CN107236027A - Echinococcus granulosus TSP1 recombinant proteins and its solubility expression method and purification process - Google Patents
Echinococcus granulosus TSP1 recombinant proteins and its solubility expression method and purification process Download PDFInfo
- Publication number
- CN107236027A CN107236027A CN201710393428.0A CN201710393428A CN107236027A CN 107236027 A CN107236027 A CN 107236027A CN 201710393428 A CN201710393428 A CN 201710393428A CN 107236027 A CN107236027 A CN 107236027A
- Authority
- CN
- China
- Prior art keywords
- tsp1
- echinococcus granulosus
- recombinant proteins
- tris
- post
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43536—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms
- C07K14/4355—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes
- C07K14/43554—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from worms from cestodes from Taenia
-
- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
-
- 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
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/43504—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates
- G01N2333/43526—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms
- G01N2333/43539—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from cestodes
- G01N2333/43543—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans from invertebrates from worms from cestodes from Taenia
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Microbiology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Immunology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- Analytical Chemistry (AREA)
- Plant Pathology (AREA)
- Pathology (AREA)
- General Physics & Mathematics (AREA)
- Food Science & Technology (AREA)
- Cell Biology (AREA)
- Toxicology (AREA)
- Gastroenterology & Hepatology (AREA)
- Peptides Or Proteins (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The present invention provides Echinococcus granulosus TSP1 recombinant proteins, and the amino acid sequence of the Echinococcus granulosus TSP1 recombinant proteins is sequence table SEQ ID No.2.Present invention also offers a kind of solubility expression method, the solution expression with high efficiency of TSP1 recombinant proteins is realized:The recombinant protein of expression is largely soluble protein, accounts for bacterium coli solubility total protein 90%;Then Ni NTA His Bind are used(Clontech)Prepacked column purification of Recombinant Echinococcus granulosus TSP1, obtains TSP1 purifying proteins.Using elution buffer(500mM/L imidazoles, 20mM/L Tris, 500mM/L NaCI, PH8.0)When can obtain the Echinococcus granulosus TSP1 of high-purity a kind of.Using the purifying protein as envelope antigen, a kind of ELISA kit for being used to detect echinococcosis granulosa can be prepared.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to Echinococcus granulosus TSP1 recombinant proteins and its solubility
Expression and purification process.
Background technology
Transmembrane protein 1(TSP1)It is an antigen gene in Echinococcus granulosus Schistosoma japonicum.TSP1 is a kind of
Important Echinococcus granulosus antigen, may take part in Echinococcus granulosus signal transduction path, growth, differentiation for cell
With important physiological significance.TSP1 is probably homologous gene with annexin family (Annexin family) member, is belonged to
Annexin families.The physiological function of the antigen is studied, not only it will be seen that it is for the parasitic of Echinococcus Granulosus Cysts, growth, hair
The significance in vital movement such as educate, new drug target and candidate vaccine can also be provided to treat and prevent echinococcosis.
Based on above research background, the present invention constructs the prokaryotic expression plasmid of coding Echinococcus granulosus TSP1 genes, is transformed into big
Enterobacteria expression system, induces its solubility expression, and carries out protein purification, has obtained the albumen, is its immunogene from now on
Property, Analysis of Biochemical Characteristics and functional study are laid a good foundation.
The content of the invention
There is provided a kind of Echinococcus granulosus TSP1 prokaryotic expression carrier by technique for gene engineering by the present invention
PET30a- TSP1, Escherichia coli are converted using the carrier(BL21-DE3)Realize TSP1 high-level solubility expression.And
There is provided a kind of affinity chromatographic purification process, the restructuring TSP1 of substantial amounts of high-purity is purified into, for TSP1 immunogenicities, life
Change specificity analysis and functional study, anti-Echinococcus hydatid cyst vaccine, the research and development of anti-hydatid drugs and the immunodiagnosis of cystic echinococcosis patient.
The present invention provides Echinococcus granulosus TSP1 recombinant proteins, the amino of the Echinococcus granulosus TSP1 recombinant proteins
Acid sequence is sequence table SEQ ID No.2.
Preferably, the nucleotides sequence of the Echinococcus granulosus TSP1 recombinant proteins coding is classified as sequence table SEQ ID
27th -836 bit bases in No.1.
The present invention also provides the solubility expression of above-mentioned Echinococcus granulosus TSP1 recombinant proteins, and step is as follows:
(1)Target gene TSP1 amplification;
(2)Build TSP1 expression plasmids:By step(1)Target gene TSP1 digestions of preparation and after purification, are built into pET-30a
Corresponding polyclone enzyme enzyme site, builds plasmid pET30a-TSP1;
(3)By step(2)The plasmid pET30a-TSP1 of preparation is transformed intoE.Coli, will in BL21 (DE3) competent cellE.ColiBL21 (DE3)-pET30a-TSP1 is enlarged culture, then adds 0.2 mmol/L IPTG in 25 DEG C, 120
R/min inductions shaken cultivation 8 hours;Ultrasound after centrifugation is resuspended, collects supernatant.
