CN109485725A - A kind of Echinococcus hydatid cyst recombinant protein and its preparation method and application - Google Patents
A kind of Echinococcus hydatid cyst recombinant protein and its preparation method and application Download PDFInfo
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Abstract
The invention discloses a kind of Echinococcus hydatid cyst recombinant proteins and its preparation method and application, are related to biomedicine technical field, including echinococcous antigen and Echinococcus hydatid cyst single-chain antibody；The echinococcous antigen is the AgB8/2 antigen containing major antigenic determinant；The Echinococcus hydatid cyst single-chain antibody is scFv, comprising the following steps: (1) searches the AgB8/2 antigen containing major antigenic determinant in Echinococcus hydatid cyst；(2) optimize the codon of Echinococcus hydatid cyst AgB8/2 antigen gene, synthetic nucleic acid sequence is simultaneously expressed；(3) building is directed to the single-chain antibody of Echinococcus hydatid cyst AgB8/1 antigen, transfers the gene of single-chain antibody scFv；(4) gene of the Echinococcus hydatid cyst AgB8/2 antigen gene and the single-chain antibody scFv for Echinococcus hydatid cyst AgB8/1 antigen of recombination optimization, and expressed；(5) purifying and renaturation Echinococcus hydatid cyst recombinant protein.The present invention on the basis of developing monoclonal antibody again carry out recombinant antibodies technological development single-chain antibody, be easy to express in cell, can be expressed in eukaryon or prokaryotic cell, can mass production, cost is relatively low.
Description
Technical field
The present invention relates to biomedicine technical field, in particular to a kind of Echinococcus hydatid cyst recombinant protein and preparation method thereof and answer
With.
Background technique
Echinococcosis is divided into capsule echinococcosis and alveolar echinococcosis, wherein 70% alveolar echinococcosis patient is dead in 5 years
It dies, the death rate in 10 years is up to 92%.Because of its high infection rate and high lethality rate, echinococcosis has the title of " worm cancer " again.Water purification work
Journey and Controlling dogs are considered as cutting off the key in worm's ovum source.
Echinococcosis is that people infects chronic parasitic disease caused by the larva (echinococcus) of echinococcus.The clinical table of this disease
Now regard hydatid position, size and different whether there is or not complication.For a long time, echinococcosis is considered as that a kind of infecting both domestic animals and human is posted
Infested disease, referred to as zoonosis.According to epidemiological survey in recent years, referred to as region parasitic disease；In Endemic Area
The characteristics of with occupational adverse effect, is listed in the occupational disease of certain crowds；From global range say echinococcosis be ethnic group or
A kind of common disease and frequently-occurring disease specific to religion clan.
The exploitation of in-vitro diagnosis core material is to develop the premise of corresponding diagnostic reagent, only develop with high activity,
High performance antigen-antibody, external diagnosis reagent product can just become a reality.Therefore, it is developed for Echinococcus hydatid cyst antibody and antigen
Can be extremely urgent to produce the raw material of diagnostic reagent, to some degree, the exploitation of raw material, which will also become, reduces packet
Primary the solving the problems, such as of parasitosis sprawling.
Summary of the invention
The object of the present invention is to provide a kind of Echinococcus hydatid cyst recombinant proteins and its preparation method and application.
Technical solution of the present invention: a kind of Echinococcus hydatid cyst recombinant protein, including echinococcous antigen and Echinococcus hydatid cyst single-chain antibody；
The echinococcous antigen is the AgB8/2 antigen containing major antigenic determinant；
The Echinococcus hydatid cyst single-chain antibody is scFv.
A kind of preparation method of Echinococcus hydatid cyst recombinant protein, comprising the following steps:
(1) the AgB8/2 antigen containing major antigenic determinant in Echinococcus hydatid cyst is searched；
(2) optimize the codon of Echinococcus hydatid cyst AgB8/2 antigen gene, synthetic nucleic acid sequence is simultaneously expressed；
(3) building is directed to the single-chain antibody of Echinococcus hydatid cyst AgB8/1 antigen, transfers the gene of single-chain antibody scFv；
(4) the Echinococcus hydatid cyst AgB8/2 antigen gene of recombination optimization and the single-chain antibody scFv for Echinococcus hydatid cyst AgB8/1 antigen
Gene, and expressed；
(5) purifying and renaturation Echinococcus hydatid cyst recombinant protein.
Expression system of the invention is escherichia expression system, it has the period short, and expense is low, and expression quantity is big to wait spies
Point.
In order to enable recombinant protein preferably keeps active site, the comparatively gentle culture of present invention selection and induction item
Part, expression when inducing temperature be 20 DEG C, 25 DEG C, 28 DEG C, 37 DEG C, further preferred inducing temperature be 25 DEG C, 37 DEG C,
Inducing temperature still more preferably is 25 DEG C.Induction revolving speed is 250rpm, IPTG (the isopropyl-beta D-thio gala of induction
Glucosides) concentration be 0.1mM.Make recombinant protein have more slowly expression in this way, there is the sufficient time to carry out space structure
The formation of elephant.
