CN105461805B - Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus and its application - Google Patents
Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus and its application Download PDFInfo
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- CN105461805B CN105461805B CN201510948571.2A CN201510948571A CN105461805B CN 105461805 B CN105461805 B CN 105461805B CN 201510948571 A CN201510948571 A CN 201510948571A CN 105461805 B CN105461805 B CN 105461805B
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
Abstract
The present invention provides the variable region sequences of the mouse monoclonal antibody of specific binding Porcine epidemic diarrhea virus, the antibody or antibody fragment of its preparation, and vaccine and kit using the Antibody preparation.Pharmaceutical composition of the invention can be effectively prevented and treated Porcine epidemic diarrhea virus infection.Kit of the invention can effectively detect Porcine epidemic diarrhea virus, and high sensitivity, detection is quickly.
Description
Technical field
The present invention relates to Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus, and the drug prepared using the monoclonal antibody
The application such as composition, kit, belongs to biomedicine field.
Background technique
Pig epidemic diarrhea (porcine epidemic diarrhea, PED) is by Porcine epidemic diarrhea virus
A kind of enteric infectious disease caused by (porcine epidemic diarrhea virus, PEDV), mainly causes suckling pig to be vomitted
It spits, watery diarrhea, the symptoms such as dehydration, the death of piglet can be caused when serious, the death rate can reach 50%-100%.In recent years, certainly
Winter in 2010, the most area eruption and prevalence in China are in succession also a wide range of in the area such as South Korea, Japan, Taiwan later
Break out this disease.The first half of the year in 2013 also breaks out this disease in north America region, and propagates rapidly, causes seriously to the aquaculture in the whole world
Loss.
Currently, the method for detection Porcine epidemic diarrhea virus includes Virus Isolation and serum inspection, but take long time；
PCR method is although sensitive, but needs expensive instrument and professional operator.Thus, it needs to develop a kind of letter in the prior art
Product that is single, quickly, accurately detecting Porcine epidemic diarrhea virus.
Summary of the invention
In order to solve the deficiencies in the prior art, the present invention provides two Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus,
And kit, pharmaceutical composition and its application containing monoclonal antibody.
The present invention relates to it is a kind of specifically bind Porcine epidemic diarrhea virus mouse monoclonal antibody PEDV-McAB1 can
Become region sequence, wherein 1) heavy chain variable amino acid sequence is that amino acid sequence shown in SEQ ID No.2 or the sequence are passed through
One or more amino acid additions are deleted, the conservative variant of replacement or modification conservative mutation acquisition；2) light chain variable region
Amino acid sequence be amino acid sequence or sequence shown in SEQ ID No.4 by one or more amino acid additions, delete,
The conservative variant that replacement or modification conservative mutation obtain.
The invention further relates to antibody or its segment with above-mentioned variable region sequences, the antibody or its segment still keep special
The opposite sex combines the ability of Porcine epidemic diarrhea virus.
The invention further relates to a kind of mouse monoclonal antibody PEDV-McAB2's for specifically binding Porcine epidemic diarrhea virus
Variable region sequences, wherein 1) heavy chain variable amino acid sequence is amino acid sequence shown in SEQ ID No.6 or sequence warp
One or more amino acid additions are crossed, deletes, replace or modify the conservative variant that conservative mutation obtains；2) light chain variable
Region amino acid sequence is that amino acid sequence shown in SEQ ID No.8 or the sequence are added by one or more amino acid, deleted
Remove, replace or modify the conservative variant of conservative mutation acquisition.
The invention further relates to antibody or its segment with above-mentioned variable region sequences, the antibody or its segment still keep special
The opposite sex combines the ability of Porcine epidemic diarrhea virus.
The invention further relates to a kind of pharmaceutical compositions, wherein described pharmaceutical composition includes immune amount containing described
The segment and pharmaceutically acceptable load of the antibody of the variable region sequences of mouse monoclonal antibody PEDV-McAB2 or the antibody
Body.
It is related in preparation prevention and/or treatment Porcine epidemic diarrhea virus infection that the invention further relates to described pharmaceutical compositions
Application in the drug of disease.
The invention further relates to a kind of kits, wherein the kit includes a effective amount of with mouse monoclonal antibody
The segment of the antibody of the variable region sequences of PEDV-McAB1 or the antibody and/or a effective amount of there is mouse monoclonal antibody
The antibody of the variable region sequences of PEDV-McAB2 or the segment of the antibody, and for Porcine epidemic diarrhea virus
Carry out detection reagent, the negative control, positive control that antigen-antibody reaction is detected.
The invention further relates to a kind of kits, wherein the kit includes a effective amount of with mouse monoclonal antibody
The segment of the antibody of the variable region sequences of PEDV-McAB1 or the antibody and/or a effective amount of there is mouse monoclonal antibody
The segment of the antibody of the variable region sequences of PEDV-McAB2 or the antibody, and for resisting to Porcine epidemic diarrhea virus
Detection reagent that antigen-antibody reaction is detected, negative control, positive control, wherein described for Porcine Epidemic Diarrhea
It is the enzyme mark two in conjunction with the segment of the antibody or the antibody that poison, which carries out the detection reagent that antigen-antibody reaction is detected,
Anti- and enzyme with the label generates the substrate of color reaction, and the ELIAS secondary antibody includes anti-, enzyme mark sheep more than enzyme mark sheep anti mouse
Anti- mouse secondary antibody.
The invention further relates to a kind of kits, wherein the kit includes a effective amount of monoclonal antibody PEDV-
McAB1, a effective amount of monoclonal antibody PEDV-McAB2, and for carrying out antigen-antibody to Porcine epidemic diarrhea virus
React the detection reagent detected；Wherein, the kit includes colloidal gold colloidal gold detection test paper strip, the colloidal gold test
Item includes bottom plate, and the bottom plate has a first end and a second end, and is successively had along the first end on the direction of second end
Filter paper, sample pad, gold-labelled pad, nitrocellulose filter and water absorption pad, the nitrocellulose filter is contacted with gold-labelled pad or and sample
Pad, gold-labelled pad contact are so that the combination physical efficiency of antigen and the monoclonal antibody PEDV-McAB2 are moved to bottom plate second end on it
It moves；The monoclonal antibody PEDV-McAB2 in the gold-labelled pad containing colloid gold label, it is close on the nitrocellulose filter
The position of the bottom plate second end includes a detection line and a nature controlling line, and immobilization has the monoclonal in the detection line
Antibody PEDV-McAB1, immobilization has sheep anti mouse secondary antibody or sheep anti mouse mostly anti-on the nature controlling line.
The invention further relates to a kind of kits, wherein the kit includes a effective amount of monoclonal antibody PEDV-
McAB1, a effective amount of monoclonal antibody PEDV-McAB2, and for carrying out antigen-antibody to Porcine epidemic diarrhea virus
React the detection reagent detected；Wherein, the kit includes buffer supply unit and enzyme immunity chromatography detection test paper
Item；The buffer supply unit is used to buffer supplying the enzyme immunochromatographydetecting detecting test strip；The enzyme immunochromatography
Test strip includes nitrocellulose filter (1), and the enzyme immunochromatographydetecting detecting test strip successively includes that substrate supplies in the longitudinal direction
Answer area, sample supply area, detection zone；The substrate supply area includes substrate pad (3), is adsorbed with dry zymolyte, institute thereon
It states substrate pad (3) to contact with nitrocellulose filter (1), the zymolyte is dissolved in buffer and on nitrocellulose filter (1)
To the distal migration away from the buffer supply unit；The sample supply area includes enzyme mark pad (2), is adsorbed with enzyme label thereon
The monoclonal antibody PEDV-McAB2, the zymolyte can produce with the enzyme that marks on the monoclonal antibody PEDV-McAB2
Raw chromogenic reaction, the enzyme mark pad (2) contact with nitrocellulose filter (1), and the monoclonal antibody PEDV-McAB2 is dissolved in slow
To the distal migration away from the buffer supply unit in fliud flushing and on nitrocellulose filter (1)；And the detection zone is solid
Fixedization has the monoclonal antibody PEDV-McAB1；Wherein, the buffer supply unit is slow including expansion fluid cushion (5), substrate
Fliud flushing slot (8), substrate buffer solution (9) and buffer button, the substrate buffer solution (9) are placed in substrate buffer liquid bath (8),
The buffer button is located at the top of substrate buffer solution slot (8), and buffer (9) can be immersed for the expansion fluid cushion (5) by pressing
In；The detection zone includes detection line (6), nature controlling line (7), wherein the nature controlling line (7) is compared with detection line (6) further from described
Sample supply area, immobilization has the monoclonal antibody PEDV-McAB1 on the detection line (6), in the nature controlling line (7)
It is mostly anti-that upper immobilization has sheep anti mouse secondary antibody or sheep anti mouse, and the monoclonal antibody for the enzyme label being adsorbed on enzyme mark pad (2)
PEDV-McAB2 is excessive for the monoclonal antibody PEDV-McAB1 being fixed on detection line (6)；The nitre
Acid cellulose film (1) full section is adhered on above support (10), and supporter (10) connects the buffer supply unit, substrate
Supply area, sample supply area, detection zone and water absorption pad (4), the water absorption pad (4) away from the buffer supply unit most
Distally；And the position of test sample (11) addition is the position of the enzyme mark pad (2).