Preferably, step(3)Described in expand culture be byE.ColiBL21 (DE3)-pET30a-TSP1 is first inoculated with
To LB solid mediums, 37 DEG C of overnight incubations;Then monoclonal is into LB fluid nutrient mediums on picking culture medium, in 37 DEG C,
180r/min shaken cultivations 3 hours, make OD values to 0.5;Bacterium solution is finally taken to be seeded in LB fluid nutrient mediums, in 37 DEG C, 180r/
Min shaken cultivations, make OD values to 0.5.
The present invention also provides the purification process of above-mentioned Echinococcus granulosus TSP1 recombinant proteins, is to use Ni-NTA His-
Bind(Clontech)Purification system purification of recombinant proteins, is then eluted using elution buffer;The elution buffer
Formula be:500mM/L imidazoles, 20mM/L Tris, 500mM/L NaCl, pH8.0.
Preferably, step is as follows:
(1)Ultra-pure water is added in Ni-NTA His-Bind prepacked columns, the medium for making it slowly flow through filling in post;
(2)Add combination buffer;The formula of the combination buffer is:20mM/L Tris, 500mM/L NaCl, 20mM/L
Imidazoles, pH8.0;
(3)Loading, adds ultrasonicationE.ColiMake supernatant slow after the supernatant that BL21 (DE3)-TSP1 is extracted, standing
Flow through medium in post;
(4)Combination buffer is added, it is slowly flowed across medium in post;The formula of the combination buffer is:20mM/L
Tris, 500mM/L NaCl, 20mM/L imidazoles, pH8.0;
(5)No. 1 eluent is added, it is slowly flowed across medium in post, the formula of No. 1 eluent is:100 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(6)No. 2 eluents are added, it is slowly flowed across medium in post, the formula of No. 2 eluents is:200 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(7)No. 3 eluents are added, it is slowly flowed across medium in post, the formula of No. 3 eluents is:300 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(8)No. 4 eluents are added, it is slowly flowed across medium in post, the formula of No. 4 eluents is:400 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(9)No. 5 elution buffers are added, it is slowly flowed across medium in post, eluent is collected;No. 5 elution buffers
It is formulated and is:500mM/L imidazoles, 20mM/L Tris, 500mM/L NaCl, pH8.0.
Preferably, the ultrasonicationE.ColiSupernatant and elution buffer that BL21 (DE3)-TSP1 is extracted
Volume ratio is 1:1.
The present invention also provides above-mentioned Echinococcus granulosus TSP1 recombinant proteins and is preparing the ELISA of detection echinococcosis granulosa
Application in kit.
The present invention, which also provides to be coated with a kind of ELISA kit for detecting echinococcosis granulosa, the ELISA kit, to be resisted
Originally it was the Echinococcus granulosus TSP1 recombinant proteins described in claim 1 or 2.
Preferably, the envelope antigen is diluted to the μ g/ of final concentration 8 with pH 9.6 0.05M/L carbonate buffer solutions
ml;
Preferably, the kit also includes confining liquid, the confining liquid is the TBST containing 5% skimmed milk power, the percentage
The unit of ratio is g/ml.
The invention provides a kind of new Echinococcus granulosus transmembrane protein 1(TSP1)Gene and Echinococcus granulosus TSP1
The protein of coded by said gene, and there is provided the prokaryotic expression carrier pET30a- TSP1 of Echinococcus granulosus TSP1 a kind of.Should
Carrier contains T7 promoters and terminator, bacterial ribosome binding site and TSP1 genes and one histidine-tagged, from particulate
TSP1 genes are cloned in echinococcus Schistosoma japonicum, its expression in Escherichia coli is controlled with T7 promoters, are utilized
Carrier conversion Escherichia coli (BL21-DE3), and under the conditions of lower temperature, relatively low inducer concentrations and shorter induction time
The bacterium is induced, TSP1 solution expression with high efficiency can be achieved:The recombinant protein of expression is largely soluble protein, accounts for large intestine
Bacillus total soluble protein 90%.Then with Ni-NTA superflow affinitive layer purifications restructuring Echinococcus granulosus TSP1, obtain
To TSP1 purifying proteins.Using elution buffer(500mM/L imidazoles, 20mM/L Tris, 500mM/L NaCI, PH8.0)
A kind of Echinococcus granulosus TSP1 of new high-purity is can obtain during 10ml.TSP1 expression of recombinant proteins amount is high, and specific
It is in solubility expression under inductive condition, the operation for purifying the albumen is comparatively simple, and cost is also very low, easily reuses.This hair
Bright prepared recombinant protein can be used for the immunodiagnosis of cystic echinococcosis patient, and it can be by bladder type bag as immunizing antigen
Parasitosis patients serum recognizes, when being detected applied to indirect ELISA, possesses higher specificity and sensitivity, and clinical detection meets
Rate is up to 90%.
