CN109468337A - Recombinant dna fragment and its application, the antigen and its production method of diagnosing autoimmune diseases - Google Patents
Recombinant dna fragment and its application, the antigen and its production method of diagnosing autoimmune diseases Download PDFInfo
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- CN109468337A CN109468337A CN201710806344.5A CN201710806344A CN109468337A CN 109468337 A CN109468337 A CN 109468337A CN 201710806344 A CN201710806344 A CN 201710806344A CN 109468337 A CN109468337 A CN 109468337A
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/36—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Actinomyces; from Streptomyces (G)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
- C07K2319/41—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a Myc-tag
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/36—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Actinomyces; from Streptomyces (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/24—Immunology or allergic disorders
Abstract
The present invention relates to a kind of recombinant dna fragment of diagnosing autoimmune diseases and its applications, antigen and its production method.The recombinant dna fragment include autoimmune disease antigen DNA fragmentation, autoimmune disease antigen DNA fragmentation 5 ' end connection Myc and the corresponding DNA fragmentation of Histag and autoimmune disease antigen DNA fragmentation 3 ' end connection the corresponding DNA fragmentations of Streptavidin.By the antigen of autoimmune disease molecular diagnosis by way of recombination fusion protein, it takes Streptavidin, Myc and Histag label and great expression and is purified in bacterium, greatly facilitate the production of related antigen and subsequent with magnetic micro-beads coupling process, convenient for the production of liquid-phase chip, so that the diagnosis for autoimmune disease provides new technical support.
Description
Technical field
The present invention relates to medicals diagnosis on disease and therapy field, more particularly, to a kind of recombination of diagnosing autoimmune diseases
DNA fragmentation and its application, antigen and its production method.
Background technique
Autoimmune disease refers to that body occurs immune response to autoantigen and causes caused by damaged self tissue
Disease.What autoimmune disease molecule diagnosis kit generallyd use at present is immunofluorescence technique, as indirect immunofluorescence,
ELISA etc., these methods are that antigen is fixed on to solid support surface, also referred to as solid phase chip technology.Solid phase chip technology
Complex manufacturing technology, chip stability are poor, detection flux is low, poor repeatability.And liquid-phase chip technology is used, it can replace traditional
Solid phase chip detection technique, and have the characteristics that high-throughput, speed is fast, required sample size is few.
Summary of the invention
Based on this, it is necessary to provide a kind of recombinant DNA piece of diagnosing autoimmune diseases convenient for liquid-phase chip production
Section and its application, antigen and its production method.
The technical solution that the present invention solves above-mentioned technical problem is as follows.
A kind of recombinant dna fragment of diagnosing autoimmune diseases, DNA fragmentation including autoimmune disease antigen,
It holds the Myc connected and the corresponding DNA fragmentation of Histag and described in the 5 ' of the DNA fragmentation of the autoimmune disease antigen
The corresponding DNA fragmentation of Streptavidin of 3 ' end connections of the DNA fragmentation of autoimmune disease antigen.
The structure of the recombinant dna fragment is that strepto- is affine for 5 '-Myc-Histag- antigens-in one of the embodiments,
Element -3 ', wherein the sequence of the DNA fragmentation of Myc-Histag is as shown in SEQ ID No.2.
The codon of the DNA fragmentation of the autoimmune disease antigen is the password of bacterium in one of the embodiments,
Son.
In one of the embodiments, the autoimmune disease antigen include systemic loupus erythematosus antigen, drying it is comprehensive
It is simulator sickness antigen, systemic sclerosis antigen, mixed connective tissue disease antigen, primary biliary cirrhosis antigen, multiple
Myositis antigen, dermatomyositis antigen and rheumatoid arthritis antigen.
A kind of recombinant expression carrier of diagnosing autoimmune diseases, the recombinant expression carrier is interior to contain any of the above-described reality
Apply recombinant dna fragment described in example.