The invention further relates to a kind of kits, wherein the kit includes being coated with the monoclonal antibody PEDV-
The elisa plate of McAB1, the reaction solution that the monoclonal antibody PEDV-McAB2 is marked containing enzyme, to Porcine epidemic diarrhea virus into
Detection reagent that row antigen-antibody reaction is detected, negative control, positive control.
The invention further relates to a kind of kits, wherein the kit includes coating Porcine epidemic diarrhea virus antigen
Elisa plate, the reaction solution containing the monoclonal antibody, cleaning solution, dilution, substrate developing solution, terminate liquid, negative control, sun
Property control；Wherein, the reaction solution containing the monoclonal antibody for the reaction solution containing the monoclonal antibody PEDV-McAB1 or contains
The reaction solution of the monoclonal antibody PEDV-McAB2 contains the monoclonal antibody PEDV-McAB1 and the monoclonal antibody
The mixed reaction solution of PEDV-McAB2.
The invention further relates to application of the kit in the Porcine epidemic diarrhea virus detection for non-diagnostic purpose.
Beneficial effects of the present invention: the kit containing Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus of the present invention can be used for non-
Application or Epitope Identification research in the Porcine epidemic diarrhea virus detection of diagnostic purpose；Containing porcine epidemic diarrhea resisting of the present invention
The pharmaceutical composition of viral monoclonal antibodies can solve the piglet pig epidemic caused when existing vaccine, maternal antibody deficiency
The problem of diarrhea virus infects.
Detailed description of the invention
Fig. 1 is PAGE gel electroresis appraisal monoclonal antibody PEDV-McAB1's and monoclonal antibody PEDV-McAB2
Purity, wherein swimming lane 1 is albumen Marker；Swimming lane 2 is monoclonal antibody PEDV-McAB1, heavy chain and lower end including upper end
Light chain, swimming lane 3 are monoclonal antibody PEDV-McAB2, the light chain of heavy chain and lower end including upper end.
Fig. 2 is the side schematic view of enzyme immunochromatographydetecting detecting test strip, in figure: (1) nitrocellulose filter, (2) enzyme mark pad,
(3) substrate pad, (4) water absorption pad, (5) be unfolded fluid cushion, (6) detection line, (7) nature controlling line, (8) substrate buffer liquid bath, and (9) substrate is slow
Fliud flushing, (10) supporter, (11) test sample.
Specific embodiment
Hereinafter, embodiments of the present invention will be described.
The present invention relates to it is a kind of specifically bind Porcine epidemic diarrhea virus mouse monoclonal antibody PEDV-McAB1 can
Become region sequence, wherein 1) heavy chain variable amino acid sequence is that amino acid sequence shown in SEQ ID No.2 or the sequence are passed through
One or more amino acid additions are deleted, the conservative variant of replacement or modification conservative mutation acquisition；2) light chain variable region
Amino acid sequence be amino acid sequence or sequence shown in SEQ ID No.4 by one or more amino acid additions, delete,
The conservative variant that replacement or modification conservative mutation obtain.
Term " Porcine epidemic diarrhea virus " (porcine epidemic diarrhea virus, PEDV) belongs to coronal
Viraceae (Coronaviridae) coronavirus genus (Coronavirus), be single strand plus RNA virus, easily cause with vomit,
The pig epidemic diarrhea disease that diarrhea, dehydration, high death are characterized.
Term " monoclonal antibody " refers to the antibody obtained from substantially homologous antibody population, that is, forms the antibody of the group
Body is all identical, in addition to there may be a small amount of possible spontaneous mutations.Therefore, modifier " monoclonal " refers to the property of the antibody not
It is the mixture of discrete antibody.Preferably, the monoclonal antibody includes unit price or single-chain antibody, double-chain antibody, chimeric
Antibody, the derivative of humanized antibody and above-mentioned antibody, functional equivalent and homologue also include antibody fragment and are contained anti-
Any polypeptide of former binding structural domain.Antibody be cover any specific binding of the binding structural domain with required specificity because
Son, thus, the function that this term covers antibody fragment homologous therewith, derivative, humanized antibody and antibody is equivalent
Object and homologue also include any polypeptide containing antigen-binding domains, either natural to be still synthetically produced.Antibody
Example be immunoglobulin hypotype (such as IgG, IgE, IgM, IgD and IgA) and its isotype sub-classes；It is also possible to comprising antigen knot
Close segment such as Fab, scFv, Fv, dAb, Fd of structural domain；With double-chain antibody (diabodies).It is fused to another polypeptide, packet
Chimer molecules or equivalent containing antigen-binding domains are also included within wherein.The cloning and expression of chimeric antibody exists
It is described in EP.A.0120694 and EP.A.0125023.Antibody can be modified in many ways, can be produced with DNA recombinant technique
The raw other antibody or chimeric molecule for retaining original antibody specificity.This technology may include by the immune globulin of encoding antibody
The DNA of white variable region or complementarity-determining region (CDRs) introduces the constant region of different immunoglobulins or constant region adds framework region,
Referring to EP.A.184187, GB2188638A or EP.A.239400.To hybridoma or the other thin of antibody can also be generated
Born of the same parents carry out genetic mutation or other changes, the binding specificity of antibody produced by this can change or does not change.For this hair
Bright " monoclonal antibody " can also be made with hybridoma method, because the DNA sequence dna of coding source of mouse antibody of the present invention can use this
Conventional means known to the technical staff of field, the artificial synthesized nucleotide sequence of amino acid sequence as disclosed according to the present invention or use
PCR method expands to obtain, thus can also use recombinant DNA method, can be connected into the sequence properly with various methods well known in the art
Expression vector in.Finally, culture converts resulting host cell under conditions of being suitble to antibody expression of the present invention, then originally
Field technical staff isolates and purifies means using well known routine and purifies to obtain monoclonal antibody of the invention.Antibody includes to pass through
The polypeptide chain solid that disulfide-bridged, two is connected together, referred to as the two of light chain and heavy chain polypeptide backbone constitute all main of antibody
Structured sort (isoreagent).Heavy chain and light chain all can further be divided into some subprovinces of referred to as variable region and constant region.Heavy chain packet
Single variable region and three different constant regions are included, light chain then includes single variable region (different from the variable region of heavy chain) and list
A constant region (different from the constant region of heavy chain).It is responsible for the binding specificity of antibody in the variable region of heavy chain and light chain.
