CN106699895A - Novel fusion antigen and detection kit comprising same and application - Google Patents
Novel fusion antigen and detection kit comprising same and application Download PDFInfo
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- CN106699895A CN106699895A CN201611102245.0A CN201611102245A CN106699895A CN 106699895 A CN106699895 A CN 106699895A CN 201611102245 A CN201611102245 A CN 201611102245A CN 106699895 A CN106699895 A CN 106699895A
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- C07—ORGANIC CHEMISTRY
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
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- 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
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Abstract
The invention provides a brand new fusion antigen and an immunodetection kit comprising the same. The fusion antigen comprises a fusion protein and a target antigen fragment. The expression quantity of the fusion antigen in cell supernatant is relatively high. The sensitivity and stability of the immunodetection kit prepared by use of the recombinant antigen are remarkably improved.
Description
Technical field
The invention belongs to medical science detection and diagnostic field.Specifically, the present invention relates to a kind of new fused antigen, bag
Immunity detection reagent and its application containing the fused antigen.
Background technology
Hepatitis E (abbreviation viral hepatitis type E) is also referred to as intestinal transmitted non-A and non-B hepatitis, by HEV
Caused by (Hepatitis E ViRus, HEV) infection.HEV is rare in America and Europe, but is widely current in the Far East Area, in nearly 20%
Compatriots carry hev antibody, it is meant that once infected.Pig and other animals can carry HEV, it may be possible to
This viral source of bank savings., easily there is liver failure in infection of pregnant women HEV state of an illness weights, especially third trimester of pregnancy case fatality rate is high.HEV forms
It is more similar to calicivirus with genome structure, mainly through transmission.In the acute viral hepatitis phase, occur in blood
IgM hev antibodies, can be replaced, and kept for the several years after three to the several months by IgG antibody.
HEV is a kind of RNA virus, and genome is made up of noncoding region, non-structural district and structural area, there is 3 open readings
Framework (ORF1, ORF2 and ORF3) encodes virus protein.ORF-1 holds positioned at viral genome 5 ', encodes virus nonstructural protein；
ORF2 holds positioned at viral genome 3 ', encoding virus coat proteins；ORF3 is located between ORF1 and ORF2, and both has difference
The overlap of degree, encodes viral regulatory proteins.The Serologic detection of current Hepatitis E is high still to detect based on Anti-HEV antibody
The development of the HEV immunity detection reagents of quality needs high-quality recombinant antigen.
Various countries are in terms of the research emphasis of Hepatitis E prevention and control field are concentrated mainly on vaccine and diagnosis.For many years, effectively
The work of HEV vaccine developments it is still failed, the timely and accurately diagnosis to HEV infection becomes current Hepatitis E prevention and control
Emphasis in work.In terms of Hepatitis E diagnosis, HEV antibody tests are the universal method and important means of current HEV diagnosis.
HEV Antibody screenings are carried out by various blood products and by the blood or other body fluid of inspection crowd, penta type can be effectively detected
Hepatites virus infections.
Being most widely used with indirect method and prize law in HEV immune antiboidy detection kits at present.Indirect method master
IgG is surveyed, principle is：HEV antigens are coated with enzyme exempts from reaction plate, sample to be checked is added, marked enzyme is added after incubation
, such as there is HEV antibody in secondary antibody, then HEV antibody will be adsorbed onto above solid support in sample, then by the enzyme knot on secondary antibody
Compound amplifies signal, and result of determination is obtained with ELIASA.Prize law predominantly detects IgM, and principle is to use to be coated with anti-human IgM
The reaction plate of μ chains, adds sample diluting liquid and sample to be measured, is added after incubation and resisted through the marked of enzyme dilution suitably dilution
Protoenzyme conjugate, adds nitrite ion and terminate liquid after incubation, ELIASA reads OD values.
HEV immunity detection reagent quality critical indexs are sensitivity and stability, are substantially by for the anti-of kit
Former quality is determined, and high-quality antigen needs to show outstanding in terms of activity and stability.Antigen active and stability
Constitute what is determined by the amino acid of antigen, general rule is that the recombinant antigen of vivoexpression is expressed if in supernatant
Amount is high, it is abundant to fold, and its conformation will be closer to native antigen, and activity and stability aspect are more preferably.
Due to HEV antigens, hydrophobicity is strong in itself, and expression quantity is low and poor activity, and the amount of antigen of acquisition is few, therefore most of expression
The method of HEV antigens is exactly one section of fusion protein of amalgamation and expression on antigen, forms the chimeric antigen of fusion protein-antigen, is made
The recombinant antigen expressed with the method has preferably performance in terms of soluble and expression quantity, generally adopts in the prior art
Fusion protein has the fusion protein such as TRX (thioredoxin) and GST (glutathione transferase).These fusion proteins are used
Be conducive to improving the expression quantity of antigen, but fusion protein on HEV antigen presentations for there is sensitivity not after detection reagent
The high, situation of less stable.
Therefore, HEV field of immunodetection is badly in need of the weight that can possess antigen active high, high detection sensitivity and high stability
Group antigen, to detect HEV antibody as detection reagent as envelope antigen or labelled antigen, lifts reagent quality.