The present invention is crushed bacterium by the way of ultrasound.When broken thallus, in order to avoid drastic conditions, it will break
Fringe part is set to 200w, each ultrasound 6s, and interval 3s ultrasound is primary, and totally 180 times.
The way of purification of recombinant protein of the present invention has an ion-exchange chromatography, the methods of gel permeation chromatography and affinity chromatography,
Further preferably affinity chromatography.
The present invention joined 6XHis label in recombinant protein, and affinitive layer purification mode is enable to reach higher pure
Degree.
Recombinant protein of the invention utilize genetic engineering recombinant technique, in E. coli system express echinococcous antigen and
The recombinant protein method of single-chain antibody has many advantages, such as that with short production cycle, yield is high, at low cost
In a kind of Echinococcus hydatid cyst recombinant protein above-mentioned and preparation method thereof, the step (2) and step (4) are in Escherichia coli
It is expressed in system.
Induction when a kind of Echinococcus hydatid cyst recombinant protein above-mentioned and preparation method thereof, the step (2) and step (4) are expressed
Temperature is 20 DEG C, 25 DEG C, 28 DEG C or 37 DEG C, and induction revolving speed is 250rpm, and the IPTG concentration of induction is 0.1mM.
A kind of Echinococcus hydatid cyst recombinant protein above-mentioned and preparation method thereof, the inducing temperature are 25 DEG C or 37 DEG C.
In a kind of Echinococcus hydatid cyst recombinant protein above-mentioned and its preparation method and application, the inducing temperature is 25 DEG C.
In a kind of Echinococcus hydatid cyst recombinant protein above-mentioned and its preparation side, the way of purification of the step (5) is selected from ion exchange
Chromatography, gel permeation chromatography or affinity chromatography.
In a kind of Echinococcus hydatid cyst recombinant protein above-mentioned and preparation method thereof, the way of purification of the step (5) is affine layer
Analysis.
A kind of application of Echinococcus hydatid cyst recombinant protein, the recombinant protein are used to prepare Echinococcus hydatid cyst detection kit.
Compared with prior art, the present invention carries out recombinant antibodies technological development again on the basis of developing monoclonal antibody
Single-chain antibody, 1) single-chain antibody, which has the advantage that, has complete antigen binding site, is free of antibody constant region；2) molecule
It measures small, reduces the non-specific binding of antibody, reduce the false positive risk of diagnostic products；3) structure is simple, is easy to gene
It is engineered；4) be easy to express in cell, can be expressed in eukaryon or prokaryotic cell, can mass production, cost compared with
It is low.
(2) Echinococcus hydatid cyst Main Antigenic and the scFv recombinant protein for AgB8/2 antigen are developed for the first time both at home and abroad.
Since infection Echinococcus hydatid cyst initial stage does not generate antibody, us in this time is caused to can't detect corresponding Echinococcus hydatid cyst antibody,
So as to cause missing inspection, the detection to antigen must be increased while detecting Echinococcus hydatid cyst antibody in order to solve this problem, exploitation is directed to
The antibody of Echinococcus hydatid cyst AgB8/2 antigen becomes key.
(3) it is prepared for the recombination egg in combination with the high activity of Echinococcus hydatid cyst antibody and antigen, high sensitivity and high specific
It is white.
After developing in Main Antigenic and for the antibody of Echinococcus hydatid cyst AgB8/2 antigen, utilize technique for gene engineering will
The efficient epitope (Echinococcus hydatid cyst AgB8/2 antigen) filtered out and the single-chain antibody scFv (3- for Echinococcus hydatid cyst AgB8/1 antigen
It 10aa) recombinates, is prepared into the recombination in combination with the high activity of Echinococcus hydatid cyst antibody and antigen, high sensitivity and high specific
Albumen, 1) which, which has the advantage that, only needs in reagent strip using a kind of raw material examining fastly, in this way can be with
Avoid the cross reaction between plurality of raw materials；2) in the production of raw material, it is only necessary to stablize a kind of production technology of raw material,
The investment of production cost can be reduced in this way, shortens the raw material production cycle, reduce production difference between batch；3) exploitation Echinococcus hydatid cyst is saved
The cost of diagnostic reagent.
Specific embodiment
Below with reference to embodiment, the present invention is further illustrated, but is not intended as the foundation limited the present invention.
Embodiment: a kind of Echinococcus hydatid cyst recombinant protein, including echinococcous antigen and Echinococcus hydatid cyst single-chain antibody；
The echinococcous antigen is the AgB8/2 antigen containing major antigenic determinant；
The Echinococcus hydatid cyst single-chain antibody is scFv.