Term " heavy chain variable region " refers to a kind of polypeptide, and the length is 110 to 125 amino acid, amino acid sequence phases
It should be in the monoclonal antibody of the present invention sequence of the heavy chain amino since heavy chain N-terminal amino acid.Equally, term " light chain variable
Area " refers to a kind of polypeptide, the length is 95 to 115 amino acid, amino acid sequence corresponding to monoclonal antibody of the present invention from
The light chain amino acid sequence that light chain N-terminal amino acid starts.Those of ordinary skill in the art obviously know, specific in institute of the invention
In the heavy chain variable region and chain variable region amino acid sequence basis of disclosed monoclonal antibody, conventional gene engineering can be passed through
The modifications such as addition, deletion, the replacement of one or more amino acid are carried out with protein engineering method, obtain conservative variant,
And it still is able to keep specifically binding with Porcine epidemic diarrhea virus.Monoclonal antibody in the present invention further includes its active fragment
Or conservative variant.
Term " conservative variant ", which refers to, substantially remains its maternal characteristic, and such as basic immunology biology is special
The variant of property, architectural characteristic, control characteristic or biochemical characteristic.Generally, the amino acid sequence of the conservative variant of polypeptide
Different from maternal polypeptide, but difference is limited, so that generally very with the sequence of maternal polypeptide and conservative variant
It is similar, and be identical in many regions.Difference on conservative variant and maternal polypeptid acid sequence can be example
Such as: replacement, addition and the deletion of one or more amino acid residues and any combination thereof.The amino acid residue of replacement or insertion can
It, can not also be by genetic code encoding by genetic code encoding.The conservative variant of polypeptide can generate naturally or it can be with
It is non-spontaneous variant.The conservative variant of the non-natural generation of polypeptide can synthesize by induced-mutation technique or directly
It generates.
The present invention relates to a kind of by weight chain variabl area sequence or its conservative in the PEDV-McAB1 variable region sequences
The antibody of light-chain variable sequence or its conservative variant composition in variant and/or the variable region sequences；It is described anti-
Body can be monoclonal antibody, genetic engineering antibody；Wherein, the genetic engineering antibody includes single-chain antibody, chimeric monoclonal
Antibody, reshaping monoclonal antibody, the segment of pig resource monoclonal antibody or the antibody；The segment of the antibody or the antibody is still
Keep the ability of specific binding Porcine epidemic diarrhea virus.
As one embodiment of the present invention, the antibody is monoclonal antibody PEDV-McAB1, and the monoclonal is anti-
The amino acid sequence of body PEDV-McAB1 heavy chain variable region is SEQ ID No.2, and the amino acid sequence of light chain variable region is SEQ
ID No.4。
The relative affinity constant of the monoclonal antibody PEDV-McAB1 is 0.78 μ g/ml, that is to say, that the Dan Ke
The bond strength of the antigenic determinant of grand antibody PEDV-McAB1 and antigen is very high, popular better than anti-pig in the prior art
The bond strength of diarrhea virus monoclonal antibody；The neutralize antibody titers of the monoclonal antibody are greater than 1:512, that is to say, that institute
Monoclonal antibody PEDV-McAB1 is stated with good neutralization activity, it is thin to can inhibit Porcine epidemic diarrhea virus superinfection target
Born of the same parents.
Term " genetic engineering antibody " can be by the method for genetic engineering by host cell expression appropriate.The present invention
A variety of expression host cells, such as prokaryotic host cell, including but not limited to Escherichia coli, bacillus, streptomycete can be used
The bacterial strain of category etc.；The bacterial strain and mammalian cell of eucaryon host, including but not limited to aspergillus, saccharomycete etc., plant are thin
Born of the same parents etc..The invention is not limited to specific expression vector and expressive hosts, as long as it can express gene of the present invention
Engineered antibody, conservative variant.
Term " neutralization activity " refers to that neutralizing antibody has the function of neutralizing virus, wherein " neutralizing antibody " is in this paper with most
Broad sense uses, and refers to any antibody for inhibiting Porcine epidemic diarrhea virus superinfection target cell, neutralizes but regardless of realization
Mechanism.Thus, for example, it can be realized and be neutralized by inhibiting virus to be affixed or adhered to cell surface, such as pass through design
Antibody, the antibody are bonded directly to, or close to, it is responsible for the site of virus attachment or adherency, it can also be by being directed to
The antibody on the surface virion (Virion) neutralizes to realize, leads to the aggregation of virion, can be by inhibiting virus to be attached to target
Cell is with restrovirus and cell membrane fusion, by inhibiting encytosis (endocytosis) to inhibit the progeny virus etc. of infection, into
One step neutralizes.Neutralizing antibody of the invention is not only restricted to realize the mechanism neutralized.
The present invention relates to it is a kind of specifically bind Porcine epidemic diarrhea virus mouse monoclonal antibody PEDV-McAB2 can
Become region sequence, wherein 1) heavy chain variable amino acid sequence is that amino acid sequence shown in SEQ ID No.6 or the sequence are passed through
One or more amino acid additions are deleted, the conservative variant of replacement or modification conservative mutation acquisition；2) light chain variable region
Amino acid sequence be amino acid sequence or sequence shown in SEQ ID No.8 by one or more amino acid additions, delete,
The conservative variant that replacement or modification conservative mutation obtain.
The present invention relates to a kind of by weight chain variabl area sequence or its conservative in the PEDV-McAB2 variable region sequences
The antibody of light-chain variable sequence or its conservative variant composition in variant and/or the variable region sequences；It is described anti-
Body can be monoclonal antibody, genetic engineering antibody；Wherein, the genetic engineering antibody includes single-chain antibody, chimeric monoclonal
Antibody, reshaping monoclonal antibody, the segment of pig resource monoclonal antibody or the antibody；The segment of the antibody or the antibody is still
Keep the ability of specific binding Porcine epidemic diarrhea virus.
As one embodiment of the present invention, the antibody is monoclonal antibody PEDV-McAB2, and the monoclonal is anti-
The amino acid sequence of body PEDV-McAB2 heavy chain variable region is SEQ ID No.6, and the amino acid sequence of light chain variable region is SEQ
ID No.8。
The relative affinity constant of the monoclonal antibody PEDV-McAB2 is 0.44 μ g/ml, that is to say, that the Dan Ke
The bond strength of the antigenic determinant of grand antibody PEDV-McAB2 and antigen is very high, popular better than anti-pig in the prior art
The bond strength of diarrhea virus monoclonal antibody.
The present invention relates to a kind of pharmaceutical compositions, wherein described pharmaceutical composition includes that having for immune amount is described
The antibody of PEDV-McAB1 variable region sequences or the segment and pharmaceutically acceptable carrier of the antibody.
Term " immune amount " be when being interpreted as " prevention effective dose " refer to be adequate to bring about in the individual of inoculation it is immune
Protect the amount of reaction.As known to those skilled in the art, described " prevention effective dose " is with the mode of immunity inoculation, opportunity, administration pair
As and the difference of the monoclonal antibody or its segment and it is different, in conjunction with document known in the art and introduction and corresponding
Clinical procedure, those skilled in the art should can obtain " preventing effective for monoclonal antibody used by limited test
Amount ".
Term " immune amount " be when being interpreted as " therapeutically effective amount " refer to generate individual test subjects effective protection and
Neutralize the amount of virus.As known to those skilled in the art, described " therapeutically effective amount " with therapeutic scheme, the course of disease, treatment object shape
The difference of condition and monoclonal antibody used or its segment and it is different.In conjunction with document known in the art and introduction and accordingly
Clinical procedures, clinical technician should can obtain " therapeutically effective amount " of monoclonal antibody used by its experience.
Term " pharmaceutically acceptable carrier " refer to do not stimulate body do not hinder using compound biological activity and
The carrier or diluent of characteristic.
As one embodiment of the present invention, described pharmaceutical composition includes: the monoclonal antibody of immune amount
PEDV-McAB1 and pharmaceutically acceptable carrier.
As one embodiment of the present invention, described pharmaceutical composition includes the monoclonal antibody of immune amount
The single-chain antibody and pharmaceutically acceptable carrier of the heavy chain variable region preparation of PEDV-McAB1.