The recombinant expression carrier is but not limited to pET30b (+) or pBluescript in one of the embodiments,
SK(+/-)。
A kind of expression cell of diagnosing autoimmune diseases, the expression cell are interior containing described in any of the above-described embodiment
Recombinant expression carrier.
The cell is bacterial cell in one of the embodiments,.The bacterial cell in one of the embodiments,
It is BL21 Escherichia coli.
A kind of diagnosing autoimmune diseases antigen, including autoimmune disease antigen, it is connected to the autoimmunity disease
The Myc and Histag of the N-terminal of sick antigen and be connected to the autoimmune disease antigen C-terminal Streptavidin.
A kind of production method of diagnosing autoimmune diseases antigen, includes the following steps:
The DNA fragmentation of autoimmune disease antigen is synthesized, and in the corresponding DNA fragmentation of 5 ' end connection Myc and Histag, is obtained
To the recombination fusion dna segment of Myc-Histag- antigen；
The corresponding DNA fragmentation of Streptavidin is synthesized, and the corresponding DNA fragmentation of the Streptavidin is connected to Myc-
3 ' ends of the recombination fusion dna segment of Histag- antigen obtain the purpose recombination fusion dna piece of Myc-Histag- antigen-SA
Section；
Purpose recombination fusion dna segment is cloned into recombinant expression carrier, bacterium is converted；
Picking positive transformants clone carries out the inducing expression of purpose recombination fusion dna segment；
Broken thallus is collected by centrifugation the inclusion body precipitating containing recombination fusion protein, obtains Myc-Histag- itself after purification
The recombination fusion protein of immunological diseases antigen-SA.
The autoimmune disease includes systemic loupus erythematosus, Sjogren syndrome, system in one of the embodiments,
Property sclerosis, mixed connective tissue disease, primary biliary cirrhosis, polymyositis, dermatomyositis and rheumatoid joint
It is scorching.
The codon of the DNA fragmentation of the autoimmune disease antigen is the password of bacterium in one of the embodiments,
Son.
The corresponding DNA fragmentation of the Streptavidin is connected to Myc-Histag- antigen in one of the embodiments,
Fusion dna segment 3 ' end be to be connected corresponding DNA fragmentation by overlap PCR method.
The recombinant expression carrier is pET30b (+) in one of the embodiments,.
The bacterium is BL21 Escherichia coli in one of the embodiments,.
The picking positive transformants clone carries out the inducing expression of recombination fusion dna segment in one of the embodiments,
Include the following steps: that picking positive transformants are cloned, culture is added final concentration of 2mM's to OD 600 to 0.6 in LB culture medium
IPTG inducing expression, thalline were collected by centrifugation after 4 hours.
The inclusion body precipitating containing recombination fusion protein is collected by centrifugation in the broken thallus in one of the embodiments, pure
The recombination fusion protein that Myc-Histag- autoimmune disease antigen-SA is obtained after change includes the following steps:
Ultrasonic oscillation is crushed thallus, and precipitating is collected by centrifugation, and is precipitated as containing the inclusion body for having purpose recombination fusion protein；
Precipitating is dissolved using the urea that concentration is 8M, is placed 12 hours or more at 4 DEG C, centrifugation removal insoluble matter, with Ni
Affinity column mixing combines 1 hour；
In conjunction with rear dress column, using 20 times of column volumes of 8M urea washes chromatographic column of pH6.5, then urinated using the 8M of pH5.9
10 times of column volumes of plain elution chromatography column, fraction collection finally use 8M urea elution chromatography 10 times of column volumes of column of pH4.5, point
It collects in portion；
It elutes each component difference loading and carries out SDS-PAGE analysis, the component where selecting purpose recombination fusion protein carries out
Renaturation；
Adjustment protein concentration delays urea containing 2M to 0.25mg/ml after component where recombination fusion protein is merged
Fliud flushing dialysis, 4 DEG C are dialysed 12 hours, then are dialysed to the buffer of the urea containing 0.2M, and 4 DEG C are dialysed 12 hours, then are urinated containing 0.02M
The buffer dialysis of element, 4 DEG C are dialysed 12 hours, then are dialysed to not urea-containing buffer, and 4 DEG C are dialysed 12 hours；
Using the method concentration of PEG20000 water suction concentration, centrifugation goes to precipitate, and obtains purpose recombination fusion protein.