The content of the invention
The invention aims to provide a kind of good, folded conformation of solubility close to natural HEV genetic engineerings restructuring
Antigen, sensitivity, the stability of existing HEV antibody assay kits are improved with it.
Another object of the present invention is to provide for a kind of expression quantity for improving recombinant protein, solubility, folding
Correct fusion protein, containing the nucleotide sequence for encoding the fusion protein, and the plasmid vector containing above-mentioned nucleotide sequence, gram
The grand host cell clone for having above-mentioned plasmid vector is sub.
In a first aspect, the present invention provides a kind of fused antigen, the fused antigen includes fusion protein and purpose antigen
Fragment,
Wherein, the fusion protein is：
(a) amino acid sequence such as SEQ ID NO：Albumen shown in 1；Or
B () is by SEQ ID NO:Amino acid sequence shown in 1 by one or several amino acid residues substitution, lack or add
Plus and formed and with the derived protein of the function of albumen (a) described.
In a particular embodiment, the purpose antigen fragment includes SEQ ID NO：Amino acid sequence shown in 2.
In a preferred embodiment, the derived protein described in (b) is by SEQ ID NO:Amino acid sequence shown in 1 passes through
1-10, more preferably 1-6, more typically 1-3, most preferably 1 substitution of amino acid residue, missing or addition and formed
And with the derived protein of the function of albumen (a) described.
In a preferred embodiment, the derived protein described in (b) is by SEQ ID NO:Amino acid sequence shown in 1 passes through
1-10, more preferably 1-6, more typically 1-3, missing or the addition of most preferably 1 amino acid residue and formed and tool
There is the derived protein of the function of (a) described albumen.
In a preferred embodiment, the derived protein described in (b) is in SEQ ID NO:The C of amino acid sequence shown in 1
End and/or N-terminal addition or missing 1-10, more preferably 1-6, more typically 1-3 is individual, most preferably 1 amino acid residue
And formed and with the derived protein of the function of albumen (a) described.
In a preferred embodiment, the derived protein described in (b) is in SEQ ID NO:The C of amino acid sequence shown in 1
End and/or N-terminal addition 1-10, more preferably 1-6, more typically 1-3, most preferably 1 amino acid residue and formed
And with the derived protein of the function of albumen (a) described.
In second aspect, the invention provides the coding nucleotide sequence of the fused antigen described in first aspect present invention.
In the third aspect, the present invention provides a kind of expression vector, and the expression vector is comprising described in second aspect present invention
Coding nucleotide sequence.
In fourth aspect, the present invention provides a kind of host cell, and the host cell is comprising described in third aspect present invention
Expression vector or coding nucleotide sequence described in second aspect present invention is integrated with genome.
At the 5th aspect, the present invention provides a kind of fusion protein, and the fusion protein is：
(a) amino acid sequence such as SEQ ID NO：Albumen shown in 1；Or
B () is by SEQ ID NO:Amino acid sequence shown in 1 by one or several amino acid residues substitution, lack or add
Plus and formed and with the derived protein of the function of albumen (a) described.
In a preferred embodiment, the derived protein described in (b) is by SEQ ID NO:Amino acid sequence shown in 1 passes through
1-10, more preferably 1-6, more typically 1-3, most preferably 1 substitution of amino acid residue, missing or addition and formed
And with the derived protein of the function of albumen (a) described.
In a preferred embodiment, the derived protein described in (b) is by SEQ ID NO:Amino acid sequence shown in 1 passes through
1-10, more preferably 1-6, more typically 1-3, missing or the addition of most preferably 1 amino acid residue and formed and tool
There is the derived protein of the function of (a) described albumen.
In a preferred embodiment, the derived protein described in (b) is in SEQ ID NO:The C of amino acid sequence shown in 1
End and/or N-terminal addition or missing 1-10, more preferably 1-6, more typically 1-3 is individual, most preferably 1 amino acid residue
And formed and with the derived protein of the function of albumen (a) described.
In a preferred embodiment, the derived protein described in (b) is in SEQ ID NO:The C of amino acid sequence shown in 1
End and/or N-terminal addition 1-10, more preferably 1-6, more typically 1-3, most preferably 1 amino acid residue and formed
And with the derived protein of the function of albumen (a) described.
At the 6th aspect, the present invention provides the coding nucleotide sequence of fusion protein described in fifth aspect present invention.
At the 7th aspect, the present invention provides a kind of immunity detection reagent, and the kit is equipped with：
Recombinant antigen described in (a) first aspect present invention；With
B () is used for other reagents of immune detection.
In a preferred embodiment, the immunity detection reagent is HEV immunity detection reagents.
In a preferred embodiment, the immunity detection reagent is also equipped with operation instructions.
In eighth aspect, the present invention is provided described in fused antigen or fifth aspect present invention described in first aspect present invention
Purposes of the fusion protein in immunity detection reagent or immunologic function test reagent is prepared.
In a preferred embodiment, the immunity detection reagent is HEV immunity detection reagents, the immune detection
Reagent is HEV immunologic function test reagents.