As a kind of preferred embodiment of the invention, described pharmaceutical composition includes the monoclonal antibody of immune amount
The single-chain antibody and pharmaceutically acceptable carrier of heavy chain variable region and the light-chain variable sequence preparation of PEDV-McAB1.
As a kind of preferred embodiment of the invention, described pharmaceutical composition includes the monoclonal antibody of immune amount
The single-stranded of the light-chain variable sequence of the heavy chain variable region of PEDV-McAB1 and the monoclonal antibody PEDV-McAB2 preparation resists
Body and pharmaceutically acceptable carrier.
As one embodiment of the present invention, described pharmaceutical composition is through intramuscular injection or intraperitoneal injection.
As one embodiment of the present invention, described pharmaceutical composition includes but is not limited to powder agent, granule, ball
Agent, tablet, capsule.
Term " prevention and/or treatment " refers to inhibition Porcine Epidemic Diarrhea when being related to Porcine epidemic diarrhea virus infection
The duplication of poison, the propagation for inhibiting Porcine epidemic diarrhea virus prevent Porcine epidemic diarrhea virus from settling down in its host, with
And mitigate the disease of Porcine epidemic diarrhea virus infection or the symptom of illness.If viral loads are reduced, illness mitigates and/or takes the photograph
Appetite and/or growth increase, then can think that the treatment has reached therapeutic effect.
Term " pig " refers to any animal for belonging to Suidae (Suidae) member, such as pig.
The present invention relates to described pharmaceutical compositions to infect related disease in preparation prevention and/or treatment Porcine epidemic diarrhea virus
Application in the drug of disease.
As one embodiment of the present invention, the present invention provides the monoclonal antibody PEDV- containing immune amount
The pharmaceutical composition of McAB1 answering in the drug of preparation prevention and/or treatment Porcine epidemic diarrhea virus infection related disease
With.
As one embodiment of the present invention, the present invention provides the monoclonal antibody PEDV- containing immune amount
The single-chain antibody of the heavy chain variable region preparation of McAB1 infects related disease in preparation prevention and/or treatment Porcine epidemic diarrhea virus
Application in the drug of disease.
As one embodiment of the present invention, the present invention provides the monoclonal antibody PEDV- containing immune amount
The single-chain antibody of heavy chain variable region and the light-chain variable sequence preparation of McAB1 is in preparation prevention and/or treatment pig epidemic
Diarrhea virus infects the application in the drug of related disease.
As one embodiment of the present invention, the present invention provides the monoclonal antibody PEDV- containing immune amount
The heavy chain variable region of McAB1 and the single-chain antibody prepared with the light-chain variable sequence of the monoclonal antibody PEDV-McAB2 exist
Application in the drug of preparation prevention and/or treatment Porcine epidemic diarrhea virus infection related disease.
It is anti-that pharmaceutical composition containing Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus of the present invention can solve existing vaccine, source of parents
The problem of piglet Porcine epidemic diarrhea virus infection caused when body deficiency.
The present invention relates to a kind of kits, wherein the kit includes a effective amount of with the PEDV-McAB1
The antibody of variable region sequences or the segment of the antibody and/or a effective amount of have the PEDV-McAB2 variable
The antibody of region sequence or the segment of the antibody, and for carrying out antigen-antibody reaction to Porcine epidemic diarrhea virus
The detection reagent that is detected, negative control, positive control；Wherein, described for carrying out antigen with Porcine epidemic diarrhea virus
The detection reagent that antibody response is detected is the substrate that color reaction is generated with the enzyme of the label.
As one embodiment of the present invention, the kit includes a effective amount of monoclonal antibody PEDV-
McAB1 and/or a effective amount of monoclonal antibody PEDV-McAB2, and for resisting to Porcine epidemic diarrhea virus
Detection reagent that antigen-antibody reaction is detected, negative control, positive control.
Term " effective quantity " is to refer to have using monoclonal antibody of the present invention when being interpreted as " diagnosis effective quantity "
Imitate the amount that whether there is Porcine epidemic diarrhea virus in test sample.According to known immunochemistry detection method, this field skill
Art personnel understand that the amount of monoclonal antibody used is different with the difference of the specific immunologic detection method of use, according to public affairs
Know the introduction of document, how to select monoclonal antibody dosage of the present invention appropriate, as known to those skilled in the art to be used for
It diagnoses and whether there is Porcine epidemic diarrhea virus in sample.As known to those skilled in the art, suitably should also in the kit
Including carrier appropriate, buffer/agent, and reagent and operation instructions for signal produced by detecting.Examination of the present invention
Used detection method can be used enzyme-linked when whether there is the amount of Porcine epidemic diarrhea virus in the effective test sample of agent box
Immunosorbent assay (ELISA), chemiluminescence immunoassay detection, Placenta function, fluorescence immunoassay detection, is exempted from enzyme immune detection
Epidemic disease chromatography, competition law and similar detection method.
Term " enzyme " is include horseradish peroxidase, alkaline phosphatase and beta-D-galactosidase any.
Term " buffer " includes " phosphate buffer ", refers to containing phosphoric acid or its salt and is brought to the molten of desired pH
Liquid is the most widely used a kind of buffer in biochemical research.Generally, phosphate buffer is from phosphoric acid or phosphoric acid
Salt (including but not limited to sodium and sylvite) preparation.Some phosphate, such as sodium dihydrogen phosphate and phosphoric acid has been known in the art
Potassium dihydrogen, disodium hydrogen phosphate and dipotassium hydrogen phosphate, sodium phosphate and potassium phosphate.Know that phosphate is in the form of the hydrate of salt
It is existing.Due to the second level dissociation of buffer, the pH value range of buffering is very wide, the range of for example, about pH4 to about pH10, excellent
Select the range of about pH5 to pH9, more preferably from about pH6 to the range of about pH8, most preferably from about pH7.4.It is further preferred that the phosphorus
Phthalate buffer is the phosphate buffer of sodium chloride-containing and potassium chloride.
The present invention relates to a kind of kits, wherein the kit includes a effective amount of with the PEDV-McAB1
The antibody of variable region sequences or the segment of the antibody and/or a effective amount of have the PEDV-McAB2 variable
The antibody of region sequence or the segment of the antibody, and for carrying out antigen-antibody reaction to Porcine epidemic diarrhea virus
The detection reagent that is detected, negative control, positive control；Wherein, described for carrying out antigen to Porcine epidemic diarrhea virus
The detection reagent that antibody response is detected in conjunction with the segment of the antibody or the antibody ELIAS secondary antibody and with institute
The enzyme for stating label generates the substrate of color reaction, and the ELIAS secondary antibody includes anti-, enzyme mark sheep anti mouse secondary antibody more than enzyme mark sheep anti mouse.
As one embodiment of the present invention, the kit includes a effective amount of monoclonal antibody PEDV-
McAB1 and/or a effective amount of monoclonal antibody PEDV-McAB2, and for resisting to Porcine epidemic diarrhea virus
Detection reagent that antigen-antibody reaction is detected, negative control, positive control.
The present invention relates to a kind of kit, the kit include a effective amount of monoclonal antibody PEDV-McAB1,
A effective amount of monoclonal antibody PEDV-McAB2, and for Porcine epidemic diarrhea virus carry out antigen-antibody reaction into
The detection reagent of row detection；Wherein, the kit includes colloidal gold colloidal gold detection test paper strip, and the colloidal gold colloidal gold detection test paper strip includes
Bottom plate, the bottom plate have a first end and a second end, and successively have filter paper, sample on the direction of second end along the first end
Product pad, gold-labelled pad, nitrocellulose filter and water absorption pad, the nitrocellulose filter contacted with gold-labelled pad or with sample pad, Jin Biao
Pad contact to bottom plate second end so that migrate on it with the combination physical efficiency of antigen and the monoclonal antibody PEDV-McAB2；Institute
State the monoclonal antibody PEDV-McAB2 in gold-labelled pad containing colloid gold label, the closely described bottom plate of the nitrocellulose filter
It include a detection line and a nature controlling line on the position of second end, immobilization has the monoclonal antibody in the detection line
PEDV-McAB1, immobilization has sheep anti mouse secondary antibody or sheep anti mouse mostly anti-on the nature controlling line.