It in one of the embodiments, further include the recombination fusion dna segment for the Myc-Histag- antigen-SA that will be obtained
The step of being cloned into pBluescript SK (+/-) procaryotic clone carrier to be saved.
By the antigen of autoimmune disease molecular diagnosis by recombination fusion protein by way of, it is affine to take strepto-
Element, Myc and Histag label simultaneously great expression and purify in bacterium, greatly facilitate related antigen production and after
The continuous coupling process with magnetic micro-beads, convenient for the production of liquid-phase chip, thus for autoimmune disease diagnosis provide it is new
Technical support.
Detailed description of the invention
Fig. 1 is the flow diagram of the production method of the diagnosing autoimmune diseases antigen of an embodiment of the present invention；
Fig. 2 is the structural schematic diagram of recombination fusion protein described in Fig. 1；
Fig. 3 is the protein chip detection schematic diagram using recombination fusion protein shown in Fig. 2；
Fig. 4 is the expression and purification result schematic diagram of PCNA fusion protein antigen；PCNA fusion protein (Histag-Myc-
PCNA-SA) by the way that after IPTG (2mM) inducing expression, fusion protein forms inclusion body precipitating, molten by urea in Escherichia coli
Xie Hou is integrated on nickel (Ni) affinity column, and then by different pH value elutions, different component eluent is through SDS-
PAGE glue identification mixes the component containing destination protein, then is dehydrated and is concentrated through PEG20000.
Specific embodiment
To facilitate the understanding of the present invention, a more comprehensive description of the invention is given in the following sections with reference to the relevant attached drawings.In attached drawing
Give presently preferred embodiments of the present invention.But the invention can be realized in many different forms, however it is not limited to this paper institute
The embodiment of description.On the contrary, purpose of providing these embodiments is keeps the understanding to the disclosure more thorough
Comprehensively.
Unless otherwise defined, all technical and scientific terms used herein and belong to technical field of the invention
The normally understood meaning of technical staff is identical.Term as used herein in the specification of the present invention is intended merely to description tool
The purpose of the embodiment of body, it is not intended that in the limitation present invention.Term as used herein "and/or" includes one or more phases
Any and all combinations of the listed item of pass.
As shown in Figure 1, the production method of the autoimmune disease antigen of an embodiment, includes the following steps:
Step S110: the DNA fragmentation of synthesis autoimmune disease antigen, and it is corresponding in 5 ' end connection Myc and Histag
DNA fragmentation obtains the recombination fusion dna segment of Myc-Histag- antigen.
Autoimmune disease includes systemic loupus erythematosus, Sjogren syndrome, systemic sclerosis, Combination connective tissue
Disease, primary biliary cirrhosis, polymyositis, dermatomyositis and rheumatoid arthritis etc..Table 1 shows a variety of itself exempt from
Epidemic disease and corresponding autoantibody.
Table 1
In the present embodiment, the codon that amino acid is encoded in the DNA fragmentation of autoimmune disease antigen is bacterium
Codon, to be suitable for expressing in bacterium.The DNA fragmentation of autoimmune disease antigen is obtained by way of gene chemical synthesis
?.
The amino acid sequence of Myc is EQKLISEEDL, specifically as shown in SEQ ID No.1.The DNA sequence dna of Myc-Histag
It is specific as shown in SEQ ID No.2 for GAGCAGAAACTCATCTCTGAAGAGGATCTGCATCACCATCACCATCAC,
In, the DNA sequence dna of Histag is CATCACCATCACCATCAC.