At the 9th aspect, the present invention provides a kind of immunologic detection method, and methods described is using first aspect present invention
Immunity detection reagent described in described fused antigen or seventh aspect present invention detects the antibody in sample.
In a preferred embodiment, the immunologic detection method can be with right and wrong diagnostic purpose.
In a preferred embodiment, the antibody is HEV antibody.
It should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the invention and have in below (eg embodiment)
Can be combined with each other between each technical characteristic of body description, so as to constitute new or preferred technical scheme.As space is limited, exist
This no longer tires out one by one states.
Brief description of the drawings
Fig. 1 is the schematic diagram of the building process of R carriers of the invention.
Fig. 2 is the schematic diagram of the building process of R-HEV expression plasmids of the invention.
Specific embodiment
Inventor's in-depth study by extensive, it was unexpectedly found that by specific fusion proteins of the invention (at this
Text is referred to as R albumen) and the fused antigen that obtains is respectively provided with significantly in terms of antigen active and stability after HEV Antigen Fusions are expressed
Advantage.Using the fused antigen as envelope antigen or labelled antigen be applied to HEV immunologic function test reagents detection HEV antibody when,
The sensitivity of detection and stability are significantly improved.The present invention is completed on this basis.
Definition
Unless otherwise indicated, the term for being used in the present invention is defined as follows.
The term " fusion protein " used in the present invention refers to for being expressed as fused antigen with purpose antigen segment composition
Albumen.
The term " fused antigen " used in the present invention refers to that above-mentioned fusion protein merges table together with purpose antigen fragment
The product for reaching.
The term " recombinant antigen " used in the present invention refers to that, using genetic engineering recombinant technique, will encode purpose antigen piece
Nucleotide sequence corresponding to section is cloned into expression vector, forms recombinant plasmid, and recombinant plasmid then is imported into host cell simultaneously
The antigen of expression.
Fusion protein of the invention
The invention provides a kind of new fusion protein, the amino acid sequence such as SEQ ID NO of the fusion protein：Shown in 1.
Inventors have surprisingly discovered that the hydrophilic amino acid of fusion protein of the invention is more, its amalgamation is good,
Conformational stability.This section of polypeptide is compared into other fusion proteins in terms of activity and stability as fusion protein obvious excellent
Gesture.
In view of the teachings of the present invention and prior art, those skilled in the art are also to be understood that " fusion protein of the invention "
The variant form of the albumen should also be included, the variant form has and " fusion protein of the invention " same or analogous work(
Can, but its amino acid sequence and SEQ ID NO:Amino acid sequence shown in 1 has a small amount of difference.These variant forms are included (but not
It is limited to)：One or more (usually 1-30, preferably 1-10, more preferably 1-6, most preferably 1-3) amino acid lack
Lose, insertion and/or replace, and one or more are added (usually within 20, preferably in C-terminal and/or N-terminal
Within 10, more preferably for 6 or 3 within) amino acid.For example, those skilled in the art know, with similar nature or similar
Amino acid replaced, for example, when isoleucine and leucine mutually replace, the function of gained protein will not be changed.Again
For example, adding one or several amino acid in C-terminal and/or N-terminal, the label for for example being added for ease of separation generally will not
Change the function of gained protein.For example, the fusion protein in the embodiment of the present application is the egg with 6*his labels in N-terminal
In vain.
In addition to the almost polypeptide of total length, the present invention should also include the active fragment of " fusion protein of the invention ".Generally,
The fragment has at least about 20 continuous amino acids of the amino acid sequence of " fusion protein of the invention ", typically at least about 30
Continuous amino acid, preferably at least about 50 continuous amino acids, more preferably at least about 80 continuous amino acids, most preferably at least about
100 continuous amino acids.
The present invention also provides the analog of " fusion protein of the invention ".These analogs and " fusion protein of the invention "
Difference can be difference on amino acid sequence, or not influence the difference on the modified forms of sequence, or and and
There is it.These polypeptides include natural or induction genetic variant.Induction variant can be obtained by various technologies, such as be passed through
Radiation produces random mutagenesis exposed to mutagens, can also be by site-directed mutagenesis or the skill of other known molecular biology
Art.Analog also includes the analog with the residue (such as D- amino acid) different from natural L-amino acids, and with non-day
The analog of amino acid (such as β, gamma-amino acid) so exist or synthesis.It should be understood that albumen of the invention be not limited to it is above-mentioned
The representative albumen for enumerating.
Modification (not changing primary structure generally) form includes：The chemically derived form such as acetyl of inner or in vitro polypeptide
Change or carboxylated.Modification also includes glycosylation.Modified forms also include having phosphorylated amino acid residue (such as phosphotyrosine,
Phosphoserine, phosphothreonine) sequence.Also include being modified so as to improve its anti-proteolysis performance or optimize molten
Solve the albumen of performance.
Therefore, in view of the teachings of the present invention and prior art, those skilled in the art can basis, such as carried out shown in following table
Amino acid substitution and produce the mutant of conservative variation.
In view of this, the fusion protein that the present invention is provided also includes such derived protein, and the derived protein is by SEQ
ID NO:Amino acid sequence shown in 1 is formed by the substitution of or several amino acid residues, missing or addition, and with ammonia
Base acid sequence such as SEQ ID NO:The function of the albumen shown in 1.