Preparation method includes the following steps:
1. searching the AgB8/2 antigen containing major antigenic determinant in Echinococcus hydatid cyst；
2. optimizing the codon of Echinococcus hydatid cyst AgB8/2 antigen gene, synthetic nucleic acid sequence is simultaneously expressed；
AgB8/2 antigen is searched from NCBI (US National Biotechnology Information center), protein sequence is as follows:
MRTYILLSLALVAFVAVVQAKDEPKAHMGQVVKKRWGELRDFFRNDPLGQRLVA LGNDLTAICQKLQ
LKIREVLKKYVKNSVEEKDDDSK
Sequence is as follows after codon optimization:
ATGCGTACCTATATTCTGCTGAGCCTGGCACTGGTTGCATTTGTGGCAGTTGTG CAGGCAAAAGATG
AACCGAAAGCACACATGGGTCAGGTTGTGAAAAAACGTTGGGGCG AACTGCGTGATTTTTTTCGTAATGATCCGC
TGGGTCAGCGCCTGGTGGCCCTGGGTAA TGATCTGACCGCAATTTGTCAGAAACTGCAGCTGAAAATTCGTGAAG
TGCTGAAAAAA TATGTGAAAAATAGCGTGGAAGAGAAAGATGATGATAGTAAA
Design primer is as follows:
P1u:GGCCATATGATGCGTACCTATATTCTGCTGAGCCTGGCACTGGTTGC ATTTGTGG
P1d:GTGCTTTCGGTTCATCTTTTGCCTGCACAACTGCCACAAATGCAACCA
P2u:ATGAACCGAAAGCACACATGGGTCAGGTTGTGAAAAAACGTTGGGGCG
P2d:CCAGCGGATCATTACGAAAAAAATCACGCAGTTCGCCCCAACGTTTTTT
P3u:GTAATGATCCGCTGGGTCAGCGCCTGGTGGCCCTGGGTAATGATC
P3d:ACGAATTTTCAGCTGCAGTTTCTGACAAATTGCGGTCAGATCATTACC CAGGG
P4u:CAGCTGAAAATTCGTGAAGTGCTGAAAAAATATGTGAAAAATAGCGT
Experimental method is as follows:
Primer P1u, P1d, P2u, P2d are mixed, PCR reaction is carried out, products therefrom is named as W1；By primer P3u,
P3d, P4u, P4d mixing, carry out PCR reaction, and products therefrom is named as W2；Then by above-mentioned two product W1, W2 mixing as
Template carries out the AgB8/2 gene of Echinococcus hydatid cyst after PCR reaction is optimized with primer P1u, P4d.Aforementioned four PCR (TOYOBO
Products, article No.: 02510D1) reaction condition is as follows: 94 DEG C, 2 minutes X1 circulations；(98 DEG C, 15 seconds, 55 DEG C, 30 seconds,
68 DEG C, 15 seconds) X30 circulation；72 DEG C, 7 minutes X1 circulations.After sequencing proves that sequence is correct, by this PCR product NdeI
After carrying out double digestion with XhoI restriction enzyme (being purchased from NEB company), it is inserted into identical two digestions processing
In pET30a (Novagen product, article No. 69909-3) carrier.
AgB8/1 antigen is searched from NCBI (US National Biotechnology Information center), protein sequence is as follows:
MLLALALVSFVVVTQADDGLTSTSRSVMKMFGEVKYFFERDPLGQKVVDLLKEL
EEVFQLLRKKLRMALRSHLRGLIAEGE
Sequence is as follows after codon optimization:
ATGCTGCTGGCCCTGGCACTGGTGAGCTTTGTTGTGGTGACCCAGGCCGATGAT GGCCTGACCAGCA
CCAGCCGTAGCGTGATGAAAATGTTTGGCGAAGTTAAATATTTCT TCGAACGCGATCCGCTGGGCCAGAAAGTTG
TGGATCTGCTGAAAGAACTGGAAGAAGT TTTTCAGCTGCTGCGTAAAAAACTGCGCATGGCACTGCGTAGCCATC
TGCGTGGCCTG ATTGCCGAAGGTGAA
Design primer is as follows:
P5u:GGCCATATGCTGCTGGCCCTGGCACTGGTGAGCTTTGTTGTGGTGACCCA GGCCGA
P5d:CCAAACATTTTCATCACGCTACGGCTGGTGCTGGTCAGGCCATCATCGGC CTGGGTCACC
P6u:GATGAAAATGTTTGGCGAAGTTAAATATTTCTTCGAACGCGATCCGCTGG GCCAGAA
P6d:GCTGAAAAACTTCTTCCAGTTCTTTCAGCAGATCCACAACTTTCTGGCCC AGCGGA
P7u:AAGAAGTTTTTCAGCTGCTGCGTAAAAAACTGCGCATGGCACTGCGTAGC CAT
P7d:GCCCTCGAGTTCACCTTCGGCAATCAGGCCACGCAGATGGCTACGCAGTG C
Experimental method is as follows:
Primer P5u, P5d, P6u, P6d are mixed, PCR reaction, condition are carried out are as follows: 94 DEG C, 2 minutes X1 circulations, gained
Product is named as W3；Primer P7u, P7d are mixed, PCR reaction, condition are carried out are as follows: (98 DEG C, 15 seconds, 55 DEG C, 30 seconds, 68 DEG C,
15 seconds) X30 circulation, products therefrom is named as W4；Then above-mentioned two product W3, W4 are mixed as template, uses primer
P5u, P8d carry out the AgB8/1 gene of Echinococcus hydatid cyst after PCR reaction is optimized, condition are as follows: and 72 DEG C, 7 minutes X1 circulations.It surveys
After sequence proves that sequence is correct, this PCR product is subjected to double digestion with NdeI and XhoI restriction enzyme (being purchased from NEB company)
Later, it is inserted into pET30a (Novagen product, the article No. 69909-3) carrier handled with identical two digestions.