The present invention relates to a kind of kit, the kit include a effective amount of monoclonal antibody PEDV-McAB1,
A effective amount of monoclonal antibody PEDV-McAB2, and for Porcine epidemic diarrhea virus carry out antigen-antibody reaction into
The detection reagent of row detection；Wherein, the kit includes buffer supply unit and enzyme immunochromatographydetecting detecting test strip；It is described
Buffer supply unit is used to buffer supplying the enzyme immunochromatographydetecting detecting test strip；The enzyme immunity chromatography detection test paper
Item includes nitrocellulose filter (1), and the enzyme immunochromatographydetecting detecting test strip successively includes substrate supply area, sample in the longitudinal direction
Supply area, detection zone；The substrate supply area includes substrate pad (3), is adsorbed with dry zymolyte, the substrate pad thereon
(3) it is contacted with nitrocellulose filter (1), the zymolyte is dissolved in buffer and on nitrocellulose filter (1) to away from described
The distal migration of buffer supply unit；The sample supply area includes enzyme mark pad (2), is adsorbed with the list of enzyme label thereon
Clonal antibody PEDV-McAB2, it is anti-that the zymolyte can generate colour developing with the enzyme marked on the monoclonal antibody PEDV-McAB2
It answers, the enzyme mark pad (2) contacts with nitrocellulose filter (1), and the monoclonal antibody PEDV-McAB2 is dissolved in buffer simultaneously
To the distal migration away from the buffer supply unit on nitrocellulose filter (1)；And the detection zone immobilization is
State monoclonal antibody PEDV-McAB1；Wherein, the buffer supply unit includes expansion fluid cushion (5), substrate buffer liquid bath
(8), substrate buffer solution (9) and buffer button, the substrate buffer solution (9) are placed in substrate buffer liquid bath (8), described slow
Fliud flushing button is located at the top of substrate buffer solution slot (8), and pressing can immerse the expansion fluid cushion (5) in buffer (9)；It is described
Detection zone includes detection line (6), nature controlling line (7), wherein the nature controlling line (7) is supplied compared with detection line (6) further from the sample
Area, immobilization has the monoclonal antibody PEDV-McAB1 on the detection line (6), the immobilization on the nature controlling line (7)
The monoclonal antibody PEDV- for the enzyme label that it is mostly anti-to have sheep anti mouse secondary antibody or sheep anti mouse, and is adsorbed on enzyme mark pad (2)
It is excessive for McAB2 monoclonal antibody PEDV-McAB1 described for the fixation being fixed on detection line (6)；The nitric acid
Cellulose membrane (1) full section is adhered on above support (10), and supporter (10) connects the buffer supply unit, and substrate supplies
Area, sample supply area, detection zone and water absorption pad (4) are answered, the water absorption pad (4) is away from the farthest of the buffer supply unit
End；And the position of test sample (11) addition is the position of the enzyme mark pad (2).
As one embodiment of the present invention, the enzyme immunochromatographydetecting detecting test strip in the kit includes solid
The nitrocellulose filter 1 of phase, the enzyme mark pad 2 containing labelled reagent, substrate pad 3, water absorption pad 4, expansion fluid cushion 5, detection line 6, matter
Line 7, substrate buffer liquid bath 8, substrate buffer solution 9, supporter 10 are controlled, wherein 2 belong to sample supply area, 3 belong to substrate supply area,
5,8 belong to buffer supply unit, and 6,7 belong to detection zone.The position that test sample 11 is added is the position of enzyme mark pad 2.It is described
Test strip is fixed in a plastic housing, from left to right successively fixed thereon that fluid cushion 5, substrate pad 3, enzyme mark pad 2, water suction is unfolded
Pad 4.Nitrocellulose filter 1 is adhered to the full section of supporter 10；Water absorption pad 4 is stuck in the top of nitrocellulose filter 1, and and nitric acid
Cellulose membrane 1 has overlapping；Enzyme mark pad 2 is located at the middle section of nitrocellulose filter 1, and the dry monoclonal for having enzyme to mark is anti-above
Body 2；Substrate pad 3 is stuck in the bottom end of nitrocellulose filter 1, there is dry zymolyte thereon.The upper end of expansion fluid cushion 5 covers bottom
Object pad 3, and its lower end is located at the bottom end of substrate buffer liquid bath 8.The upper surface of substrate buffer liquid bath 8 covers one layer of aluminium-foil paper, with
It prevents substrate buffer solution 9 from leaking, there is buffer button thereon, aluminium-foil paper can be punctured by pressing buffer button, and fluid cushion 5 will be unfolded
Lower end be immersed in substrate buffer solution 9.Detection line 6 is located at the middle upper end of nitrocellulose filter 1, and immobilization thereon has the list
Clonal antibody PEDV-McAB1.Nature controlling line 7 is located at the upstream of the middle upper end of nitrocellulose filter 1, detection line 6, thereon immobilization
There are sheep anti mouse secondary antibody or sheep anti mouse mostly anti-.
The present invention relates to a kind of kits, wherein the kit includes being coated with the monoclonal antibody PEDV-McAB1
Elisa plate, mark containing enzyme the monoclonal antibody PEDV-McAB2 reaction solution, for being carried out to Porcine epidemic diarrhea virus
Detection reagent that antigen-antibody reaction is detected, negative control, positive control.
As one embodiment of the present invention, the kit includes being coated with the monoclonal antibody PEDV-McAB1
Elisa plate, reaction solution, cleaning solution, dilution, the substrate developing solution, end that the monoclonal antibody PEDV-McAB2 is marked containing enzyme
Only liquid, negative control, positive control；Wherein, the cleaning solution is phosphate buffer, and the dilution is containing bovine serum albumin
White solution, the substrate developing solution are tetramethyl benzidine TMB developing solution, and the terminate liquid is the dense H of 2mol/l2SO4It is molten
Liquid, the negative control are phosphate buffer, and the positive control is comprising inactivating Porcine epidemic diarrhea virus after purification
Solution.
The present invention relates to a kind of kit, the kit include be coated with Porcine epidemic diarrhea virus antigen elisa plate,
Reaction solution, cleaning solution, dilution, substrate developing solution containing the monoclonal antibody, the sheep anti mouse secondary antibody of enzyme label, terminate liquid,
Negative control, positive control；Wherein, the reaction solution containing the monoclonal antibody is containing the monoclonal antibody PEDV-McAB1's
Reaction solution or reaction solution containing the monoclonal antibody PEDV-McAB2 contain the monoclonal antibody PEDV-McAB1 and institute
State the mixed reaction solution of monoclonal antibody PEDV-McAB2；Wherein, the cleaning solution is phosphate buffer, and the dilution is
Solution containing bovine serum albumin(BSA), the substrate developing solution are tetramethyl benzidine TMB developing solution, and the terminate liquid is 2mol/l
Dense H2SO4Solution, the negative control are phosphate buffer, and the positive control is comprising inactivating pig prevalence after purification
The solution of property diarrhea virus.
The invention further relates to the methods of Porcine epidemic diarrhea virus in the kit test sample, which comprises
(1) it will test sample to be contacted with the monoclonal antibody PEDV-McAB1 and/or the monoclonal antibody PEDV-McAB2,
(2) pig epidemic diarrhea in the monoclonal antibody PEDV-McAB1 and/or monoclonal antibody PEDV-McAB2 and sample is detected
The reaction of virus；Wherein, the monoclonal antibody PEDV-McAB1 in the method step (1) or the monoclonal antibody
PEDV-McAB2 is attached in solid phase, the solid phase be preferably titer plate, magnetic particle, latex particle, in nitrocellulose membrane
It is any；Wherein, reaction can be by enzyme colour developing, fluorescence, colloidal gold, chemiluminescence described in the method step (2)
Any method is measured.
