。
preferably, the configuration is a butterfly double-stranded dimer.
In a third aspect, the application provides an application of a double-stranded dimer recombinant protein of a cat dander allergen component feld1, which adopts the following technical scheme:
the application of the cat dander allergen component feld1 double-chain dimer recombinant protein in preparing a cat allergy detection kit.
The application develops a structural framework of a polymer based on a C4 binding protein (C4 bp). C4bp is a plasma glycoprotein, which consists of 6-7 identical alpha-chains, the C-terminal residues of which are 541-597. C4bp- α contains 2 cysteine residues and a two parent α -helical region, due to its self-assembly ability and unique structure, by generating 1:1 are capable of forming a natural state double-stranded dimer conformation.
In summary, the application has the following beneficial effects:
1. the application changes the complex natural extraction of medical allergen detection raw materials into rapid, simple and stable recombinant expression, and obtains target protein which is quite similar to the natural conformation.
2. The definite allergen component detection is difficult to be achieved by natural extracts, and the detection result of the recombinant protein is more accurate and stable and is better used as the accompanying diagnosis of clinical vaccine application.
Drawings
Fig. 1: cloning the bands by molecules;
fig. 2: coating a plate result;
fig. 3: protein expression SDS-PAGE reduction electrophoresis;
fig. 4: verifying the potency;
fig. 5: a clinical detection result graph;
fig. 6: the method is a flow chart.
Detailed Description
EXAMPLE 1 calling and Synthesis of target Gene
Ch1 target Gene (accession No. NP-001041618.1) (SEQ ID NO. 1)
SEQ:ATGTTAGACGCAGCCCTCCCACCCTGCCCTACTGTTGCGGCCACAGCAGATTGTGA AATTTGCCCAGCCGTGAAGAGGGATGTTGACCTATTCCTGACGGGAACCCCCGACGAATATGTTGAGCAAGTGGCACAATACAAAGCACTACCTGTAGTATTGGAAAATGCCAGAATACTGAAGAACTGCGTTGATGCAAAAATGACAGAAGAGGATAAGGAGAATGCTCTCAGCTTGCTGGACAAAATATACACAAGTCCTCTGTGT
AA:MDAACTVAATADCCAVKRDVDTGTDYVVAYKAVVNARKNCVDAKMTDKNASDKYT SC
Ch2 target Gene (accession No. NP-001041618.1) (SEQ ID NO. 2)
SEQ:AGGGGGGCACTGCTTGTGCTGGCATTGCTGGTGACCCAAGCGCTGGGCGTCAAGA TGGCGGAAACTTGCCCCATTTTTTATGACGTCTTTTTTGCGGTGGCCAATGGAAATGAATTACTGTTGGACTTGTCCCTCACAAAAGTCAATGCTACTGAACCAGAGAGAACAGCCATGAAAAAAATCCAGGATTGCTACGTGGAGAACGGACTCATATCCAGGGTCTTGGATGGACTAGTCATGACAACCATCAGCTCCAGCAAAGATTGCATGGGTGAAGCAGTTCAGAACACCGTAGAAGATCTCAAGCTGAACACTTTGGGGAGA
AA:RGAVAVTAGVKMATCYDVAVANGNDSTKVNATRTAMKKDCYVNGSRVDGVMTTSSS KDCMGAVNTVDKNTGR
3.Linker(C4bpαGenebank NM_001419982.1 541-597AA)(SEQ ID NO.3)
DNA:ATGCTGGGCAGCCAGAGCATTAGCTGCAGCGAAAGCGGCACCTGGTATCCGGAAG TGCCG CGCTGCGAACAGGAAGCGAGCGAAGATCTGAAACCGGCGCTGACCGGC AA:MLGSQSISCSESGTWYPEVPRCEQEASEDLKPALTG
ch2-C4bp alpha-ch 1 concatemer target fragment (SEQ ID NO. 4)
AGGGGGGCACTGCTTGTGCTGGCATTGCTGGTGACCCAAGCGCTGGGCGTCAAGATGG
CGGAAACTTGCCCCATTTTTTATGACGTCTTTTTTGCGGTGGCCAATGGAAATGAATTAC
TGTTGGACTTGTCCCTCACAAAAGTCAATGCTACTGAACCAGAGAGAACAGCCATGAA
AAAAATCCAGGATTGCTACGTGGAGAACGGACTCATATCCAGGGTCTTGGATGGACTAG
TCATGACAACCATCAGCTCCAGCAAAGATTGCATGGGTGAAGCAGTTCAGAACACCGTA
GAAGATCTCAAGCTGAACACTTTGGGGAGAatgctgggcagccagagcattagctgcagcgaaagcggcacctg
gtatccggaagtgccgcgctgcgaacaggaagcgagcgaagatctgaaaccggcgctgaccggcATGTTAGACGCAGCCCT
CCCACCCTGCCCTACTGTTGCGGCCACAGCAGATTGTGAAATTTGCCCAGCCGTGAAGA
GGGATGTTGACCTATTCCTGACGGGAACCCCCGACGAATATGTTGAGCAAGTGGCACAA
TACAAAGCACTACCTGTAGTATTGGAAAATGCCAGAATACTGAAGAACTGCGTTGATGC
AAAAATGACAGAAGAGGATAAGGAGAATGCTCTCAGCTTGCTGGACAAAATATACACA
AGTCCTCTGTGT
Example 2ch2- -C4 bp. Alpha. -ch1 fusion ligation PCR
The system is configured according to the following table:
ddH 2 o was made up to a total volume of 1000 ul-100 ul/tube split, 10 tubes total, and the PCR procedure was as follows:
1% agarose gel electrophoresis, cutting gel, recovering concatemers (approximately 1200bp in size) and detecting the concentration.