3. the single-chain antibody that building is directed to Echinococcus hydatid cyst core protein Echinococcus hydatid cyst AgB8/1 antigen；
(1) acquisition of monoclonal antibody (for the antibody for combining AgB8/1 antigen protein 3-10aa) cell strain:
A. it will construct after successful pET30a-AgB8/1 carrier is converted to BL21 expression strain expression and purification and obtain AgB8/
1 antigen protein；
B. mouse is immunized with the AgB8/1 antigen protein obtained；
C. it is screened with monoclonal antibody technique just for the antibody cell strain for combining AgB8/1 antigen protein 3-10aa.
(2) extraction of cell RNA
With TRIzol LS (invitrogen product, article No. 10296-010) reagent, operating procedure is extracted to specifications
Cell total rna.
The synthesis of the first chain of cDNA:
1 μ gRNA is taken to do reverse transcribing template, 1 μ l of Oligo (d) T does reverse transcriptase primer,
Add DEPC water to 12 μ l of total volume, mixes；
Then,
70 DEG C of denaturation 5min, rapid ice bath are cooling；
Then
5 × buffer, 4 μ l, RNase inhibitor, 12 μ l of μ l, dNTP (10mM each) is sequentially added,
It mixes
37 DEG C of incubation 5min；
Then,
1 μ l of AMV reverse transcriptase is added,
37 DEG C of reverse transcription 60min；
Then,
70 DEG C of 10min terminate reaction；
Finally,
Cooled on ice.
(3) using reverse transcription cDNA as template, according to following primer, heavy chain light chain gene is expanded with RT-PCR.
mHVu1:GATGTGAAGCTTCAGGAGTC
mHVu2:CAGGTGCAGCTGAAGGAGTC
mHVu3:CAGGTGCAGCTGAAGCAGTC
mHVu4:CAGGTTACTCTGAAAGAGTC
mHVu5:AAGGTCCAGCTGCAACAATC
mHVu6:GAGGTCCAGCTGCAGCAGTC
mHVu7:CAGGTCCAACTGCAGCAGCC
mHVu8:GAGGTGAAGCTGGTGGAGTC
mHVu9:GAGGTGAAGCTGGTGGAATC
mHVu10:GATGTGAACTTGGAAGTGTC
mHVd1:TGCAGAGACAGTGACCAGAGT
mHVd2:TGAGGAGACTGTGAGAGTGGT
mHVd3:TGAGGAGACGGTGACTGAGGT
mHVd4:TGAGGAGACGGTGACCGTGGT
mKVu1:GATGTTTTGATGACCCAAACT
mKVu2:GATATTGTGATGACGCAGGCT
mKVu3:GATATTGTGATAACCCAG
mKVu4:GACATTGTGCTGACCCAATCT
mKVu5:GACATTGTGATGACCCAGTCT
mKVu6:GATATTGTGCTAACTCAGTCT
mKVu7:GATATCCAGATGACACAGACT
mKVu8:GACATCCAGCTGACTCAGTCT
mKVu9:CAAATTGTTCTCACCCAGTCT
mKVd1:CCGTTTCAGCTCCAGCTTG
mKVd2:CCGTTTTATTTCCAGCTTGGT
mKVd3:CCGTTTTATTTCCAACTTTG
It is combined respectively with mHVd1-mHVd4 with primer mHVu1-mHVu10；MKVu1-mKVu9 respectively with mKVd1-
PCR reaction is done in mKVd4 combination.
25 μ l of PCR reaction system, wherein 0.5 μ l of cDNA, 0.25 μ l downstream primer of upstream primer, 0.25 μ l, Taq enzyme 0.2
μl dNTP 2μl 10×buffer 2.5μl。
PCR (TOYOBO Products, article No.: 02510D1), PCR condition are as follows: 94 DEG C, 2 minutes X1 circulations；(98 DEG C,
5 seconds, 55 DEG C, 30 seconds, 68 DEG C, 15 seconds) X30 circulation；72 DEG C, 7 minutes X1 circulations.
Heavy chain and light chain gene are determined after sequence verification, have then been connected heavy chain with light chain gene by recursive PCR
Come, this recombination is rscFv.