Step S120: synthesis Streptavidin (Streptavidin, SA) corresponding DNA fragmentation, and by Streptavidin
Corresponding DNA fragmentation is connected to 3 ' ends of the recombination fusion dna segment of Myc-Histag- antigen, and it is anti-to obtain Myc-Histag-
The purpose of original-SA recombinates fusion dna segment.
In the present embodiment, described that the corresponding DNA fragmentation of Streptavidin is connected to melting for Myc-Histag- antigen
3 ' the ends for closing DNA fragmentation are but not limited to through the side overlap PCR (overlap polymerase chain reaction)
Method connects corresponding DNA fragmentation.It further, can also primer two on the outside in overlap PCR connection DNA fragmentation
End adds the restriction enzyme site of restriction enzyme, as both ends add Nde I and Xho I restriction enzyme site respectively.
Step S130: purpose recombination fusion dna segment is cloned into recombinant expression carrier, bacterium is converted.
In the present embodiment, recombinant expression carrier can be but not limited to pET30b (+).Bacterium can be but not limited to
BL21 Escherichia coli.
Step S140: picking positive transformants clone carries out the inducing expression of purpose recombination fusion dna segment.
In the present embodiment, picking positive transformants clone carries out the inducing expression of recombination fusion dna segment and includes
Following steps: picking positive transformants clone, the IPTG of final concentration of 2mM is added to OD 600 to 0.6 in culture in LB culture medium
Inducing expression, thalline were collected by centrifugation after 4 hours.
Step S150: broken thallus is collected by centrifugation the inclusion body precipitating containing recombination fusion protein, obtains SA- after purification certainly
The recombination fusion protein of body immunological diseases Myc-Histag- antigen-SA.
The structure of recombination fusion protein is as shown in Fig. 2, include the Streptavidin in the C-terminal of autoimmune disease antigen
(SA), and N-terminal Myc and Histag.
In the present embodiment, the inclusion body precipitating containing recombination fusion protein is collected by centrifugation, after purification in the broken thallus
The recombination fusion protein for obtaining Myc-Histag- autoimmune disease antigen-SA includes the following steps:
Ultrasonic oscillation is crushed thallus, and precipitating is collected by centrifugation, and is precipitated as containing the inclusion body for having purpose recombination fusion protein；
Precipitating is dissolved using the urea that concentration is 8M, is placed 12 hours or more at 4 DEG C, centrifugation removal insoluble matter, with Ni
Affinity column mixing combines 1 hour；
In conjunction with rear dress column, using 20 times of column volumes of 8M urea washes chromatographic column of pH6.5, then urinated using the 8M of pH5.9
10 times of column volumes of plain elution chromatography column, fraction collection finally use 8M urea elution chromatography 10 times of column volumes of column of pH4.5, point
It collects in portion；
It elutes each component difference loading and carries out SDS-PAGE analysis, the component where selecting purpose recombination fusion protein carries out
Renaturation；
Adjustment protein concentration delays urea containing 2M to 0.25mg/ml after component where recombination fusion protein is merged
Fliud flushing dialysis, 4 DEG C are dialysed 12 hours, then are dialysed to the buffer of the urea containing 0.2M, and 4 DEG C are dialysed 12 hours, then are urinated containing 0.02M
The buffer dialysis of element, 4 DEG C are dialysed 12 hours, then are dialysed to not urea-containing buffer, and 4 DEG C are dialysed 12 hours；
Using the method concentration of PEG20000 water suction concentration, centrifugation goes to precipitate, and obtains purpose recombination fusion protein.
Further, in the present embodiment, the production method of the autoimmune disease antigen further includes that will obtain
The recombination fusion dna segment of Myc-Histag- antigen-SA be cloned into pBluescript SK (+/-) procaryotic clone carrier with
The step of being saved.