Brief description of the drawings
Accompanying drawing is used for providing a further understanding of the present invention, and constitutes a part for specification, the reality with the present invention
Applying example is used to explain the present invention together, is not construed as limiting the invention.In the accompanying drawings:
Fig. 1 is Echinococcus granulosus TSP1 gene PCR amplified production electrophoresis results;
Fig. 2 is Echinococcus granulosus TSP1 prokaryotic expression;
Fig. 3 is Echinococcus granulosus TSP1 affinitive layer purification results.
Embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, is conventional method unless otherwise specified.Test material used, is city unless otherwise specified in following embodiments
Sell.
The protein sequence of the Echinococcus granulosus TSP1 gene orders of embodiment one and its coding
1047 nucleotides of Echinococcus granulosus TSP1 total lengths, maximum ORFs(ORF)Positioned at 27-836, contain
810bp, initiation codon is atg, and terminator codon is tga, encodes the ammonia of 269 amino acid, its nucleotide sequence and coding
Base acid sequence is shown in sequence table SEQ ID No.1 and 2 respectively.
Nucleotide sequence:
Amino acid sequence:
The Echinococcus granulosus TSP1 of embodiment two prokaryotic expression carrier pET30a-TSP1 clone builds
1. target gene TSP1 amplification
With the SK-TSP1 of Echinococcus granulosus pBluescript II(The SK-TSP1 of Echinococcus granulosus pBluescript II are from sea
Southern medical college's parasitology teaching and research room obtains, and the public can also obtain from the teaching and research room)For template, according to particulate spine ball silk ribbon
Worm TSP1 gene orders design following two pairs of primers:
Upstream primer sequence is:CGCGATATCATGACCACTGG, containsEcoRV restriction enzyme sites,
Downstream primer sequence is:GAGTCGACTCATATTGGGGAAGC, containsSalI restriction enzyme sites,
Expand the maximum ORFs of TSP1 genes(ORF)In gene order(That is 27-836 gene order), PCR reactions
Condition:98 DEG C of pre-degeneration 5min;96 DEG C of denaturation 35s, 65 DEG C of renaturation 35s, 72 DEG C of extension 45s, 35 circulations;72 DEG C of extensions
10min.Amplified production electrophoresis result is shown in Fig. 1.
Fig. 1 is Echinococcus granulosus TSP1 gene PCR amplified production electrophoresis results;Wherein, M, Marker III;1, TSP1
Pcr amplification product.
2. build TSP1 expression plasmids
After PCR primer is purified, amplification obtains fragment quiltEcoRV andSalI digestions and after purification, are built into pET-30a corresponding
Polyclone enzyme enzyme site, build plasmid pET30a-TSP1, through sequencing identification, TSP1 sequence information and target gene sequence
Unanimously:Nucleotides sequence is classified as in sequence table SEQ ID No.1 27-836, encodes 269 amino acid, the amino acid sequence of coding
It is classified as sequence table SEQ ID No.2.
The Echinococcus granulosus TSP1 of embodiment three prokaryotic expression
Under different inductive conditions, the amount of soluble expression of recombinant protein is different, is below optimal abductive approach:
PrepareE.ColiBL21 (DE3) competent cell, the plasmid pET30a-TSP1 that embodiment 2 is prepared is transformed intoE.ColiIn BL21 (DE3) competent cell, streak inoculationE.ColiBL21 (DE3)-pET30a-TSP1 to LB solids train
Support on base, 37 DEG C of overnight incubations;Monoclonal is into 5ml LB fluid nutrient mediums on picking culture medium, and in 37 DEG C, 180r/min shakes
Culture 3 hours is swung, makes OD values to 0.5;1ml bacterium solutions are taken to be seeded in 200ml LB fluid nutrient mediums, in 37 DEG C, 180r/min
Shaken cultivation, makes OD values to 0.5;Add IPTG(0.2 mmol/L)In 25 DEG C, 120 r/min inductions shaken cultivation 8 hours;4
DEG C 12000g/min, centrifugation 10min collects bacterium, is resuspended in 40ml combination buffers(20mM/L Tris, 500mM/L
NaCl, 20mM/L imidazoles, pH8.0);Supernatant is extracted after ultrasonication bacterium, power 200W, ultrasonic 8S, interval 8S, 25mins,
And carrying out SDS-PAGE electrophoresis, it is seen that albumen expression quantity in supernatant is very high, in solubility expression(See Fig. 2).Recombinant protein
Matter is largely soluble protein, accounts for bacterium coli solubility total protein 90%.
In order to search out optimal inductive condition, applicant carried out many experiments, Fig. 2 is Echinococcus granulosus TSP1 not
Prokaryotic expression under the conditions of isogeneous induction.
Wherein, in A figures, M:Marker;1-4, BL21(DE3)- pET30a- TSP1 are in 37 DEG C, and IPTG concentration is respectively
1st, the electrophoretogram of supernatant after 0.8,0.5,0.2 mmol/L, 18 h of induction;5-8, BL21(DE3)- pET30a- TSP1 are in 30
DEG C, IPTG concentration is respectively 1,0.8,0.5,0.2 mmol/L, the electrophoretogram of supernatant after 12 h of induction;
In B figures, M:Marker;1-3, BL21(DE3)- pET30a- TSP1 are in 25 DEG C, and IPTG is respectively 0.5,0.2,0.1
The electrophoretogram of supernatant after mmol/L, induction 8h.