4. containing Echinococcus hydatid cyst AgB8/2 antigen gene and for single-chain antibody gene (3-10aa) weight of Echinococcus hydatid cyst AgB8/1 antigen
Group, and expressed:
(1) there will be Echinococcus hydatid cyst AgB8/2 antigen gene and for the single-chain antibody base of Echinococcus hydatid cyst AgB8/1 antigen by recursive PCR
Because (3-10aa) links together, after carrying out double digestion with NdeI and XhoI restriction enzyme (being purchased from NEB company), insert
Enter into pET30a (Novagen product, the article No. 69909-3) carrier handled with identical two digestions.
(2) expression vector of above-mentioned recombinant protein gene is converted into e. coli bl21, is coated on containing 100ug/ml
On the LB plate of kanamycin sulfate (the raw work bioengineering Services Co., Ltd in Shanghai, article No.: KB0286), 37 DEG C of trainings overnight
It supports, picking monoclonal colonies, is cultivated with 37 DEG C of 300mlLB culture medium of the kanamycin sulfate containing same concentrations to OD600
Up to 0.6 or so, the IPTG for being 0.1mM with concentration (raw work, article No.: IB0168) carries out inducing expression, inductive condition are as follows: 37
DEG C, revolving speed 250rpm, 5h.After induction, 4 DEG C of 5000rpm centrifugation 20min of culture solution are collected into thallus.
5. purifying and renaturation containing Echinococcus hydatid cyst recombinant protein:
By thallus with 50ml inclusion body extract (20mM Tris-HCl, 0.5MUrea pH 7.5,0.5M NaCl, 2%
Triton X-100) it is resuspended, then ultrasonication, condition is each ultrasound 3s, interval 6s, totally 180 times, 12000rpm, 4 DEG C
Inclusion body precipitating is collected by centrifugation.With sample-loading buffer Binding Buffer (50mM Tris, 8M Urea, 0.5M Nacl,
20mM imidazoles pH8.0) 50ml be crushed inclusion body, upper column purification, with elution buffer Elution Buffer (50mM Tris,
8M Urea, 0.5M Nacl, 300mM imidazoles pH8.0) elution destination protein.By recombinant protein elution buffer after purification
(1mM oxidized form of glutathione GSSH, 2mM reduced glutathione GSH, 2mMEDTA, 20mM Tris, pH8.5) dialysis, often
A dialyzate is changed every 48h.Protein liquid after taking out dialysis is saved backup through being concentrated according to ethyl alcohol -20000 in -20 DEG C.
Sequence table
<110>hundred million minot Biotechnology Co., Ltd of Hangzhou
<120>a kind of Echinococcus hydatid cyst recombinant protein and its preparation method and application
<130> 2
<160> 43
<170> SIPOSequenceListing 1.0
<210> 1
<211> 90
<212> PRT
<213>echinococcous antigen (AgB8/2)
<400> 1
Met Arg Thr Tyr Ile Leu Leu Ser Leu Ala Leu Val Ala Phe Val Ala
1 5 10 15
Val Val Gln Ala Lys Asp Glu Pro Lys Ala His Met Gly Gln Val Val
20 25 30
Lys Lys Arg Trp Gly Glu Leu Arg Asp Phe Phe Arg Asn Asp Pro Leu
35 40 45
Gly Gln Arg Leu Val Ala Leu Gly Asn Asp Leu Thr Ala Ile Cys Gln
50 55 60
Lys Leu Gln Leu Lys Ile Arg Glu Val Leu Lys Lys Tyr Val Lys Asn
65 70 75 80
Ser Val Glu Glu Lys Asp Asp Asp Ser Lys
85 90
<210> 2
<211> 270
<212> PRT
<213>echinococcous antigen codon optimization (AgB8/2)
<400> 2
Ala Thr Gly Cys Gly Thr Ala Cys Cys Thr Ala Thr Ala Thr Thr Cys
1 5 10 15
Thr Gly Cys Thr Gly Ala Gly Cys Cys Thr Gly Gly Cys Ala Cys Thr
20 25 30
Gly Gly Thr Thr Gly Cys Ala Thr Thr Thr Gly Thr Gly Gly Cys Ala
35 40 45
Gly Thr Thr Gly Thr Gly Cys Ala Gly Gly Cys Ala Ala Ala Ala Gly
50 55 60
Ala Thr Gly Ala Ala Cys Cys Gly Ala Ala Ala Gly Cys Ala Cys Ala
65 70 75 80
Cys Ala Thr Gly Gly Gly Thr Cys Ala Gly Gly Thr Thr Gly Thr Gly