In liquid phase protein chip production, the recombination fusion protein and magnetic micro-beads that above-mentioned production obtains can be passed through into life
Object element-Streptavidin is indirectly connected with, and correspondingly, the surface of magnetic micro-beads is keyed biotinylated bovine serum albumin by peptide
White (biotin-BSA) such as has-COOH in the surface modification of magnetic micro-beads, is connected biotin-BSA by amino coupled reagent
On magnetic micro-beads surface.Pass through between Avidin and biotinylated bovine serum albumin(BSA) in succession between antigen fragment and magnetic micro-beads
It connects.It is indirectly connected between antigen fragment and magnetic micro-beads (hereinafter referred to as magnetic bead), is directly connected to compared with absorption etc., egg can be improved
The detection sensitivity of white chip.In the detection process, after the completion of magnetic bead coating, the tested blood sample of addition is mixed with magnetic bead is incubated
It educates, so that the antigen binding of corresponding with purpose antigen antibody and magnetic bead surfaces in tested blood sample, to adsorb indirectly
In magnetic bead surfaces.The foreign protein for washing away non-specific adsorption through the various detergent that usual immunological technique uses again, in magnetic bead table
Face leaves corresponding human antibody.Finally, by magnetic bead respectively with marked containing fluorescent material secondary antibody (such as anti-human igg Fc-PE and
Anti-human IgM Fc-PE) mixing is incubated for, so that the secondary antibody of fluorescent material label is in conjunction with the antibody of magnetic bead surfaces.Streaming can be used
The method detection of sorting is visited from above-mentioned different antigen reactive serum antibodies, flow sorter green light source (such as 532nm light source)
The serum antibody with antigen binding is surveyed, can be quantified by the fluorescence intensity of secondary antibody in conjunction with the serum antibody of antigen.Single antigen
In conjunction with serum antibody whether be it is positive, according to the reading of fluorescent quantitation, song that serum antibody extension rate and standard items are drawn
Line is judged.After determining whether single antigen is the positive, the corresponding IgG and IgM autoantibody of Conjoint Analysis antigen as a result,
It is compared again with corresponding judgment criteria, all can be judged as greater than required standard value person has correspondingly autoimmune disease
Possibility, then synthesis combine other testing results that can finally be judged as whether there is autoimmune disease.
By the antigen of autoimmune disease molecular diagnosis by recombination fusion protein by way of, it is affine to take strepto-
Element, Myc and Histag label simultaneously great expression and purify in bacterium, greatly facilitate related antigen production and after
The continuous coupling process with magnetic micro-beads, convenient for the production of liquid phase protein chip, so that the diagnosis for autoimmune disease provides
New technical support.
Below the recombination fusion protein mainly in combination with the proliferating cell nuclear antigen (PCNA) containing systemic loupus erythematosus and
Its production process is specifically described.
The production method of Myc-Histag-PCNA-SA recombination fusion protein is as follows:
The amino acid sequence of 1.PCNA is MFEGRLVQGSILKKVLEALKDLINEACWDISSSGVNLQSMDSSHVSLVQ
LTLRSEGFDTYRCDRNLAMGVNLTSMSKILKCAGNEDIITLRAEDNADTLALVFEAPNQEKVSDYEMKLMDLDVEQL
GIPEQEYSCVVKMPSGEFARICRDLSHIGDAVVISCAKDGVKFSASGELGNGNIKLSQTSNVDKEEEAVTIEMNEPV
QLTFALRYLNFFTKATPLSSTVTLSMSADVPLVVEYKIADMGHLKYNLAPKIEDEE GS, it is specific such as SEQ ID No.3
It is shown.