As seen from Figure 2, it is 37 DEG C in inducing temperature, IPTG is respectively 1,0.8,0.5,0.2 mmol/L, induces 18 h
Afterwards, the extremely low TSP1 in solubility expression of visible expression quantity in supernatant;Inducing temperature is down to 30 DEG C, IPTG is respectively
1st, 0.8,0.5,0.2 mmol/L, induces 12 h, the visible TSP1 in solubility expression in supernatant, its expression quantity is compared with 37 DEG C
When increased;Further reduction inducing temperature is to 25 DEG C, and IPTG concentration is respectively 0.5,0.2,0.1 mmol/L, induces 8 h
Afterwards, can make the expression quantity of TSP1 in supernatant increases, and IPTG concentration is 0.2 mmol/L, and soluble T SP1 expression quantity is most
It is many, thus using this condition as TSP1 protokaryon induced expressions optimal conditions.
Example IV Echinococcus granulosus TSP1 affinitive layer purification
Using Ni-NTA His-Bind(Clontech)Purification system purifies destination protein.Ni-NTA His-Bind prepacked columns are purchased
From Novagen companies of the U.S., model:70691-3.
When various materials are added dropwise, rate of addition is 1 drop/10 seconds.
The purification effect difference for the recombinant protein that different purification process is obtained to purification process, it is necessary to optimize.With
It is part optimization process down:
20ml ultra-pure waters are added in Ni-NTA His-bind prepacked columns, the medium for making it slowly flow through filling in post;Add
20ml combination buffers(50mM/L NaH2PO4, 300mM/L NaCl, pH8.0), Jie for making it slowly flow through filling in post
Matter;Loading, adds 4ml and passes through ultrasonicationE.ColiThe supernatant that BL21 (DE3)-pET30a- TSP1 are extracted, controlling stream
Speed makes supernatant slowly flow across in post medium and collect;Add 40ml wash buffers(50mM/L NaH2PO4, 300mM/L
NaCl, 20mM/L imidazoles, pH8.0), coutroi velocity makes it slowly flow across medium in post, to elute foreign protein;Add 50mM/L
Imidazole elution(50 mM/L imidazoles, 50mM/L NaH2PO4, 300mM/L NaCl, pH8.0)2ml, makes it slowly flow across in post
Medium is simultaneously collected;Add 100mM/L imidazole elutions(100 mM/L imidazoles, 50mM/L NaH2PO4, 300mM/L NaCl,
pH8.0)2ml, makes it slowly flow across in post medium and collect;Add 200 mM/L imidazole elutions(200mM/L imidazoles,
50mM/L NaH2PO4, 300mM/L NaCl, pH8.0)2ml, makes it slowly flow across in post medium and collect;Add 300mM/L
Imidazole elution(300 mM/L imidazoles, 50mM/L NaH2PO4, 300mM/L NaCl, pH8.0)2ml, makes it slowly flow across post
Interior medium is simultaneously collected;Add 400 mM/L imidazole elutions(400 mM/L imidazoles, 50mM/L NaH2PO4, 300mM/L
NaCl, pH8.0)2ml, makes it slowly flow across in post medium and collect.The eluent of collection is subjected to SDS-PAGE analyses respectively,
The degree of purification of albumen is checked, the A figures seen in Fig. 3.
Fig. 3 is Echinococcus granulosus TSP1 affinitive layer purification results.
Wherein, in A figures, M:Marker;1-2, BL21(DE3)- pET30a- TSP1 supernatants are as former state;3, albumen loading mistake
Liquid is flowed through after post;4,50mM/L imidazole elutions;5,100mM/L imidazole elutions;6,200mM/L imidazole elutions;7,
300mM/L imidazole elutions;8,400mM/L imidazole elutions.
From the A figures in Fig. 3:Respectively with 50 mM/L, 100 mM/L, 200 mM/L, 300 mM/L, 400 mM/L miaows
Azoles eluent can elute destination protein, and the destination protein amount under the elution of especially 200 mM/L imidazole elutions is most
It is many, but purity of protein is not high, foreign protein content is very high, therefore further optimization protein purification condition, is carried out using following methods
Purifying.