85 90 95
Ala Ala Ala Ala Ala Ala Cys Gly Thr Thr Gly Gly Gly Gly Cys Gly
100 105 110
Ala Ala Cys Thr Gly Cys Gly Thr Gly Ala Thr Thr Thr Thr Thr Thr
115 120 125
Thr Cys Gly Thr Ala Ala Thr Gly Ala Thr Cys Cys Gly Cys Thr Gly
130 135 140
Gly Gly Thr Cys Ala Gly Cys Gly Cys Cys Thr Gly Gly Thr Gly Gly
145 150 155 160
Cys Cys Cys Thr Gly Gly Gly Thr Ala Ala Thr Gly Ala Thr Cys Thr
165 170 175
Gly Ala Cys Cys Gly Cys Ala Ala Thr Thr Thr Gly Thr Cys Ala Gly
180 185 190
Ala Ala Ala Cys Thr Gly Cys Ala Gly Cys Thr Gly Ala Ala Ala Ala
195 200 205
Thr Thr Cys Gly Thr Gly Ala Ala Gly Thr Gly Cys Thr Gly Ala Ala
210 215 220
Ala Ala Ala Ala Thr Ala Thr Gly Thr Gly Ala Ala Ala Ala Ala Thr
225 230 235 240
Ala Gly Cys Gly Thr Gly Gly Ala Ala Gly Ala Gly Ala Ala Ala Gly
245 250 255
Ala Thr Gly Ala Thr Gly Ala Thr Ala Gly Thr Ala Ala Ala
260 265 270
<210> 3
<211> 55
<212> DNA
<213>artificial sequence (P1u)
<400> 3
ggccatatga tgcgtaccta tattctgctg agcctggcac tggttgcatt tgtgg 55
<210> 4
<211> 48
<212> DNA
<213>artificial sequence (P1d)
<400> 4
gtgctttcgg ttcatctttt gcctgcacaa ctgccacaaa tgcaacca 48
<210> 5
<211> 48
<212> DNA
<213>artificial sequence (P2u)
<400> 5
atgaaccgaa agcacacatg ggtcaggttg tgaaaaaacg ttggggcg 48
<210> 6
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<212> DNA
<213>artificial sequence (P2d)
<400> 6
ccagcggatc attacgaaaa aaatcacgca gttcgcccca acgtttttt 49
<210> 7
<211> 45
<212> DNA
<213>artificial sequence (P3u)
<400> 7
gtaatgatcc gctgggtcag cgcctggtgg ccctgggtaa tgatc 45
<210> 8
<211> 53
<212> DNA
<213>artificial sequence (P3d)
<400> 8
acgaattttc agctgcagtt tctgacaaat tgcggtcaga tcattaccca ggg 53
<210> 9
<211> 47
<212> DNA
<213>artificial sequence (P4u)
<400> 9
cagctgaaaa ttcgtgaagt gctgaaaaaa tatgtgaaaa atagcgt 47
<210> 10
<211> 81
<212> PRT
<213>echinococcous antigen (AgB8/1)
<400> 10
Met Leu Leu Ala Leu Ala Leu Val Ser Phe Val Val Val Thr Gln Ala
1 5 10 15
Asp Asp Gly Leu Thr Ser Thr Ser Arg Ser Val Met Lys Met Phe Gly
20 25 30
Glu Val Lys Tyr Phe Phe Glu Arg Asp Pro Leu Gly Gln Lys Val Val
35 40 45
Asp Leu Leu Lys Glu Leu Glu Glu Val Phe Gln Leu Leu Arg Lys Lys
50 55 60
Leu Arg Met Ala Leu Arg Ser His Leu Arg Gly Leu Ile Ala Glu Gly
65 70 75 80
Glu
<210> 11
<211> 243
<212> PRT
<213>echinococcous antigen codon optimization (AgB8/1)
<400> 11
Ala Thr Gly Cys Thr Gly Cys Thr Gly Gly Cys Cys Cys Thr Gly Gly
1 5 10 15
Cys Ala Cys Thr Gly Gly Thr Gly Ala Gly Cys Thr Thr Thr Gly Thr
20 25 30
Thr Gly Thr Gly Gly Thr Gly Ala Cys Cys Cys Ala Gly Gly Cys Cys
35 40 45
Gly Ala Thr Gly Ala Thr Gly Gly Cys Cys Thr Gly Ala Cys Cys Ala
50 55 60
Gly Cys Ala Cys Cys Ala Gly Cys Cys Gly Thr Ala Gly Cys Gly Thr
65 70 75 80
Gly Ala Thr Gly Ala Ala Ala Ala Thr Gly Thr Thr Thr Gly Gly Cys
85 90 95
Gly Ala Ala Gly Thr Thr Ala Ala Ala Thr Ala Thr Thr Thr Cys Thr
100 105 110
Thr Cys Gly Ala Ala Cys Gly Cys Gly Ala Thr Cys Cys Gly Cys Thr
115 120 125
Gly Gly Gly Cys Cys Ala Gly Ala Ala Ala Gly Thr Thr Gly Thr Gly
130 135 140
Gly Ala Thr Cys Thr Gly Cys Thr Gly Ala Ala Ala Gly Ala Ala Cys
145 150 155 160
Thr Gly Gly Ala Ala Gly Ala Ala Gly Thr Thr Thr Thr Thr Cys Ala
165 170 175
Gly Cys Thr Gly Cys Thr Gly Cys Gly Thr Ala Ala Ala Ala Ala Ala
180 185 190
Cys Thr Gly Cys Gly Cys Ala Thr Gly Gly Cys Ala Cys Thr Gly Cys
195 200 205
Gly Thr Ala Gly Cys Cys Ala Thr Cys Thr Gly Cys Gly Thr Gly Gly
210 215 220
Cys Cys Thr Gly Ala Thr Thr Gly Cys Cys Gly Ala Ala Gly Gly Thr
225 230 235 240
Gly Ala Ala
<210> 12
<211> 56
<212> DNA
<213>artificial sequence (P5u)
<400> 12