The sequence of the DNA fragmentation of the coding PCNA of selection are as follows: ATGTTTGAGGGTCGTCTTGTTCAGGGTTCTATTCTTA
AAAAAGTTCTTGAGGCTCTTAAAGATCTTATTAATGAGGCTTGCTGGGATATTTCTTCTTCTGGTGTTAATCTTCAG
TCTATGGATTCTTCTCATGTTTCTCTTGTTCAGCTTACTCTTCGTTCTGAGGGTTTTGATACTTATCGTTGCGATCG
TAATCTTGCTATGGGTGTTAATCTTACTTCTATGTCTAAAATTCTTAAATGCGCTGGTAATGAGGATATTATTACTC
TTCGTGCTGAGGATAATGCTGATACTCTTGCTCTTGTTTTTGAGGCTCCTAATCAGGAGAAAGTTTCTGATTATGAG
ATGAAACTTATGGATCTTGATGTTGAGCAGCTTGGTATTCCTGAGCAGGAGTATTCTTGCGTTGTTAAAATGCCTTC
TGGTGAGTTTGCTCGTATTTGCCGTGATCTTTCTCATATTGGTGATGCTGTTGTTATTTCTTGCGCTAAAGATGGTG
TTAAATTTTCTGCTTCTGGTGAGCTTGGTAATGGTAATATTAAACTTTCTCAGACTTCTAATGTTGATAAAGAGGAG
GAGGCTGTTACTATTGAGATGAATGAGCCTGTTCAGCTTACTTTTGCTCTTCGTTATCTTAATTTTTTTACTAAAGC
TACTCCTCTTTCTTCTACTGTTACTCTTTCTATGTCTGCTGATGTTCCTCTTGTTGTTGAGTATAAAATTGCTGATA
TGGGTCATCTTAAATATAATCTTGCTCCTAAAATTGAGGATGAGGAGGGTTCT, specifically as shown in SEQ ID No.4.
2. in the recombinant dna fragment of Hua Da gene chemical synthesis Myc-Histag-PCNA.
3. the DNA fragmentation of Streptavidin to be connected to the recombination of Myc-Histag-PCNA by overlap PCR method
3 ' ends of DNA fragmentation, constitute the recombinant dna fragment of Myc-Histag-PCNA-SA.
Primer sequence used in specific overlap PCR is as follows:
1#: 5 'CATATGATGGAACAAAAACTGATTTC3 ' (underscore is NdeI sequence) (SEQ ID.5)
2#: 5 'CTCGAGTCACTGCTGTACCGCGTCCAGCGG3 ' (underscore is XhoI sequence) (SEQ ID.6)
3#: 5 ' GATGAGGAGGGTTCTGGCGATCCGTCCAAGGA3 ' (SEQ ID.7)
4#: 5 ' TCCTTGGACGGATCGCCAGAACCCTCCTCATC3 ' (SEQ ID.8)
Overlap PCR system is as shown in table 2 below.
Table 2
The reaction step of overlap PCR:
1) 95 DEG C, 2min；
2)95℃,45Sec；
3)50℃,30Sec；
4)72℃,1min；
5) step 2), 30 circulations are returned to；
6)72℃,10min。
Invitrogen sequencing company is sent to be sequenced, sequencing sequence is consistent with building sequence, as a result accurately and reliably.
4. the recombinant dna fragment of Myc-Histag-PCNA-SA is existed using Nde I and Xho I enzyme clone
In pBluescript SK (+/-) procaryotic clone carrier (source: Stratagen company)；
NdeI and Xho I double digestion recombinant expression carrier pET30b (+) (source Novagen company) recycles double digestion piece
Section is spare；
PBluescript SK (+/-) carrier of Nde I and Xho I double digestion containing each antigen fragment gene recycles antigen
Corresponding double digestion segment, is cloned into respectively in pET30b (+) expression vector of recycling；
Nde I and Xho I double digestion screening recombinant clone is respectively adopted again, send endonuclease bamhi to Ying Jieji
(Invitrogen) sequencing company is sequenced, verified, and sequencing sequence is consistent with building sequence, as a result accurately；
Using alkaline lysis large quantity extracting plasmid, BL21 Escherichia coli are converted with the DNA of 0.1 μ g；
Picking transformed clone, the IPTG induction of final concentration of 2mM is added to OD 600 about 0.6 in culture in LB culture medium
Thallus is harvested by centrifugation in expression after 4 hours；
Ultrasonic oscillation is crushed thallus, and precipitating is collected by centrifugation, and is precipitated as the inclusion body containing purpose antigen albumen；
8M urea dissolution precipitating, 4 DEG C are placed 12 hours or more, and centrifugation removal insoluble matter mixes knot with Ni affinity column
It closes 1 hour；
In conjunction with rear dress column, using 20 times of column volumes of 8M urea washes chromatographic column of pH 6.5, the 8M of pH 5.9 is then used
10 times of column volumes of urea elution chromatography column, fraction collection finally use 8M urea elution chromatography 10 times of column volumes of column of pH 4.5,
Fraction collection；
It elutes each component difference loading and carries out SDS-PAGE analysis, the component where selecting destination protein carries out renaturation.