In a preferred embodiment, described derived protein is by SEQ ID NO:Amino acid sequence is by 1- shown in 1
10, more preferably 1-6, more typically 1-3, most preferably 1 substitution of amino acid residue, missing or addition and formed；
In further embodiment, described derived protein is by SEQ ID NO:Amino acid sequence shown in 1 is more excellent by 1-10
Select 1-6, more typically 1-3, missing or the addition of most preferably 1 amino acid residue and formed；In further preferred reality
Apply in mode, described derived protein is in SEQ ID NO:C-terminal and/or the N-terminal addition of amino acid sequence shown in 1 lack
Lose 1-10, more preferably 1-6, more typically 1-3, most preferably 1 amino acid residue and formed；In most preferred embodiment party
In formula, described derived protein is in SEQ ID NO:C-terminal and/or N-terminal addition 1-10 of amino acid sequence shown in 1,
More preferably 1-6, more typically 1-3, most preferably 1 amino acid residue and formed.
On the basis of fusion protein of the invention, the present invention also provides the coding nucleotide sequence of the fusion protein.
Based on present disclosure, those skilled in the art various methods can obtain fusion of the invention known to
Albumen (R albumen).Such as, but not limited to：Full genome synthesis SEQ ID NO：1 coding nucleotide sequence, by synthetic core
Nucleotide sequence is gene constructed to turning into R carriers in a carrier without antigen-4 fusion protein gene by this by digestion, connection.
Extracting vector plasmid carries out restriction enzyme digestion and electrophoresis identification and obtains positive strain.Induction destination protein expression, carries out SDS-PAGE electrophoresis sights
Examine the molecular weight and expression quantity of expressing protein.Purify corresponding genetic engineering recombinant protein R.
Fused antigen of the invention
On the basis of fusion protein of the invention, the present inventor further melts above-mentioned fusion protein and target protein
Expression is closed, discovery can improve expression quantity and solubility of the target protein in supernatant.Therefore, by the fusion protein and purpose antigen
The fused antigen that segment composition expression is obtained, sensitivity and the stability of detection can be significantly improved for immunoreagent.
In a particular embodiment, by fusion protein of the invention and such as (but not limited to) HEV ORF2 albumen
366-630 amino acids (SEQ ID NO:2) amalgamation and expression obtains fused antigen.The fused antigen is applied to the immune inspections of HEV
Survey, sensitivity and the stability of HEV detections can be significantly improved.
On the basis of fused antigen of the invention, present invention also offers the coding nucleotide sequence of the fused antigen
Row；Expression vector comprising described coding nucleotide sequence；And comprising described expression vector or the integration in genome
There is the host cell of described coding nucleotide sequence.
Those skilled in the art can prepare fused antigen of the invention using well known various modes.For example, to build
Good R carriers are template, select suitable restriction enzyme site, while expanding SQE ID NO：HEV ORF2 genetic fragments shown in 2,
And the fragment is with the restriction enzyme site for being connectable to together with R carriers, according to general molecular cloning process carrier construction.
After vector construction is good, by clone, strain culturing obtains corresponding plasmid.Suitable host strain is chosen, culture expression is simultaneously pure
Change corresponding albumen.
For example, in a particular embodiment, the preparation method includes：
A () will encode and contain SEQ ID NO：1 and SEQ ID NO：The nucleotide sequence of 2 protein fragments is connected in plasmid load
Body, forms recombinant plasmid；
B recombinant plasmid in step (a) is transferred to host cell by (), form clone's daughter cell；
C the clone's daughter cell in () incubation step (b), expresses the recombinant protein；
(d) purification of recombinant proteins.
Immunity detection reagent of the invention
Based on fusion protein of the invention and fused antigen, of the invention melt it will be appreciated by those skilled in the art that described
Hop protein and fused antigen can be used to prepare corresponding immunity detection reagent or immunologic function test reagent.
The immunity detection reagent is equipped with recombinant antigen of the invention and other reagents for immune detection.Ability
Field technique personnel should be understood that if the purpose antigen fragment in the recombinant antigen is the 366-630 of above-mentioned HEV ORF2 albumen
Amino acids (SEQ ID NO:2), then the immune detection is HEV immunity detection reagents, but not limited to this.
In further embodiment, immunity detection reagent of the invention is also equipped with operation instructions.
Those skilled in the art can be using fused antigen of the invention or immunity detection reagent or come in detecting sample
Antibody, such as, but not limited to HEV antibody.In a preferred embodiment, the immunologic detection method can be non-diagnostic mesh
.
Advantages of the present invention：
1. after fusion protein of the invention and other purposes protein fusion expression, the expression quantity of destination protein and molten can be improved
Xie Xing, is conducive to destination protein soluble-expression, makes the space conformation of purpose antigen close to native antigen；
2. using fusion protein of the invention and the activity and stability of destination protein amalgamation and expression recombinant antigen out
Etc. the antigen being superior to expressed by other modes conventional at present, so as to significantly improve the sensitivity of immune detection.