ggccatatgc tgctggccct ggcactggtg agctttgttg tggtgaccca ggccga 56
<210> 13
<211> 60
<212> DNA
<213>artificial sequence (P5d)
<400> 13
ccaaacattt tcatcacgct acggctggtg ctggtcaggc catcatcggc ctgggtcacc 60
<210> 14
<211> 57
<212> DNA
<213>artificial sequence (P6u)
<400> 14
gatgaaaatg tttggcgaag ttaaatattt cttcgaacgc gatccgctgg gccagaa 57
<210> 15
<211> 56
<212> DNA
<213>artificial sequence (P6d)
<400> 15
gctgaaaaac ttcttccagt tctttcagca gatccacaac tttctggccc agcgga 56
<210> 16
<211> 53
<212> DNA
<213>artificial sequence (P7u)
<400> 16
aagaagtttt tcagctgctg cgtaaaaaac tgcgcatggc actgcgtagc cat 53
<210> 17
<211> 51
<212> DNA
<213>artificial sequence (P7d)
<400> 17
gccctcgagt tcaccttcgg caatcaggcc acgcagatgg ctacgcagtg c 51
<210> 18
<211> 20
<212> DNA
<213>artificial sequence (mHVU1)
<400> 18
gatgtgaagc ttcaggagtc 20
<210> 19
<211> 20
<212> DNA
<213>artificial sequence (mHVu2)
<400> 19
caggtgcagc tgaaggagtc 20
<210> 20
<211> 15
<212> DNA
<213>artificial sequence (mHVu3)
<400> 20
caggctgaag cagtc 15
<210> 21
<211> 20
<212> DNA
<213>artificial sequence (mHVu4)
<400> 21
caggttactc tgaaagagtc 20
<210> 22
<211> 20
<212> DNA
<213>artificial sequence (mHVu5)
<400> 22
aaggtccagc tgcaacaatc 20
<210> 23
<211> 20
<212> DNA
<213>artificial sequence (mHVu6)
<400> 23
gaggtccagc tgcagcagtc 20
<210> 24
<211> 20
<212> DNA
<213>artificial sequence (mHVu7)
<400> 24
caggtccaac tgcagcagcc 20
<210> 25
<211> 20
<212> DNA
<213>artificial sequence (mHVu8)
<400> 25
gaggtgaagc tggtggagtc 20
<210> 26
<211> 20
<212> DNA
<213>artificial sequence (mHVu9)
<400> 26
gaggtgaagc tggtggaatc 20
<210> 27
<211> 20
<212> DNA
<213>artificial sequence (mHVu10)
<400> 27
gatgtgaact tggaagtgtc 20
<210> 28
<211> 21
<212> DNA
<213>artificial sequence (mHVd1)
<400> 28
tgcagagaca gtgaccagag t 21
<210> 29
<211> 21
<212> DNA
<213>artificial sequence (mHVd2)
<400> 29
tgaggagact gtgagagtgg t 21
<210> 30
<211> 21
<212> DNA
<213>artificial sequence (mHVd3)
<400> 30
tgaggagacg gtgactgagg t 21
<210> 31
<211> 21
<212> DNA
<213>artificial sequence (mHVd4)
<400> 31
tgaggagacg gtgaccgtgg t 21
<210> 32
<211> 21
<212> DNA
<213>artificial sequence (mKVu1)
<400> 32
gatgttttga tgacccaaac t 21
<210> 33
<211> 21
<212> DNA
<213>artificial sequence (mKVu2)
<400> 33
gatattgtga tgacgcaggc t 21
<210> 34
<211> 18
<212> DNA
<213>artificial sequence (mKVu3)
<400> 34
gatattgtga taacccag 18
<210> 35
<211> 21
<212> DNA
<213>artificial sequence (mKVu4)
<400> 35
gacattgtgc tgacccaatc t 21
<210> 36
<211> 21
<212> DNA
<213>artificial sequence (mKVu5)
<400> 36
gacattgtga tgacccagtc t 21
<210> 37
<211> 21
<212> DNA
<213>artificial sequence (mKVu6)
<400> 37
gatattgtgc taactcagtc t 21
<210> 38
<211> 21
<212> DNA
<213>artificial sequence (mKVu7)
<400> 38
gatatccaga tgacacagac t 21
<210> 39
<211> 21
<212> DNA
<213>artificial sequence (mKVu8)
<400> 39
gacatccagc tgactcagtc t 21
<210> 40
<211> 21
<212> DNA
<213>artificial sequence (mKVu9)
<400> 40
caaattgttc tcacccagtc t 21
<210> 41
<211> 19
<212> DNA
<213>artificial sequence (mKVd1)
<400> 41
ccgtttcagc tccagcttg 19
<210> 42
<211> 21
<212> DNA
<213>artificial sequence (mKVd2)
<400> 42
ccgttttatt tccagcttgg t 21
<210> 43
<211> 20
<212> DNA
<213>artificial sequence (mKVd3)
<400> 43
ccgttttatt tccaactttg 20
Claims (9)
1. a kind of Echinococcus hydatid cyst recombinant protein, it is characterised in that: including echinococcous antigen and Echinococcus hydatid cyst single-chain antibody；
The echinococcous antigen is the AgB8/2 antigen containing major antigenic determinant；
The Echinococcus hydatid cyst single-chain antibody is scFv.