20ml ultra-pure waters are added in Ni-NTA His-Bind prepacked columns, the medium for making it slowly flow through filling in post
With scouring media;Add 20ml Tris- combination buffers(20 mM/L Tris, 500 mM/L NaCl, 20 mM/L imidazoles,
pH8.0)With balance media;Loading, adds 10ml and passes through ultrasonicationE.ColiThe supernatant that BL21 (DE3)-TSP1 is extracted,
Coutroi velocity makes supernatant slowly flow across medium in post;Add 40ml Tris- combination buffers(20 mM/L Tris, 500
MM/L NaCl, 20 mM/L imidazoles, pH8.0), coutroi velocity makes it slowly flow across in post medium to elute foreign protein;Add 1
Number eluent(100 mM/L imidazoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0)2 ml, make it slowly flow across in post
Medium is simultaneously collected;Add No. 2 eluents(200 mM/L imidazoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0)2 ml,
It is set to slowly flow across in post medium and collect;Add No. 3 eluents(300 mM/L imidazoles, 20 mM/L Tris, 500 mM/L
NaCl, pH8.0)2 ml, make it slowly flow across in post medium and collect;Add No. 4 eluents(400 mM/L imidazoles, 20 mM/
L Tris, 500 mM/L NaCl, pH8.0)2 ml, make it slowly flow across in post medium and collect;Add No. 5 eluents(500
MM/L imidazoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0)2 ml, make it slowly flow across in post medium and collect;Will
The eluent of collection carries out SDS-PAGE analyses respectively, checks the degree of purification of albumen.It can be seen that being obtained when with No. 5 elutions
To purity very high TSP1(See Fig. 3 B), the molecular weight of albumen is about 29.7KD.The B figures that SDS-PAGE results are shown in Fig. 3.
In Fig. 3 B figures, M:Marker ;1-2, BL21(DE3)- pET30a- TSP1 supernatants are as former state;3, albumen loading
Cross and liquid is flowed through after post;4, No. 1 eluents;5, No. 2 eluents;6, No. 3 eluents;7, No. 4 eluents;8, No. 5 eluents(Purifying
Preferably, purity is high for effect).
Embodiment five detects the ELISA kit of echinococcosis granulosa
Detecting the ELISA kit of echinococcosis granulosa includes following components:
1st, envelope antigen:The recombinant protein of the purifying obtained using embodiment 4 is envelope antigen, with pH 9.6 0.05M/L carbonic acid
The recombinant protein of salt buffer dilution purifying is to the μ g/ml of final concentration 8;
2nd, confining liquid:5 g skimmed milk powers are dissolved in 100 ml TBST;
3rd, negative control:Healthy Human Serum, 1:200 dilutions;
4th, ELIAS secondary antibody:The goat anti-human igg of peroxidase labelling, 1:10000 dilutions;
5th, TMB nitrite ions;
6th, terminate liquid:The 2 mol/L concentrated sulfuric acids.
The Echinococcus granulosus antigen TSP1 of embodiment six ELISA detections
Using recombinant protein after purification as antigen, to 20 parts of infection hydatidosis human serum samples and 20 parts of Healthy Human Serum samples
Product carry out indirect ELISA detection.Specific method is as follows:
The recombinant protein purified with pH 9.6 0.05M/L carbonate buffer solutions dilution is to the μ g/ml of final concentration 8, per hole
20096 hole elisa Plates are coated with, 4 DEG C overnight;Liquid in hole is got rid of, with pH 7.4 3 (5 min/ of PBST board-washings
It is secondary) 200 μ l confining liquids are added per hole afterwards, 37 DEG C are closed 1 hour.PBST board-washings 3 times (5 min/ times), per hole 100It is separately added into Echinococcus Granulosus Cysts patient and Healthy Human Serum(1: 200 dilution), 37 DEG C incubate 1 hour;PBST is washed
Plate 3 times, the goat anti-human igg of peroxidase labelling is added per the μ l of hole 100( 1:10000 dilutions), 37 DEG C incubate 1 hour;
PBST board-washings 3 times, TMB nitrite ions are added per the μ l of hole 100, and 37 DEG C of lucifuges are reacted 30 minutes, and 2 are added per the μ l of hole 50
Mol/L concentrated sulfuric acid terminating reactions, determine absorbance()Value.With Healthy Human SerumThe extraordinarily standard deviation of average 2 is
Positive cutoff value.ELISA testing results as shown in table 1, the diagnosis of kit of the invention to echinococcosis granulosa patients serum
Sensitiveness is 90%(18/20), there is preferable Immunodiagnosis to echinococcosis granulosa.
The Echinococcus granulosus TSP1 ELISA testing results of table 1
Finally it should be noted that:The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the invention, although
The present invention is described in detail with reference to the foregoing embodiments, for those skilled in the art, it still can be right
Technical scheme described in foregoing embodiments is modified, or carries out equivalent substitution to which part technical characteristic.It is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements made etc. should be included in the protection of the present invention
Within the scope of.