As one embodiment of the present invention, the antibody with the PEDV-McAB1 variable region sequences or
The segment of the segment of the antibody and the antibody or the antibody with the PEDV-McAB2 variable region sequences
Marked using enzyme, it is described for and the Porcine epidemic diarrhea virus detection reagent that is detected of progress antigen-antibody reaction for institute
The enzyme for stating label generates the substrate of color reaction.
As one embodiment of the present invention, it is described for and Porcine epidemic diarrhea virus carry out antigen-antibody reaction into
The detection reagent of row detection is the ELIAS secondary antibody in conjunction with the segment of the antibody or the antibody and the enzyme with the label
Generate the substrate of color reaction.
Term " test sample " includes but is not limited to the excreta of animal or patient, mouth nasal secretion, zooblast training
Feeding intact virus or lytic virus liquid etc..
As one embodiment of the present invention, which comprises the microorganism swab of acquisition to be inserted at sample
It manages in pipe, dissolves sample as far as possible in the solution, sample is added dropwise in colloidal gold colloidal gold detection test paper strip well by treated
The heart determines as a result, result judgement standard after ten minutes are as follows: (1) if without band at nature controlling line, no matter whether has item at detection line
Band, detection process is invalid, in the case that (2) have band at nature controlling line, if there is band at detection line, for the positive, otherwise
For feminine gender, it may be assumed that whether the presence or absence of nature controlling line determines detection process effective, if having no band at nature controlling line, no matter at detection line
Whether have band, detection process is invalid, be in the case where having band at nature controlling line, if having band at the detection line it is positive,
It otherwise is feminine gender.
As one embodiment of the present invention, which comprises sample processing tube pretreatment sample is used, it will be described
The position of enzyme mark pad described in the enzyme immunochromatographydetecting detecting test strip is added in pretreatment sample, presses buffer button, and 30 points
Observation is as a result, result judgement standard after clock are as follows: (1) if having no band at nature controlling line, no matter whether has band at detection line, detect
Process is invalid, is otherwise feminine gender for the positive if there is band at detection line in the case that (2) have band at nature controlling line,
That is: whether effective the presence or absence of nature controlling line determines detection process, if having no band at nature controlling line, no matter whether has item at detection line
Band, detection process is invalid, in the case where having band at nature controlling line, is positive if having band at the detection line, is otherwise yin
Property.
As one embodiment of the present invention, which comprises the monoclonal antibody PEDV-McAB1 will be coated with
Elisa plate closed with confining liquid, washed with cleaning solution；It, will be described pre- using sample processing tube pretreatment sample
Processing sample is added in ELISA reacting hole after diluting in dilution and carries out detection reaction, while it is right to do positive control, feminine gender
According to being washed after reaction；Diluted enzyme is added and marks the monoclonal antibody PEDV-McAB2, it is anti-in 37 DEG C of effect 40-80min
Should after wash；Substrate developing solution is added, terminate liquid is added after 37 DEG C of reaction 10min；With microplate reader read data, to result into
Row determines.
As one embodiment of the present invention, which comprises Porcine epidemic diarrhea virus antigen will be coated with
Elisa plate is closed with confining liquid, while doing negative control, positive control, and washed with cleaning solution；At sample
Pipe pretreatment sample is managed, is added in ELISA reacting hole and detects after the pretreatment sample is diluted in dilution
Reaction, and be added in ELISA reacting hole and examined after carrying out gradient dilution with the Porcine epidemic diarrhea virus antigen of known concentration
Reaction is surveyed, is washed after reaction；By the reaction solution containing the monoclonal antibody PEDV-McAB1, or contain the monoclonal antibody
The reaction solution of PEDV-McAB2, or mixed containing the monoclonal antibody PEDV-McAB1 and monoclonal antibody PEDV-McAB2
Reaction solution, ELISA reacting hole is added after dilution, in 37 DEG C of reaction 20-40min, washs, while setting blank control；By enzyme mark
It is added in ELISA reacting hole after the sheep anti mouse secondary antibody dilution of note, in 37 DEG C of reaction 20-40min, washing；Substrate developing solution is added,
Terminate liquid is added after 37 DEG C of reaction 10min；Data are read with microplate reader, result is determined.
The present invention relates to application of the kit in the Porcine epidemic diarrhea virus detection for non-diagnostic purpose.
As one embodiment of the present invention, the present invention provides include a effective amount of monoclonal antibody PEDV-
Application of the kit of McAB1 in the Porcine epidemic diarrhea virus detection for non-diagnostic purpose.
As one embodiment of the present invention, the present invention provides include a effective amount of monoclonal antibody PEDV-
Application of the kit of McAB2 in the Porcine epidemic diarrhea virus detection for non-diagnostic purpose.
As one embodiment of the present invention, the present invention provides include a effective amount of monoclonal antibody PEDV-
The kit of McAB1 and a effective amount of monoclonal antibody PEDV-McAB2 are in the Porcine epidemic diarrhea virus for non-diagnostic purpose
Application in detection.
As one embodiment of the present invention, the present invention provides use the monoclonal antibody PEDV- of the invention
The kit that the McAB1 and monoclonal antibody PEDV-McAB2 carries out sandwich method detection is popular in the pig for non-diagnostic purpose
Property diarrhea virus detection in application.
The pig that kit containing Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus of the present invention can be used for non-diagnostic purpose is popular
Property diarrhea virus detection in application；The non-diagnostic purpose Porcine epidemic diarrhea virus detection include epidemiological analysis,
In vitro tissue is detected, Epitope Identification research and qualitative and quantitative diagnostic test antigen containing Porcine epidemic diarrhea virus and
The detection of Porcine epidemic diarrhea virus antigen in the vaccine composition of other antigens.
As one embodiment of the present invention, other described antigens include transmissible gastro-enteritis virus antigen, pig wheel
Shape viral antigen, pig circular ring virus antigen, porcine reproductive and respiratory syndrome virus antigen, haemophilus parasuis antigen, pig are tiny
Viral antigen, Actinobacillus pleuropneumoniae antigen, bacillus coli antigen, atrophic rhinitis antigen, porcine pseudorabies virus
One of antigen, hog cholera disease antigen, swine flue antigen are a variety of；It is further preferred that other described antigens are pig
Infectious gastroenteritis virus antigen.
The invention will now be further described with reference to specific embodiments, and the advantages and features of the present invention will be with description more
It is clear.But examples are merely exemplary for these, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art
It should be understood that without departing from the spirit and scope of the invention can details to technical solution of the present invention and form carry out
Modifications or substitutions, but these modifications and replacement are fallen within the protection scope of the present invention.
Experimental method of the present invention, if being conventional method without specified otherwise；The biomaterial, if without spy
Different explanation, commercially obtains.
Preparation, purifying, identification and the inspection of 1 Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus of embodiment
The preparation and purification of 1.1 Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus
By HN1301 plants of pig fluidity diarrhea virus (referring to Chinese patent CN104513827A) according to (Zhang Li such as Zhang Liyan
The development Sichuan Agricultural University Master's thesis of swallow Porcine epidemic diarrhea virus monoclonal antibody) in method purified virus, exempt from
Epidemic disease mouse, clone obtain hybridoma, and 2 plants of monoclonal antibodies are prepared in Mice Body.
By 2 plants of monoclonal antibodies of preparation, according to Chen Dan etc., (Chen Dan, Sun Guangrui, it is heavy that willow increases kind octanoic acid-ammonium sulfate joint
Application Agriculture of Anhui science of the shallow lake method in monoclonal antibody-purified, 2007,35 (26): octanoic acid-ammonium sulfate 8105,8108)
The operating method of co-precipitation method monoclonal antibody purification purifies Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus PEDV- respectively
McAB1, Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus PEDV-McAB2.
The identification of 1.2 Monoclonal Antibodies against Porcine Epidemic Diarrhea Virus
1.2.1 the identification of monoclonal antibody type and subclass
With Pierce Rapid ELISA Mouse mAb Isotyping Kit and referring to specification to the Asia of antibody
Type is identified.Qualification result is shown in Table 1:
The identification of each monoclonal antibody type of table 1
Note :+indicate positive reaction ,-indicate negative reaction.