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these embodiments are merely to illustrate the present invention
Rather than limitation the scope of the present invention.The experimental technique of unreceipted actual conditions in the following example, generally according to conventional strip
Part, or document known in the art, such as condition described in various textbooks, such as Molecular Cloning:A Laboratory guide (second edition)
Condition described in (work such as J. Pehanorm Brookers)；Or according to commercially available instrument or the operation instructions of reagent, or according to
Condition proposed by manufacturer.
Embodiment 1. builds the carrier with R fusion proteins
Expression vector plasmid used in the present invention is pET-28a (+) (Novagen companies), is implementing mistake of the invention
It is not necessary to expression vector, other many expression vectors such as pQE32 (Qiagen companies) are used equally to pET-28a (+) in journey
Implement the present invention.
For SEQ ID NO：Amino acid sequence shown in 1, the password that Escherichia coli are liked is derived with biological related software
Subsequence, sequence front end adds NcoI restriction enzyme sites and 6 His sequence label CCATGGGCCATCACCACCATCACCAC,
Sequence end adds BamHI, XhoI restriction enzyme site sequence, and a terminator codon is added between two restriction enzyme sites
GGATCCTAACTCGAG, sequence send raw work biology full genome to synthesize.
It is (of the present invention each with NcoI and XhoI enzymes respectively by the synthetic vector plasmid containing target sequence
Plant molecular biology enzyme and be purchased from NEB companies) while digestion, reclaims digestion products afterwards, it is connected to NcoI and XhoI enzymes
On pET-28a carriers after cutting, by clone, PCR identification recons obtain positive clone molecule, extract plasmid, recombinated
The carrier R1 steps can be found in Fig. 1.
Embodiment 2. builds the expression plasmid containing HEV fusion antigen genes
SEQ ID NO in the gene ORF2 sections of PCR amplifications HEV：DNA fragmentation corresponding to 2, its sense primer is carried
BamHI sites, anti-sense primer carries terminator codon TAA before carrying XhoI sites and XhoI sites.The fragment of PCR is passed through back
Receive with after digestion, be connected respectively to by the R1 carriers after BamH I and XhoI digestions, by clone, PCR identification restructuring
Son, the positive colony R1-HEV for obtaining.The step can be found in Fig. 2.
Expression and purifying of the embodiment 3. containing HEV fused antigens
By R1-HEV plasmids convert BL21DE3 cells, coat kanamycin sulfate containing 50ug/ml) LB flat boards on, 37
DEG C incubated overnight, picking monoclonal, with 37 DEG C of shaken cultivations of 500ml LB culture mediums containing 50ug/ml kanamycins to OD600
About 0.8, carry out Fiber differentiation with the IPTG of final concentration of 0.5mM：37 DEG C, 3 hours.Thalline is collected by centrifugation, every liter of bacterium solution is collected
Thalline 20ml sonication buffers (50mM Tris-HCl, pH8.0,100mM NaCl) resuspended, ultrasonication, through SDS-
PAGE electroresis appraisals show fused antigen more than 70% expression in ultrasonic supernatant.
Further to prove the advantage of the active high, good stability of the fusion protein in the present invention, by above-mentioned HEV's
SEQ ID NO in ORF2：Segment fusion protein TRX, GST different with other corresponding to 2 carries out amalgamation and expression (this two respectively
The preparation process of fusion protein, similar with R1-HEV, will not be repeated here), and its result is compared into experiment carry out on
R fusion protein superiority explanations.Will R1-HEV, TRX-HEV, GST-HEV carry out induced expression and compare in identical conditions.It is right
Compare experimental procedure：R1-HEV is respectively taken, TRX-HEV, GST-HEV clone are respectively with the 500ml LB containing same concentration kanamycins
37 DEG C of shaken cultivations of culture medium are induced, inductive condition to OD600=0.8 or so using the IPTG of final concentration of 0.5mM
For：37 DEG C, 3 hours.20 minutes collects thallines of centrifugation, every liter of thalline of bacterium solution 20ml sonication buffers (50mM TRis-
HCl, pH8.0,100mM NaCl) resuspended, ultrasonication, it is centrifuged 20 minutes, supernatant fraction is collected, the warp of same volume is taken respectively
Treatment sample carries out SDS-PAGE electrophoresis, by software analysis gel, show that each fusion protein accounts for the percentage of total supernatant protein,
Result is as follows：
From the above it can be seen that, R1-HEV expression quantity of fusion protein in supernatant is better than other two kinds.
The supernatant of each recombinant protein is purified respectively：Use the 5 times of level pad of bed volume (50mM TRis-
HCl, pH8.0,100mM NaCl, 5mM imidazoles) balance Ni-NTA affinity columns, ultrasonic Supernatant samples are added, with 5 times of bed volumes
Level pad wash away uncombined albumen, then with elution buffer (50mM TRis-HCl, 100mM NaCl, 500mM miaows
Azoles, pH8.0), destination protein is eluted, protein concentration is determined, -20 DEG C save backup.The fused antigen of R1-HEV expression and purifications exists
Hereinafter abbreviated as R1-HEV Ag, other fused antigens are similar to, and are respectively designated as TRX-HEV Ag, GST-HEV Ag.