Sequence table
<110>Lanzhou University
<120>Echinococcus granulosus TSP1 recombinant proteins and its solubility expression method and purification process
<210> 1
<211> 1047
<212> DNA
<213>Echinococcus granulosus TSP1 nucleotide sequences
<400> 1
ccggggacca cctacagcgg cacacaatga ccactggtta tgatataatg cgcagttcct 60
gctcgacgtt gacgcgattc atcatcggtc tgctcaatct gattttggga gttacatttt 120
tggtgcttgg agtagttggc gtgctgctac gaaccaacaa ggaattcttg cttaaattca 180
ctaacagtct ctccgaaaag ttcctcaatg aggcttcaca agaacaggcg gatgaaatag 240
ctcaattctt gctaaaatac gatgttggaa taccggcgat attcatcgtg gtgggatttg 300
ccatcaccgg tgtctgcatt ctcgggttca tagcgacctg ctgttcatgc aataaactcc 360
tacaaatcta tgcggtgatt ttaacatcca ttgttgtgat ccaagttatc gccgttggag 420
tcatctttgg ggtcccggac atataccgca gcttagcatt tcagggcatg gagaaaacgc 480
ttgcctacta cgatccgagc actcctgagg gcaaagggtc tgccagactg tgggatctta 540
ttatgactgc aaacaaagag acctgttgcg gaatggatgg agccaatgac ttcaaaaaca 600
caccttttga tcccaagtgt ccaaaatact gttgcaaagc agacaaggac tgtttgatta 660
ctgatgcatt ggcgttgact ccaccggttg agggatgtcg tggaaagata agacgctacg 720
ctgatgaata catgttgaag attttggtca ttcttatcat cttcatcgct tgccaggtag 780
ttctttcaat cctcacctac gtggcactga catgtaagac tgcttcccca atatgagagg 840
aggggcgccg tttgctgcac caaattatct ctgcttgttt tctgcacctc atcattcact 900
actgtgttaa gagatgtggg ttcctctcat tgattcccct tcactcacaa ggaaagttaa 960
taaagcgtag agatatgtgc attttttacg taaaaaaaaa aaaaaaaaac atgtcggccg 1020
cctcggccta tgtgcggccg ccaccgc 1047
<210> 2
<211> 269
<212> PRT
<213>Echinococcus granulosus TSP1 recombinant proteins
<400> 2
Met Thr Thr Gly Tyr Asp Ile Met Arg Ser Ser Cys Ser Thr Leu Thr
1 5 10 15
Arg Phe Ile Ile Gly Leu Leu Asn Leu Ile Leu Gly Val Thr Phe Leu
20 25 30
Val Leu Gly Val Val Gly Val Leu Leu Arg Thr Asn Lys Glu Phe Leu
35 40 45
Leu Lys Phe Thr Asn Ser Leu Ser Glu Lys Phe Leu Asn Glu Ala Ser
50 55 60
Gln Glu Gln Ala Asp Glu Ile Ala Gln Phe Leu Leu Lys Tyr Asp Val
65 70 75 80
Gly Ile Pro Ala Ile Phe Ile Val Val Gly Phe Ala Ile Thr Gly Val
85 90 95
Cys Ile Leu Gly Phe Ile Ala Thr Cys Cys Ser Cys Asn Lys Leu Leu
100 105 110
Gln Ile Tyr Ala Val Ile Leu Thr Ser Ile Val Val Ile Gln Val Ile
115 120 125
Ala Val Gly Val Ile Phe Gly Val Pro Asp Ile Tyr Arg Ser Leu Ala
130 135 140
Phe Gln Gly Met Glu Lys Thr Leu Ala Tyr Tyr Asp Pro Ser Thr Pro
145 150 155 160
Glu Gly Lys Gly Ser Ala Arg Leu Trp Asp Leu Ile Met Thr Ala Asn
165 170 175
Lys Glu Thr Cys Cys Gly Met Asp Gly Ala Asn Asp Phe Lys Asn Thr
180 185 190
Pro Phe Asp Pro Lys Cys Pro Lys Tyr Cys Cys Lys Ala Asp Lys Asp
195 200 205
Cys Leu Ile Thr Asp Ala Leu Ala Leu Thr Pro Pro Val Glu Gly Cys
210 215 220
Arg Gly Lys Ile Arg Arg Tyr Ala Asp Glu Tyr Met Leu Lys Ile Leu
225 230 235 240
Val Ile Leu Ile Ile Phe Ile Ala Cys Gln Val Val Leu Ser Ile Leu
245 250 255
Thr Tyr Val Ala Leu Thr Cys Lys Thr Ala Ser Pro Ile
260 265
Claims (10)
1. Echinococcus granulosus TSP1 recombinant proteins, it is characterised in that:The amino of the Echinococcus granulosus TSP1 recombinant proteins
Acid sequence is sequence table SEQ ID No.2.
2. Echinococcus granulosus TSP1 recombinant proteins according to claim 1, it is characterised in that:The Echinococcus granulosus
The nucleotides sequence of TSP1 recombinant proteins coding is classified as the 27th -836 bit bases in sequence table SEQ ID No.1.