Example 3 construction of expression vector Using plasmid pcDNA3.1 (+) as vector
5ug of the concatemer was taken, 5ul of SfiI was added, and Buffer CutSmart Buffer and water were added to a total volume of 200 ul. The reaction was carried out at 50℃for 5 hours, and the product was recovered using a PCR product recovery kit. Meanwhile, 10ug pcDNA3.1 (+) plasmid was taken, 8ul SfiI was added, buffer CutSmart Buffer and water were added to a total volume of 200ul, reacted for 3 hours at 50℃and cut to recover. 1.5ug of the digested pcDNA3.1 (+) plasmid and 450ng of the digested concatemer were taken and 15ul of T4 DNA ligase was added and buffer and water was added to a total volume of 150ul and ligated overnight at 16 ℃. The PCR product recovery kit was used for product recovery, eluting with 20ul water. Adding 10 mu L of purified ligation product into 100 mu L of TOP10 competent cells, and ice-bathing for 30min; LB-AG liquid medium was cultured overnight at 37℃to collect the cells and extract the plasmids.
Analysis of results
(1) The ch2-C4bp alpha-ch 1 ligation product constructs the vector, and the expression vector is successfully constructed, as shown in FIG. 1. Fig. 1: the bands were cloned molecularly. The size of the empty vector target band (5428 bp), the target fragment band (1200 bp) and the connected vector band (6628 bp) is correct, thus completing the construction of the expression vector.
(2) The monoclonal was transformed and the plating results are shown in FIG. 2. Fig. 2: the conversion TOP10 competent ligation products were plated.
EXAMPLE 4ch2-C4bp alpha-ch 1 Gene transfection, expression, purification
1. Gene transfection
The plasmid DNA extraction kit is used for extracting the ch2-C4bp alpha-ch 1 plasmid, HEK293 cells are transfected by genes, and the expression is carried out for 5 days in a serum-free culture medium shaking table. Centrifugation was performed at 5000rpm at 4℃for 10min, the supernatant (cell fermentation broth) was collected, concentrated gel was prepared, and the supernatant was subjected to SDS-PAGE electrophoresis, followed by a series of membrane transfer, blocking, antibody incubation, and the like.
2. Purification of proteins
Affinity chromatography using nickel column: washing 3-5 column volumes with ultra-pure water to wash off ethanol; washing 5-10 column volumes with equilibration buffer (20 mM phosphate buffer, 150mM NaCl,pH 7.4-8.5); the sample was loaded onto a column at a loading flow rate of 0.5ml/min. The equilibration buffer rewashes 5-10 column volumes. 8ml of elution buffer was collected by elution. The column was immediately equilibrated to neutrality with 5-10 column volumes of equilibration buffer. 1M NaCl regeneration flow wash 3-5 column volumes. Washing with pure water and 20% ethanol respectively for 3-5 column volumes. The column is kept at 4-8 ℃. The collected samples were pooled, passed through a dialysis zone, and dialyzed against Tris plasma using PBS at 4deg.C.
ELISA titers detection
Coating the purified protein with 2ug/ml 96-well ELISA plate for 24h at 4 ℃ and 100ul per well; next, 100ul per well was blocked with 1% skim milk dissolved in PBS at 4 ℃; then adding 1000PBS to continuously dilute human cat allergy or human normal serum, and incubating for 30min at 25 ℃ with 100ul per hole; followed by incubation at 25℃for 30min with addition of HRP-labeled IgE Fc at 1:1000 dilution, 100ul per well; then adding 100ul of TMB substrate solution into each hole, and reacting for 10min at room temperature in a dark place; finally, the reaction was quenched with 2M sulfuric acid and the OD at 450nm was read.
4. Analysis of results
4.1 protein expression SDS-PAGE reduction electrophoresis, see FIG. 3. Fig. 3: protein bands. The molecular weight of the predicted target protein is about 34kd, the molecular weight of the actually expressed protein after glycosylation is about 38kDa, the expressed target band is clear, and the purity is more than or equal to 95%.