The fused antigen of embodiment 4. is coated with solid phase carrier
R1-HEV Ag fused antigens are added to carbonate buffer solution (50mM, pH9.6) with the concentration of 0.5ug/ml, is mixed
Even and room temperature 30 minutes is into coating buffer.Coating buffer is pressed into 100ul/ holes and adds enzyme reaction plate, 2~8 DEG C of coatings are overnight.It is secondary
Day sucks coating buffer, once, is then blotted with PBST (10mM PBS, 0.1%Tween-20, pH7.4) cleaning solution board-washing.To contain
The confining liquid for having 5% sucrose and 1%BSA fills every hole, and 37 DEG C are closed 1~2 hour, suck confining liquid, finally dry reaction plate
In residual liquid.Reaction plate vacuum is drained, is loaded aluminide-coating bag and is put into drier, sealed, put 2~8 DEG C of preservations, it is standby.
Other fused antigen method for coating are identical.
The coated reaction plate indirect ELISA detection HEV antibody of each fused antigen of embodiment 5.
The μ l of sample diluting liquid 100 are first added per hole in coated elisa plate, the μ l (positive serum) of sample to be measured 10 are added,
37 DEG C incubate 30 minutes；With PBST cleaning solutions board-washing five times, pat dry.Added by 100ul/ holes and contain 20% NBCS and one
The PB buffer solutions (10mM, pH7.4) of quantitative rabbit anti-human igg-HRP, 37 DEG C incubate 30 minutes；With PBST cleaning solutions board-washing five
It is secondary, pat dry.(the same R1-HEVAg of experimental technique of TRX-HEV Ag, GST-HEV Ag)
Added per hole containing 0.8% citric acid, the developer A of 1.7% sodium citrate and containing 0.2 ‰ TMB (raw work, article No.
TB0514), each 50ul of developer B of 5mM citric acids, 0.5mM EDTA-2Na, 4% absolute ethyl alcohol, 0.3% acetone, 37 DEG C are kept away
Light develops the color 10 minutes.Add 50ul per hole, the terminate liquid terminating reaction containing 2M sulfuric acid, ELIASA 450nm wavelength (reference wavelengths
OD values 630nm) are read behind blank well school zero.
TRX-HEV Ag, GST-HEV Ag are coated with solid phase carrier by the present inventor with same procedure, are detected with indirect ELISA
Above-mentioned HEV positive serums, have obtained the result of table 2, as a result show fusion protein connection HEV antigen coats detection activity of the present invention
Higher than other two kinds of fused antigens.
The remolding sensitivity of each fused antigen of table 2. coated reaction plate indirect ELISA detection HEV antibody compared with
The stability of the coated reaction plate of each fused antigen of embodiment 6.
The coated enzyme reaction plate of each fused antigen is placed 6 days in 37 DEG C, take out reaction plate equilibrium at room temperature 30 minutes with
On.Positive sample is together detected with 4 DEG C of reaction plates.Each reaction plate is placed 14 months in 2~8 DEG C, reaction plate room temperature is taken out and is put down
Weighing apparatus more than 30 minutes, detects 10 parts of positive bloods respectively, and method is ibid.The results are shown in Table 3.Experiment shows that fusion protein of the present invention connects
Shelf stability is better than other two kinds of coated reaction plates of fused antigen after connecing HEV antigen coat reaction plates.
The stability of the coated reaction plate of each fused antigen of table 3. compares
The recombinant antigen expressed after fusion protein R connections HEV destination proteins of the invention, is used for after coating reaction plate
ELISA detects that the indexs such as HEV antibody, activity, stability are superior to the HEV of the expression vectors such as TRX-HEV Ag, GST-HEV expression
Antigen.
The fused antigen of embodiment 7. marks HRP
Fused antigen mark HRP uses classics NaIO4 methods.(HRP, U.S. SIGMA are public to weigh 10mg horseradish peroxidases
Department, article No. P8375) it is dissolved in 1ml ultra-pure waters, then it is slowly added dropwise 5mg/ml NaIO4 (the raw works of 1ml ultra-pure water Fresh
Biology, article No. ST1244) solution, 4 DEG C of lucifuges add 5% ethylene glycol (raw work is biological, article No. 0518A60) solution after 30 minutes
1ml, 4 DEG C of lucifuges 30 minutes.The R1-HEVAg antigen 1s ml of 2mg/ml is added immediately after, and 4 DEG C of lucifuges are to carbonate buffer solution
(50mM, pH9.6) dialysed overnight.Next day, to the 5mg/ml NaBH4 (U.S. SIGMA that 0.2ml Fresh is added dropwise in mixture
Company, article No. 71321) solution, mix, 4 DEG C stand 2 hours.Above-mentioned solution is saturating to 4 DEG C of PBS (150mM, pH7.4)
Analysis is overnight.- 20 DEG C keep in dark place standby after adding the glycerine of final concentration 50% to mix, and are named as R1-HEV Ag-HRP, and other melt
Close antigen marking method identical with title.