3. the solubility expression of the Echinococcus granulosus TSP1 recombinant proteins described in claim 1 or 2, it is characterised in that:Step
It is as follows:
(1)Target gene TSP1 amplification;
(2)Build TSP1 expression plasmids:By step(1)Target gene TSP1 digestions of preparation and after purification, are built into pET-30a
Corresponding polyclone enzyme enzyme site, builds plasmid pET30a-TSP1;
(3)By step(2)The plasmid pET30a-TSP1 of preparation is transformed intoE.Coli, will in BL21 (DE3) competent cellE.ColiBL21 (DE3)-pET30a-TSP1 is enlarged culture, then adds 0.2 mmol/L IPTG in 25 DEG C, lures
Lead shaken cultivation 8 hours;Ultrasound after centrifugation is resuspended, collects supernatant.
4. solubility expression according to claim 3, it is characterised in that:Step(3)Described in expand culture be byE.ColiBL21 (DE3)-pET30a-TSP1 is first seeded on LB solid mediums, 37 DEG C of overnight incubations;Then picking culture
Monoclonal is into LB fluid nutrient mediums on base, and in 37 DEG C, 180r/min shaken cultivations 3 hours make OD values to 0.5;Finally take bacterium
Liquid is seeded in LB fluid nutrient mediums, and in 37 DEG C, 180r/min shaken cultivations make OD values to 0.5.
5. the purification process of the Echinococcus granulosus TSP1 recombinant proteins described in claim 1 or 2, it is characterised in that:It is to use
Ni-NTA His-Bind(Clontech)Purification system purification of recombinant proteins, is then eluted using elution buffer;It is described
The formula of elution buffer is:500mM/L imidazoles, 20mM/L Tris, 500mM/L NaCl, pH8.0.
6. purification process according to claim 5, it is characterised in that:Step is as follows:
(1)Ultra-pure water is added in Ni-NTA His-Bind prepacked columns, the medium for making it slowly flow through filling in post;
(2)Add combination buffer;The formula of the combination buffer is:20mM/L Tris, 500mM/L NaCl, 20mM/L
Imidazoles, pH8.0;
(3)Loading, adds ultrasonicationE.ColiMake supernatant slow after the supernatant that BL21 (DE3)-TSP1 is extracted, standing
Flow through medium in post;
(4)Combination buffer is added, it is slowly flowed across medium in post;The formula of the combination buffer is:20mM/L
Tris, 500mM/L NaCl, 20mM/L imidazoles, pH8.0;
(5)No. 1 eluent is added, it is slowly flowed across medium in post, the formula of No. 1 eluent is:100 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(6)No. 2 eluents are added, it is slowly flowed across medium in post, the formula of No. 2 eluents is:200 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(7)No. 3 eluents are added, it is slowly flowed across medium in post, the formula of No. 3 eluents is:300 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(8)No. 4 eluents are added, it is slowly flowed across medium in post, the formula of No. 4 eluents is:400 mM/L miaows
Azoles, 20 mM/L Tris, 500 mM/L NaCl, pH8.0;
(9)No. 5 elution buffers are added, it is slowly flowed across medium in post, eluent is collected;No. 5 elution buffers
It is formulated and is:500mM/L imidazoles, 20mM/L Tris, 500mM/L NaCl, pH8.0.
7. purification process according to claim 6, it is characterised in that:The ultrasonicationE.Coli BL21(DE3)-
The supernatant and the volume ratio of elution buffer that TSP1 is extracted are 1:1.
8. the Echinococcus granulosus TSP1 recombinant proteins described in claim 1 or 2 are preparing the ELISA of detection echinococcosis granulosa
Application in kit.
9. a kind of ELISA kit for detecting echinococcosis granulosa, it is characterised in that:Envelope antigen in the ELISA kit
For the Echinococcus granulosus TSP1 recombinant proteins described in claim 1 or 2.
10. kit according to claim 9, it is characterised in that:Envelope antigen pH 9.6 0.05M/L carbon
Phthalate buffer is diluted to the μ g/ml of final concentration 8;
Preferably, the kit also includes confining liquid, the confining liquid is the TBST containing 5% skimmed milk power, the percentage
The unit of ratio is g/ml.