2. a kind of based on a kind of preparation method of Echinococcus hydatid cyst recombinant protein described in claim 1, it is characterised in that: including following step
It is rapid:
(1) the AgB8/2 antigen containing major antigenic determinant in Echinococcus hydatid cyst is searched；
(2) optimize the codon of Echinococcus hydatid cyst AgB8/2 antigen gene, synthetic nucleic acid sequence is simultaneously expressed；
(3) building is directed to the single-chain antibody of Echinococcus hydatid cyst AgB8/1 antigen, transfers the gene of single-chain antibody scFv；
(4) gene of the Echinococcus hydatid cyst AgB8/2 antigen gene and the single-chain antibody scFv for Echinococcus hydatid cyst AgB8/1 antigen of recombination optimization,
And it is expressed；
(5) purifying and renaturation Echinococcus hydatid cyst recombinant protein.
3. a kind of preparation method of Echinococcus hydatid cyst recombinant protein according to claim 2, it is characterised in that: the step (2) and
Step (4) is expressed in E. coli system.
4. a kind of preparation method of Echinococcus hydatid cyst recombinant protein according to claim 3, it is characterised in that: the step (2) and
Inducing temperature when step (4) is expressed is 20 DEG C, 25 DEG C, 28 DEG C or 37 DEG C, and induction revolving speed is 250rpm, the IPTG concentration of induction
For 0.1mM.
5. a kind of preparation method of Echinococcus hydatid cyst recombinant protein according to claim 3, it is characterised in that: the inducing temperature is
25 DEG C or 37 DEG C.
6. a kind of preparation method of Echinococcus hydatid cyst recombinant protein according to claim 3, it is characterised in that: the inducing temperature is
25℃。
7. a kind of preparation method of Echinococcus hydatid cyst recombinant protein according to claim 2, it is characterised in that: the step (5)
Way of purification is selected from ion-exchange chromatography, gel permeation chromatography or affinity chromatography.
8. a kind of Echinococcus hydatid cyst recombinant protein according to claim 2 and preparation method thereof, it is characterised in that: the step (5)
Way of purification be affinity chromatography.
9. a kind of based on a kind of application of Echinococcus hydatid cyst recombinant protein described in claim 1, it is characterised in that: the recombinant protein is used
In preparation Echinococcus hydatid cyst detection kit.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811604101.4A CN109485725A (en) | 2018-12-26 | 2018-12-26 | A kind of Echinococcus hydatid cyst recombinant protein and its preparation method and application |
| CN201911284591.9A CN110790835A (en) | 2018-12-26 | 2019-12-13 | Hydatid recombinant protein and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811604101.4A CN109485725A (en) | 2018-12-26 | 2018-12-26 | A kind of Echinococcus hydatid cyst recombinant protein and its preparation method and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN109485725A true CN109485725A (en) | 2019-03-19 |
Family
ID=65712397
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|---|---|---|---|
| CN201811604101.4A Withdrawn CN109485725A (en) | 2018-12-26 | 2018-12-26 | A kind of Echinococcus hydatid cyst recombinant protein and its preparation method and application |
| CN201911284591.9A Pending CN110790835A (en) | 2018-12-26 | 2019-12-13 | Hydatid recombinant protein and preparation method and application thereof |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911284591.9A Pending CN110790835A (en) | 2018-12-26 | 2019-12-13 | Hydatid recombinant protein and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (2) | CN109485725A (en) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107033240B (en) * | 2016-02-04 | 2021-01-15 | 中国疾病预防控制中心寄生虫病预防控制所 | Echinococcus diagnostic antigen and application thereof |
| CN107286250A (en) * | 2017-06-14 | 2017-10-24 | 杭州亿米诺生物科技有限公司 | A kind of brucella fusion protein, its preparation method and application |
| CN108059684A (en) * | 2017-12-25 | 2018-05-22 | 杭州亿米诺生物科技有限公司 | A kind of bovine viral diarrhoea recombinant protein, its preparation method and application |
-
2018
- 2018-12-26 CN CN201811604101.4A patent/CN109485725A/en not_active Withdrawn
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| CN110790835A (en) | 2020-02-14 |
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