As shown in Table 1, monoclonal antibody PEDV-McAB1, monoclonal antibody PEDV-McAB2 heavy chain subgroup are all IgG2a,
Light chain type is all kappa.
1.2.2 the identification of monoclonal antibody specificity
PEDV, TGEV, PoRV, PRRSV, PRV and Vero cell are coated with reaction plate respectively, measure monoclonal antibody
Whether PEDV-McAB1, monoclonal antibody PEDV-McAB2 and PEDV, TGEV, PoRV, PRRSV, PRV and Vero cell have friendship
Fork reactivity.
Measurement result is shown: monoclonal antibody PEDV-McAB1, monoclonal antibody PEDV-McAB2 and TGEV, PoRV,
The equal no cross reaction of PRRSV, PRV and Vero cell, only reacts with PEDV and is positive, show monoclonal antibody PEDV-McAB1,
Monoclonal antibody PEDV-McAB2 is the monoclonal antibody specific of porcine epidemic diarrhea resisting virus.
The inspection of 1.3 monoclonal antibodies after purification
1.3.1 appearance test
At room temperature, monoclonal antibody PEDV-McAB1, the monoclonal antibody PEDV-McAB2 for visually observing visible purifying are equal
In achromaticity and clarification state, have no that floccule precipitates.
1.3.2 steriling test
Using steriling test method, the nutrient agar slopes of monoclonal antibody raw material inoculation 10ml/ pipe after purification are taken respectively
Culture medium, improvement Martin's culture medium and each 2 pipe of sulphur glycollate culture medium (inoculum concentration is 0.5ml/ pipe).Sulphur ethyl alcohol after inoculation
Hydrochlorate culture medium and each pipe of nutrient agar slant medium are placed in 30-35 DEG C of culture, and another pipe is cultivated in 20-25 DEG C.Improvement
Martin's culture medium is placed in 20-25 DEG C of culture.Negative control is done with method operation with sterile saline simultaneously.After culture 14 days not
Observing in culture medium has microorganism growth, observation indicate that: monoclonal antibody PEDV-McAB1, monoclonal antibody PEDV-
McAB2 raw material meets sterility requirements.
1.3.3 the purity of monoclonal antibody
Using Chen Dan etc., (Sun Guangrui, willow increase kind octanoic acid-ammonium sulfate co-precipitation method answering in monoclonal antibody-purified
With Agriculture of Anhui science, 2007,35 (26): 8105,8108) PAGE gel electrophoresis is identified, applied sample amount is 10 μ g,
Testing result is as shown in Figure 1.The result shows that: monoclonal antibody PEDV-McAB1, the purity of monoclonal antibody PEDV-McAB2 are equal
Not less than 95%.
1.3.4 the measurement of monoclonal antibody relative affinity
By the monoclonal antibody PEDV-McAB1 of preparation, monoclonal antibody PEDV-McAB2 according to (Song Shuai, the woods such as Song Shuai
It is red, Shao Junjun, the measurement veterinary immunology of Chang Huiyun resisting O-type foot and mouth disease virus monoclonal antibody relative affinity, 2009,25
(4): 333-335 the operating method in) measures its relative affinity.Wherein the peridium concentration of antigen is 5 μ g/ml, ELIAS secondary antibody
Dilution be 1:10000, measure the relative affinity of monoclonal antibody after purification.Calculated result shows: monoclonal antibody
The relative affinity of PEDV-McAB1 is 0.78 μ g/ml, and the relative affinity of monoclonal antibody PEDV-McAB2 is 0.44 μ g/
ml。
1.3.5 the measurement of monoclonal antibody neutralization activity
2 plants of monoclonal antibodies and 500TCID50Separate sources PEDV strain neutralize after be inoculated with Vero cell, in
Measuring the monoclonal antibody with test has the representative for neutralizing CV777 plants of vaccine strain PEDV, SM98 plants of vaccine strain and popular strain
The ability that HN1301 plants of strain.It measures monoclonal antibody PEDV-McAB1 neutralize antibody titers and is not less than 1:512, show Dan Ke
Grand antibody PEDV-McAB1 has good neutralization activity, and the neutralize antibody titers of monoclonal antibody PEDV-McAB2 are respectively less than
1:2 shows monoclonal antibody PEDV-McAB2 without neutralization activity.
The neutralize antibody titers that the different strains of table 2 and monoclonal antibody measure after neutralizing
The measurement of 1.4 monoclonal antibody contents
Monoclonal antibody PEDV-McAB1, monoclonal antibody PEDV-McAB2 are pressed respectively with BCA protein quantification kit
Book carries out quantitative analysis as directed, measurement result shows: monoclonal antibody PEDV-McAB1, monoclonal antibody PEDV-McAB2
Concentration is respectively 4,4.5mg/ml.
The pairing of 1.5 monoclonal antibodies
HN1301 plants of Porcine epidemic diarrhea virus are selected, are diluted to 105.0TCID50/ ml examines different collocation modes
It surveys, the results are shown in Table 3:
The collocation of 3 monoclonal antibody of table
Note: "+" indicates positive, and "-" indicates negative.
The result shows that: labeled monoclonal antibody PEDV-McAB1 and immobilized monoclonal antibody PEDV-McAB2 detection pig is popular
Property diarrhoeal diseases venom be feminine gender, remaining collocation testing result be the positive.Thus selection immobilized monoclonal antibody PEDV-McAB1,
Labeled monoclonal antibody PEDV-McAB2 is collocation mode.
The measurement of 2 variable region of mab sequence of embodiment
2.1 monoclonal antibody heavies and light chain variable region design of primers
According to the sequence signature of source of mouse monoclonal antibody, heavy chain variable region primer sequence is designed:
P1:5 '-ACTAGTTGACGTGGTCTCTAGGGTCACTTTAGTTTTCCT-3 '
P2:5 '-CGGAAGCTTCCAGCGRCCARKCCATATACIGRTGG-3 '
Design light chain variable region primer sequence:
P3:5 '-GCCATCTCATGRAGWCATTKWCYCAAGTCTTT-3 '
P4:5 '-CGGACGCTTACTGCCTGGTAAGAAGATGGA-3 '
2.2 variable region of mab sequencings
According to Zhang Aihua etc., (Zhang Aihua, closes orchid, and the anti-CD molecule monoclonal antibody light and heavy chain of the series mouse such as Wang Zhiyou is variable
The Cloned culturing Products in China magazine of area's gene, 2001,15 (2): 65-68) establish variable region sequences survey
Determine method, obtained respectively by molecule clone technology monoclonal antibody PEDV-McAB1, monoclonal antibody PEDV-McAB2 can
Become region sequence, chooses corresponding cloned plasmids and send to Suzhou Jin Wei intelligence Biotechnology Co., Ltd and be sequenced.
The heavy chain variable region of monoclonal antibody PEDV-McAB1, the gene order of light chain variable region are measured respectively such as SEQ ID
It is respectively SEQ ID No.2, SEQ ID No.4 by its amino acid sequence derived shown in No.1, SEQ ID No.3；Monoclonal
The heavy chain variable region of antibody PEDV-McAB2, the gene order of light chain variable region are respectively such as SEQ ID No.5, SEQ ID No.7
It is shown, it is respectively SEQ ID No.6, SEQ ID No.8 by its amino acid sequence derived.