Adjustment protein concentration is to 0.25mg/ml after component where destination protein is merged, to the buffer of the urea containing 2M
Dialysis, 4 DEG C are dialysed 12 hours, then are dialysed to the buffer of the urea containing 0.2M, and 4 DEG C are dialysed 12 hours, then to the urea containing 0.02M
Buffer dialysis, 4 DEG C are dialysed 12 hours, then are dialysed to not urea-containing buffer, and 4 DEG C are dialysed 12 hours；
Each antigen is concentrated to ultimate density as 1mg/ml in the method for using PEG20000 water suction concentration；Centrifugation goes to precipitate, and surveys
Determine protein content, obtains purpose recombination fusion protein, as a result as shown in Figure 4.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>Guangzhou Dan Lan Biotechnology Co., Ltd
<120>recombinant dna fragment and its application, the antigen and its production method of diagnosing autoimmune diseases
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cgttgcgatc gtaatcttgc tatgggtgtt aatcttactt ctatgtctaa aattcttaaa 240
tgcgctggta atgaggatat tattactctt cgtgctgagg ataatgctga tactcttgct 300
cttgtttttg aggctcctaa tcaggagaaa gtttctgatt atgagatgaa acttatggat 360
cttgatgttg agcagcttgg tattcctgag caggagtatt cttgcgttgt taaaatgcct 420
tctggtgagt ttgctcgtat ttgccgtgat ctttctcata ttggtgatgc tgttgttatt 480
tcttgcgcta aagatggtgt taaattttct gcttctggtg agcttggtaa tggtaatatt 540
aaactttctc agacttctaa tgttgataaa gaggaggagg ctgttactat tgagatgaat 600
gagcctgttc agcttacttt tgctcttcgt tatcttaatt tttttactaa agctactcct 660
ctttcttcta ctgttactct ttctatgtct gctgatgttc ctcttgttgt tgagtataaa 720
attgctgata tgggtcatct taaatataat cttgctccta aaattgagga tgaggagggt 780
tct 783
<210> 5
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
catatgatgg aacaaaaact gatttc 26
<210> 6
<211> 30
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ctcgagtcac tgctgtaccg cgtccagcgg 30
<210> 7
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
gatgaggagg gttctggcga tccgtccaag ga 32
<210> 8
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
tccttggacg gatcgccaga accctcctca tc 32
Claims (10)
1. a kind of recombinant dna fragment of diagnosing autoimmune diseases, which is characterized in that including autoimmune disease antigen
DNA fragmentation, the autoimmune disease antigen DNA fragmentation 5 ' end connection Myc and the corresponding DNA fragmentation of Histag,
And the corresponding DNA fragmentation of Streptavidin that 3 ' ends of the DNA fragmentation in the autoimmune disease antigen connect.
2. the recombinant dna fragment of diagnosing autoimmune diseases as described in claim 1, which is characterized in that the recombinant DNA
The structure of segment is 5 '-Myc-Histag- antigens-Streptavidin -3 ', wherein the sequence of the DNA fragmentation of Myc-Histag is such as
Shown in SEQ ID No.2.