Each fused antigen ELISA detection HEV antibody of the mark of embodiment 8. HRP
Prize law detects the activity of each enzyme conjugates, takes reaction plate (China of the Shanghai section biology work for being coated with Anti-human IgMμ chain
Journey limited company), the μ l of sample diluting liquid 100 are added per hole, the μ l (positive blood) of sample to be measured 10 are added, add dilute through enzyme
Each antigen enzyme of liquid suitably dilution is released, nitrite ion and terminate liquid is added, OD values are read after ELIASA.
The expression activitiy of HEV antibody is detected after each HEV fused antigens mark HRP of table 4.
R-HEV Ag, TRX-HEV Ag, GST-HEV Ag are marked upper HRP by the present inventor respectively with identical method, then
HEV positive samples are detected with Capture ELISA, the result of table 4 has been obtained, as a result shows fusion protein connection HEV bases of the present invention
Detect that activity is higher than other two kinds of fused antigens after cause and mark.
Each fused antigen shelf-stability of the mark of embodiment 8. HRP compares
Placed 6 days respectively at 37 DEG C and 2-8 DEG C after each fused antigen enzyme is diluted by a certain percentage through antigen enzyme dilution,
Take out, equilibrium at room temperature more than 30 minutes.HEV positive samples are together detected by prize law.Each fused antigen enzyme is dilute through antigen enzyme
Release liquid dilute by a certain percentage after 2-8 DEG C respectively place 6 months and 12 months, taking-up, equilibrium at room temperature more than 30 minutes.By catching
Obtain method and together detect HEV positive samples, method is ibid.The results are shown in Table 5.Experiment shows that fusion protein of the present invention connects HEV antigens
Shelf stability is significantly better than other two kinds of HRP of fused antigen mark after mark HRP.
Each HEV fused antigens mark HRP of table 5. compares through diluting rear stability
The present inventor is coated with and mark HRP by by each fused antigen R-HEV Ag, TRX-HEV Ag, GST-HEV Ag
R-HEV Ag-HRP, TRX-HEV Ag-HRP, GST-HEV Ag-HRP carry out quick heat endurance and long-term storage stabilization respectively
The research of property.Each reaction plate and each antigen enzyme are placed 6 days through the suitably dilution of enzyme dilution after 37 DEG C, reaction plate and enzyme is taken out
Detect identical positive serum as 37 DEG C of THERMAL STABILITYs, such as table 4 under identical conditions respectively.Equally, by each reaction plate
Placed after 2-8 DEG C 14 months through the suitably dilution of enzyme dilution with each antigen enzyme, detected under identical conditions respectively identical it is cloudy,
Positive quality control serum is studied as storage ability, such as table 5.Result shows fusion protein connection HEV genes of the present invention
Fused antigen is substantially better than other two kinds of fused antigens for HEV detection reagent stability.
The all documents referred in the present invention are all incorporated as reference in this application, independent just as each document
It is incorporated as with reference to such.In addition, it is to be understood that after above-mentioned instruction content of the invention has been read, those skilled in the art can
Made various changes or modifications with to the present invention, these equivalent form of values equally fall within the model that the application appended claims are limited
Enclose.
Sequence table
<110>Shanghai Kehua Bio-engineering Co., Ltd
<120>A kind of New Fusion antigen and the detection kit comprising it and application
<130> P2016-1948
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 146
<212> PRT
<213>Artificial sequence
<220>
<223>Fusion protein of the invention
<400> 1
Lys Lys Gly Gly Tyr Ala Gln Ser Gly Leu Leu Ala Glu Ile Thr Pro
1 5 10 15
Asp Lys Ala Phe Gln Asp Lys Leu Tyr Pro Phe Thr Trp Asp Ala Val
20 25 30
Tyr Asn Gly Lys Leu Ile Tyr Pro Ile Val Glu Ile Tyr Asn Lys Asp
35 40 45
Leu Leu Pro Asn Pro Pro Lys Thr Trp Glu Glu Ile Pro Ala Leu Asp
50 55 60
Lys Glu Leu Lys Ala Lys Gly Lys Glu Glu Leu Met Phe Asn Leu Gln
65 70 75 80
Glu Pro Tyr Phe Thr Trp Pro Leu Ile Ala Ala Asp Gly Gly Tyr Ala
85 90 95
Phe Lys Tyr Glu Asn Gly Lys Tyr Asp Asn Lys Asp Val Gly Val Asp
100 105 110
Asn Ala Gly Ala Lys Ala Gly Leu Thr Asp Asn Leu Val Asp Leu Ile
115 120 125
Lys Asn Lys His Met Asn Ala Asp Thr Asp Tyr Ser Ile Ala Gly Gly
130 135 140
Gly Glu
145
<210> 2
<211> 264
<212> PRT
<213>Artificial sequence
<220>
<223>The 366-630 amino acids of HEV ORF2 albumen
<400> 2