4.2 potency verification, results are shown in figure 4. Fig. 4: ELISA titer validation plots. Compared with negative serum, the allergic serum has high reaction intensity and meets the immune requirement.
Example 5 clinical detection application of recombinant proteins
1. The method comprises the following steps:
after biotinylation feld1 reacts with clinical serum and fusion alkaline phosphatase recombinant anti-human IgE-Fc antibody in one step, SA magnetic beads are put into the kit, and luminescent substrates are added to read the light value after the magnetic beads are cleaned.
2. The kit comprises the following components:
1. reagent 1: biotinylated feld1
2. Reagent 2: recombinant murine anti-human IgE-Fc fusion alkaline phosphate
3. Reagent 3: SA magnetic bead
4. Reagent 4: luminous liquid
3. Full-automatic chemiluminescence instrument detection flow
3.1 placing the reagent components into a full-automatic chemiluminescence apparatus, and placing the serum sample into a sample tray.
3.2 the instrument was programmed to automatically aspirate 25ul each of reagents 1,2 and add to the reaction tube.
3.3, automatically sucking 25ul of serum sample according to a program, adding the serum sample into a 3.2 reaction tube, vibrating for 5s, and reacting for 15min.
3.4, automatically sucking the reagent 3 according to a program, adding the reagent into a 3.3 reaction tube, vibrating for 5s, and reacting for 5min.
3.5 the apparatus was programmed to automatically wash the beads in the 3.4 reaction tube.
3.6, automatically sucking the reagent 4 according to a program, adding the reagent into a 3.5 reaction tube, vibrating for 5s, and reacting for 5min.
And 3.7, automatically reading the photon number according to a program, calculating a luminescence value, and displaying the luminescence value on an operation panel.
4. Analysis of results
A total of 19 positive samples and 60 negative samples are detected, and the detection is carried out by synchronously using a commercial cat allergen detection kit, wherein kU/L is more than or equal to 0.35, and the result is shown in figure 5.
Fig. 5: and (5) a clinical detection result diagram. Compared with the commercial kit, the self-test kit has high sensitivity and more accurate detection, but both have the possibility of missing detection.
The present embodiment is only for explanation of the present application and is not to be construed as limiting the present application, and modifications to the present embodiment, which may not creatively contribute to the present application as required by those skilled in the art after reading the present specification, are all protected by patent laws within the scope of claims of the present application.

Claims (8)

1. A method for preparing a double-chain dimer recombinant protein of a cat dander allergen component feld1, which is characterized by comprising the following steps:
(1) Preparation of concatemer target fragments
a. The sequences of the feld1 ch1, the feld 2 and the Linker gene are respectively called as SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.3;
b. fusion connection PCR to obtain a ch2-C4bp alpha-ch 1 concatemer target fragment with a sequence of SEQ ID NO.4;
(2) Construction of expression vectors
a. Amplifying a target fragment of the enzyme cutting site;
b. double digestion of pcDNA3.1 (+) plasmid and connection of target fragments;
(3) Protein expression
Adding the connection product into TOP10 competent cells for culturing, collecting thalli, and extracting plasmids;
(4) Transfecting HEK293 cells with ch2-C4bp alpha-ch 1 gene, expressing and purifying;
(5) ELISA titers.
2. The method for preparing the double-stranded dimer recombinant protein of the cat dander allergen component feld1 according to claim 1, which is characterized in that: and (2) constructing an expression vector by taking the plasmid pcDNA3.1 (+) as a vector.
3. The method for preparing the double-stranded dimer recombinant protein of the cat dander allergen component feld1 according to claim 1, which is characterized in that: the step (3) was added to TOP10 competent cells, ice-bath and cultured again.
4. The method for preparing the double-stranded dimer recombinant protein of the cat dander allergen component feld1 according to claim 1, which is characterized in that: the culture in the step (3) adopts LB-AG liquid culture medium.
5. The method for preparing the double-stranded dimer recombinant protein of the cat dander allergen component feld1 according to claim 1, which is characterized in that: and (3) performing affinity chromatography by using a nickel column for purification in the step (4).
6. The cat dander allergen component feld1 double-stranded dimer recombinant protein prepared by the method of any one of claims 1-5, which has the amino acid sequence of:
RGAVAVTAGVKMATCYDVAVANGNDSTKVNATRTAMKKDCYVNGSRVDGVMTTSSSKDCMGAVNTVDKNTGRMLGSQSISCSESGTWYPEVPRCEQEASEDLKPALTGMDAACTVAATADCCAVKRDVDTGTDYVVAYKAVVNARKNCVDAKMTDKNASDKYTSC。
7. the cat dander allergen component feld1 double-stranded dimer recombinant protein according to claim 6, characterized in that: the configuration is a butterfly double-stranded dimer.
8. Use of the cat dander allergen component feld1 double-stranded dimer recombinant protein according to claim 6 for preparing a cat allergy detection kit.
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