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710393428.0A CN107236027B (en) | 2017-05-27 | 2017-05-27 | Echinococcus granulosus TSP1 recombinant protein and soluble expression method and purification method thereof |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201710393428.0A CN107236027B (en) | 2017-05-27 | 2017-05-27 | Echinococcus granulosus TSP1 recombinant protein and soluble expression method and purification method thereof |
Publications (2)
Publication Number | Publication Date |
---|---|
CN107236027A true CN107236027A (en) | 2017-10-10 |
CN107236027B CN107236027B (en) | 2020-10-16 |
Family
ID=59985270
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201710393428.0A Expired - Fee Related CN107236027B (en) | 2017-05-27 | 2017-05-27 | Echinococcus granulosus TSP1 recombinant protein and soluble expression method and purification method thereof |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN107236027B (en) |
Cited By (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110655564A (en) * | 2019-10-29 | 2020-01-07 | 四川农业大学 | Combined protein and application thereof |
CN110684096A (en) * | 2019-10-29 | 2020-01-14 | 四川农业大学 | Combined protein and application thereof |
CN113185592A (en) * | 2021-04-28 | 2021-07-30 | 新疆农垦科学院 | Echinococcus granulosus antigen and application thereof |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101948521A (en) * | 2010-09-17 | 2011-01-19 | 中国疾病预防控制中心寄生虫病预防控制所 | Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof |
CN102863524A (en) * | 2012-08-28 | 2013-01-09 | 中国疾病预防控制中心寄生虫病预防控制所 | Recombinant antigen protein for diagnosing echinococcosis granulosa as well as preparation method and application thereof |
-
2017
- 2017-05-27 CN CN201710393428.0A patent/CN107236027B/en not_active Expired - Fee Related
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101948521A (en) * | 2010-09-17 | 2011-01-19 | 中国疾病预防控制中心寄生虫病预防控制所 | Recombinant antigenic protein for diagnosing echinococcosis granulosus, preparation method thereof and use thereof |
CN102863524A (en) * | 2012-08-28 | 2013-01-09 | 中国疾病预防控制中心寄生虫病预防控制所 | Recombinant antigen protein for diagnosing echinococcosis granulosa as well as preparation method and application thereof |
Non-Patent Citations (3)
Title |
---|
TSAI,I.J. ET AL.: "tetraspanin [Echinococcus granulosus]", 《GENBANK: CDS22357.1》 * |
刘田莉等: "细粒棘球蚴TSP1和TSP6基因的克隆及生物信息学分析", 《华北农学报》 * |
辛奇: "细粒棘球绦虫成虫基因表达谱分析及抗包虫药物筛选", 《中国博士学位论文全文数据库医药卫生科技辑》 * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN110655564A (en) * | 2019-10-29 | 2020-01-07 | 四川农业大学 | Combined protein and application thereof |
CN110684096A (en) * | 2019-10-29 | 2020-01-14 | 四川农业大学 | Combined protein and application thereof |
CN113185592A (en) * | 2021-04-28 | 2021-07-30 | 新疆农垦科学院 | Echinococcus granulosus antigen and application thereof |
CN113185592B (en) * | 2021-04-28 | 2022-08-09 | 新疆农垦科学院 | Echinococcus granulosus antigen and application thereof |
Also Published As
Publication number | Publication date |
---|---|
CN107236027B (en) | 2020-10-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US8357778B2 (en) | Polypeptide, an affinity chromatography material, and a method for separating and/or purifying immunoglobulin | |
CN107236027A (en) | Echinococcus granulosus TSP1 recombinant proteins and its solubility expression method and purification process | |
CN113512099B (en) | Staphylococcus protein A, purification preparation method and application thereof | |
CN109762070A (en) | For detecting fused antigen, its encoding gene, host cell and the kit of echinococcosis | |
CN108314710B (en) | Mycoplasma pneumoniae recombinant antigen and application thereof | |
CN107200776A (en) | Echinococcus granulosus antigen cC1 recombinant protein and its solubility expression method and purification process | |
CN111499726A (en) | Preparation method of gobiocyprisrarus apoA1 recombinant protein and anti-rabbit polyclonal antibody | |
CN111253478B (en) | Mycoplasma pneumoniae antigen and preparation method and application thereof | |
CN110845582B (en) | Preparation of feline parvovirus recombinant protein and monoclonal antibody thereof | |
CN111087453A (en) | Preparation method and application method of chlamydia pneumoniae recombinant antigen | |
CN108179149B (en) | S100B mutant and application thereof | |
KR100257574B1 (en) | Treponema pallidum fused antigen and assay for anti-treponema pallidum antibodies using the same fused antigen | |
CN107056915A (en) | Echinococcus granulosus TSP6 recombinant proteins and its solubility expression method and purification process | |
CN111875677A (en) | Canine parvovirus VP2 protein recombinant antigen, encoding gene thereof, expression and application thereof | |
CN110592039A (en) | Application of hybridoma cell and monoclonal antibody generated by hybridoma cell in detection of AM79 EPSPS protein | |
CN108300725B (en) | Soluble single-chain antibody superantigen fusion gene and protein, and preparation and application thereof | |
CN109055416A (en) | A kind of preparation method of soluble recombinant protein | |
CN107201348B (en) | Echinococcus granulosus Sigma-glutathione S-transferase recombinant protein and soluble expression method and purification method thereof | |
CN107253988A (en) | A kind of preparation method of people's source program death factors part PD L2 albumen | |
CN111888797B (en) | Method for purifying egg yolk antibody by using affinity immune medium | |
CN111378025A (en) | Tetrodotoxin binding protein tfPSTBP2, nucleotide sequence, polyclonal antibody thereof and preparation method thereof | |
CN107099519A (en) | Echinococcus granulosus turns dihydroxyacetone kinase recombinant protein, its solubility expression method and its purification process and application | |
CN112094853A (en) | White spot syndrome virus VP28 gene, recombinant protein, polyclonal antibody, preparation method and application | |
CN117924439B (en) | Recombinant CagA protein and preparation method and application thereof | |
CN114989270B (en) | Polypeptide with binding affinity to humanized CEA and application thereof |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20201016 Termination date: 20210527 |