3. the recombinant dna fragment of diagnosing autoimmune diseases as claimed in claim 1 or 2, which is characterized in that it is described itself
The codon of the DNA fragmentation of immunological diseases antigen is the codon of bacterium.
4. the recombinant dna fragment of diagnosing autoimmune diseases as claimed in claim 1 or 2, which is characterized in that it is described itself
Immunological diseases antigen includes systemic loupus erythematosus antigen, Sjogren syndrome antigen, systemic sclerosis antigen, Combination connective
Organize sick antigen, primary biliary cirrhosis antigen, polymyositis antigen, dermatomyositis antigen and rheumatoid arthritis anti-
It is former.
5. a kind of recombinant expression carrier of diagnosing autoimmune diseases, which is characterized in that contain in the recombinant expression carrier
Recombinant dna fragment as described in any one of claims 1 to 4.
6. the recombinant expression carrier of diagnosing autoimmune diseases as claimed in claim 5, which is characterized in that the recombination table
PET30b (+) or pBluescript SK (+/-) are but not limited to up to carrier.
7. a kind of expression cell of diagnosing autoimmune diseases, which is characterized in that the expression cell is included to be wanted just like right
Recombinant expression carrier described in asking 5 or 6.
8. the expression cell of diagnosing autoimmune diseases as claimed in claim 7, which is characterized in that the cell is bacterium
Cell.
9. a kind of diagnosing autoimmune diseases antigen, which is characterized in that including autoimmune disease antigen, be connected to it is described from
The Myc and Histag of the N-terminal of body immunological diseases antigen and be connected to the autoimmune disease antigen C-terminal strepto- parent
And element.
10. a kind of production method of diagnosing autoimmune diseases antigen, which comprises the steps of:
The DNA fragmentation of autoimmune disease antigen is synthesized, and in the corresponding DNA fragmentation of 5 ' end connection Myc and Histag, is obtained
The recombination fusion dna segment of Myc-Histag- antigen；
The corresponding DNA fragmentation of Streptavidin is synthesized, and the corresponding DNA fragmentation of the Streptavidin is connected to Myc-
3 ' ends of the recombination fusion dna segment of Histag- antigen obtain the purpose recombination fusion dna piece of Myc-Histag- antigen-SA
Section；
Purpose recombination fusion dna segment is cloned into recombinant expression carrier, bacterium is converted；
Picking positive transformants clone carries out the inducing expression of purpose recombination fusion dna segment；
Broken thallus is collected by centrifugation the inclusion body precipitating containing recombination fusion protein, obtains Myc-Histag- autoimmunity after purification
The recombination fusion protein of disease antigen-SA.
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| CN201710806344.5A CN109468337A (en) | 2017-09-08 | 2017-09-08 | Recombinant dna fragment and its application, the antigen and its production method of diagnosing autoimmune diseases |
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Citations (4)
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|---|---|---|---|---|
| CN101378783A (en) * | 2005-12-08 | 2009-03-04 | 路易斯维尔大学研究基金会有限公司 | Methods and compositions for expanding T regulatory cells |
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| WO2017112784A1 (en) * | 2015-12-22 | 2017-06-29 | The Trustees Of The University Of Pennsylvania | Spycatcher and spytag: universal immune receptors for t cells |
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| CN101378783A (en) * | 2005-12-08 | 2009-03-04 | 路易斯维尔大学研究基金会有限公司 | Methods and compositions for expanding T regulatory cells |
| CN103204921A (en) * | 2012-01-17 | 2013-07-17 | 北京大学 | Secretory protein with chemotactic activity, and coding sequence and application thereof |
| CN103869086A (en) * | 2014-04-14 | 2014-06-18 | 杭州凯保罗生物科技有限公司 | Serum autoantibody detection kit |
| WO2017112784A1 (en) * | 2015-12-22 | 2017-06-29 | The Trustees Of The University Of Pennsylvania | Spycatcher and spytag: universal immune receptors for t cells |
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