Leu His Phe Thr Gly Thr Asn Gly Val Gly Glu Val Gly Arg Gly Ile
1 5 10 15
Ala Leu Thr Leu Phe Asn Leu Ala Asp Thr Leu Leu Gly Gly Leu Pro
20 25 30
Thr Glu Leu Ile Ser Ser Ala Gly Gly Gln Leu Phe Tyr Ser Arg Pro
35 40 45
Val Val Ser Ala Asn Gly Glu Pro Thr Val Lys Leu Tyr Thr Ser Val
50 55 60
Glu Asn Ala Gln Gln Asp Lys Gly Ile Ala Ile Pro His Asp Ile Asp
65 70 75 80
Leu Gly Glu Ser Arg Val Val Ile Gln Asp Tyr Asp Asn Gln His Glu
85 90 95
Gln Asp Arg Pro Thr Pro Ser Pro Ala Pro Ser Arg Pro Phe Ser Val
100 105 110
Leu Arg Ala Asn Asp Val Leu Trp Leu Ser Leu Thr Ala Ala Glu Tyr
115 120 125
Asp Gln Thr Thr Tyr Gly Ser Ser Thr Asn Pro Met Tyr Val Ser Asp
130 135 140
Thr Val Thr Phe Val Asn Val Ala Thr Gly Ala Gln Gly Val Ser Arg
145 150 155 160
Ser Leu Asp Trp Ser Lys Val Thr Leu Asp Gly Arg Pro Leu Ile Thr
165 170 175
Ile Gln Gln Tyr Ser Lys Thr Phe Tyr Val Leu Pro Leu Arg Gly Lys
180 185 190
Leu Ser Phe Trp Glu Ala Gly Thr Thr Lys Ala Gly Tyr Pro Tyr Asn
195 200 205
Tyr Asn Thr Thr Ala Ser Asp Gln Ile Leu Ile Glu Asn Ala Ala Gly
210 215 220
His Arg Val Cys Ile Ser Thr Tyr Thr Thr Asn Leu Gly Ser Gly Pro
225 230 235 240
Val Ser Ile Ser Ala Val Gly Val Leu Ala Pro His Ser Val Leu Ala
245 250 255
Ala Leu Glu Asp Thr Val Asp Tyr
260
Claims (10)
1. a kind of fused antigen, the fused antigen includes fusion protein and purpose antigen fragment,
Wherein, the fusion protein is：
(a) amino acid sequence such as SEQ ID NO：Albumen shown in 1；Or
B () is by SEQ ID NO:Amino acid sequence shown in 1 is by the substitution of or several amino acid residues, missing or addition
Formed and with the derived protein of the function of albumen (a) described.
2. fused antigen as claimed in claim 1, it is characterised in that the purpose antigen fragment includes SEQ ID NO：2 institutes
The amino acid sequence for showing.
3. the coding nucleotide sequence of fused antigen as claimed in claim 1 or 2.
4. a kind of expression vector, the expression vector includes the coding nucleotide sequence described in claim 3.
5. a kind of host cell, the host cell is integrated with comprising the expression vector described in claim 4 or in genome
Coding nucleotide sequence described in claim 3.
6. a kind of fusion protein, the fusion protein is：
(a) amino acid sequence such as SEQ ID NO：Albumen shown in 1；Or
B () is by SEQ ID NO:Amino acid sequence shown in 1 is by the substitution of or several amino acid residues, missing or addition
Formed and with the derived protein of the function of albumen (a) described.
7. the coding nucleotide sequence of fusion protein described in claim 6.
8. a kind of immunity detection reagent, the kit is equipped with：
Recombinant antigen described in (a) claim 1 or 2；With
B () is used for other reagents of immune detection.
9. the fusion protein described in the fused antigen or claim 6 described in claim 1 or 2 is preparing immunity detection reagent
Or the purposes in immunologic function test reagent.
10. a kind of immunologic detection method, methods described is using the fused antigen or claim 8 described in claim 1 or 2
Described immunity detection reagent detects the antibody in sample.
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| US20060270595A1 (en) * | 2001-12-18 | 2006-11-30 | Denis Jullien | Nucleic acids encoding compositions of THAP-family chemokine binding domains |
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| US20140017269A1 (en) * | 2012-07-11 | 2014-01-16 | Ming Tan | Protein complex system for increased immunogenicity and functionality, and methods making and use |
| CN104857510A (en) * | 2015-04-23 | 2015-08-26 | 张澍 | Hepatitis E virus-like particle vaccine preparation method and application |
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|---|---|---|---|---|
| US20060270595A1 (en) * | 2001-12-18 | 2006-11-30 | Denis Jullien | Nucleic acids encoding compositions of THAP-family chemokine binding domains |
| CN101497891A (en) * | 2008-02-03 | 2009-08-05 | 深圳市源兴生物医药科技有限公司 | Novel recombinant viral vector and use thereof |
| CN101403746A (en) * | 2008-07-18 | 2009-04-08 | 深圳市菲鹏生物股份有限公司 | Conjugate used for immunity detection |
| CN102807621A (en) * | 2011-06-01 | 2012-12-05 | 厦门大学 | Fusion protein containing diphtheria toxin non-toxic mutant cross-reacting material 197 (CRM197) or segment thereof |
| US20140017269A1 (en) * | 2012-07-11 | 2014-01-16 | Ming Tan | Protein complex system for increased immunogenicity and functionality, and methods making and use |
| CN103235120A (en) * | 2013-04-22 | 2013-08-07 | 苏州华益美生物科技有限公司 | Kit for compound detection of hepatitis E virus antibody profile as well as application of kit |
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