The preparation and application of 3 kit of embodiment
The colloidal gold colloidal gold detection test paper strip of 3.1 kits
The preparation of colloidal gold colloidal gold detection test paper strip: according to (Li Hongmei, the glue of the immune detection such as Nie Zheng, Chen Jia such as Li Hongmei
The optimizing research food industry science and technology of body gold preparation process, 2009,30 (12): 289-291) the colloidal gold preparation method established,
Colloidal gold solution and labeled monoclonal antibody PEDV-McAB2 are prepared, monoclonal antibody PEDV-McAB1 and sheep anti-mouse igg (are purchased from
Sigma company) it is sprayed on nitrocellulose filter respectively as detection line and nature controlling line, by the nitrocellulose filter and it is impregnated with
The gold-labelled pad of gold mark monoclonal antibody PEDV-McAB2 is wired up with plastic shell, is made into colloidal gold colloidal gold detection test paper strip.And match
Corresponding phosphate buffer is set as sample treatment liquid.When detection, measuring samples are placed in sample processing tube, use up sample
It may dissolve in the solution, the sample processing tube lid head containing measuring samples is fractureed, the sample after 2-3 drop mixes is added dropwise
(about 80 μ l) is to test strips well center；It is observed and recorded in the detection zone of test strip after ten minutes as a result, and according to sentencing
Calibration standard is determined.Result judgement standard: establishment is then tested in nature controlling line colour developing, and detection line colour developing is the positive, does not develop the color i.e.
For feminine gender；Nature controlling line does not develop the color, tests invalid, and no matter whether detection line, which develops the color, is determined as null result, need to resurvey.
Quality research:
(1) sensitivity test: by PEDV HN1301 strain virus liquid (virus titer 106.5TCID50/ ml) carry out 10 times times
Than dilution, being diluted to malicious valence is 105.5TCID50/ml、104.5TCID50/ml、103.5TCID50/ ml, uses colloidal gold colloidal gold detection test paper strip
It is detected.The Monitoring lower-cut of test strips is 10 as the result is shown4.5TCID50/ml。
(2) sensitivity assays: by PEDV (HN1301 plants and CV777 plants), (virus titer is 10 to virus liquid6.5TCID50/
Ml 10 times of doubling dilutions) are carried out, being diluted to malicious valence is 105.5TCID50/ml、104.5TCID50/ml、103.5TCID50/ ml, uses glue
Body gold test strip is detected.Test strips are to HN1301 plants and CV777 plants of PEDV of Monitoring lower-cut as the result is shown
104.5TCID50/ml。
(3) (OSU plants) transmissible gastro-enteritis virus (magnificent strain) virus liquid, porcine rotavirus diseases specific assay: are taken
Venom, pig parvoviral (WH-1 plants) virus liquid, swine fever virus (C plants) virus liquid, porcine pseudorabies virus (Bartha K-61
Strain) virus liquid, and negative fecal specimens, it is detected according to colloidal gold colloidal gold detection test paper strip detection method.Test paper as the result is shown
Above 6 parts specific samples of item detection are feminine gender.
(4) repetitive test
Repeatability between batch: take 3 batches of Porcine epidemic diarrhea virus colloidal gold colloidal gold detection test paper strips by inspection described in embodiment 3.1
Survey method detects the PEDV positive and negative sample respectively.Coincidence rate between test strips batch is greater than 90%, and detects between test strips same
The colored intensity of a sample is consistent.
Repeatability in batch: taking 1 batch of Porcine epidemic diarrhea virus colloidal gold colloidal gold detection test paper strip, positive to PEDV and negative by 1 people
Property sample carry out repeat detect three times.Coincidence rate in test strips batch is greater than 90%, and is detected between test strips with a sample
Colored intensity is consistent.
Clinical application: 20 parts of clinical samples of acquisition are detected according to detection method described in embodiment 3.1, knot
Fruit is shown in Table 4.
Meanwhile with the RT-PCR of the foundation such as Zou Yong, (Zou Yong, money Yongqing, the such as Tang Yonglan pig are popular respectively for 20 parts of clinical samples
Property diarrhea, transmissible gastroenteritis of swine and porcine rotavirus RT-PCR antidiastole technical research Shanghai Agricultural journal, 2003,2:
It 82-84) detects, the results are shown in Table with colloidal gold strip (Bionote Inc. is operated referring to kit reference book)
4。
As shown in Table 4: the testing result of test strips and the coincidence rate of RT-PCR testing result have reached 90%, and do not have
Occur non-specific.The coincidence rate for pacifying prompt paper slip and RT-PCR testing result is 80%, and has the appearance of false positive results.Therefore from
The sensibility and specificity of colloidal gold strip processed is superior to the prompt colloidal gold strip of peace.
The enzyme immunochromatographydetecting detecting test strip of 3.2 kits
Enzyme immunochromatographydetecting detecting test strip is prepared according to CN104062430A, as shown in Fig. 2, list prepared by embodiment 1
Clonal antibody PEDV-McAB2 carries out enzyme label, and monoclonal antibody PEDV-McAB1 and sheep anti-mouse igg prepared by embodiment 1 divides
It is not sprayed on nitrocellulose filter as detection line and nature controlling line, the nitrocellulose filter, enzyme mark pad and substrate pad is moulded
Material outer cover packaging gets up, and is made into enzyme immunoassay test strip.And phosphate buffer is prepared as sample treatment liquid.
When detection, measuring samples are placed in sample processing tube, dissolve sample as far as possible in the solution, will be contained to be checked
The sample processing tube lid head of sample fractures, and the sample (about 80 μ l) after 2-3 drop mixes is added dropwise to test strips well center；
Detection zone after 30 minutes in test strip observes and records as a result, and being determined according to criterion.Result judgement standard:
Establishment is then tested in nature controlling line colour developing, and detection line colour developing is the positive, does not develop the color as feminine gender；Nature controlling line line does not develop the color, tests not
It sets up, no matter whether detection line, which develops the color, is determined as null result, need to resurvey.
According to 3.1 mesogen strain of embodiment and doubling dilution, the quality research of enzyme immunochromatographydetecting detecting test strip is carried out, is carried out
Sensitivity, sensibility, specificity and repetitive test, the results showed that (1) sensitivity test: the Monitoring lower-cut of test strips is shown
It is 103.5TCID50/ml；(2) sensitivity assays: test strips are to HN1301 plants and CV777 plants of PEDV of Monitoring lower-cut
103.5TCID50/ml；(3) specific assay: display test strips detect transmissible gastro-enteritis virus (magnificent strain) virus liquid, pig
Rotavirus (OSU plants) virus liquid, pig parvoviral (WH-1 plants) virus liquid, swine fever virus (C plants) virus liquid, porcine pseudorabies
Viral (K-61 plants of Bartha) virus liquid, and the specific sample of 6 parts of negative fecal specimens is feminine gender；(4) repeatability examination
Test: between display test strips batch and batch interior coincidence rate is all larger than 90%, and the colored intensity with a sample is detected between test strips
Unanimously.
Clinical application: 20 parts of clinical samples of 2~3 drop (about 80 μ l) embodiment 3.1 preparations are added dropwise and are tried in enzyme linked immunological
At the well of paper slip, while buffer button is pressed rapidly, observe note in the detection zone of test strip after standing 30 minutes
Record is as a result, the results are shown in Table 4.
As shown in Table 4: the testing result of enzyme immunochromatographydetecting detecting test strip and the coincidence rate of RT-PCR testing result reach
100%, and it is non-specific without occurring.
4 colloidal gold colloidal gold detection test paper strip of table, RT-PCR, pacifies the prompt clinical sample of test strips detection at enzyme immunochromatographydetecting detecting test strip
Product result compares
Note: "+" indicates that testing result is the positive, and "-" indicates that testing result is feminine gender
The double antibodies sandwich Sandwich-ELISA-McAB1/2 of 3.3 kits
With the PEDV in double antibodies sandwich principle detection clinical sample, Sandwich-ELISA-McAB1/2 kit is prepared:
The monoclonal antibody PEDV-McAB1 that Example 1 purifies is diluted to 4 μ g/ml with coating buffer (carbonate buffer solution of pH9.6),
100 holes μ L/ are placed in 4 DEG C of coatings overnight, take out and use PBST board-washing 3 times, pat dry；Added with 5% skimmed milk power with 100 holes μ L/
Enter, 37 DEG C are closed 1 hour, board-washing 3 times, are patted dry；Taking HN1301 plants of the PEDV of inactivation after purification, (content is before inactivating
104.0TCID50/ ml) it is used as positive control, use PBS buffer solution as negative control, while measuring samples being taken to be detected, 100 μ
The hole L/ is placed in 37 DEG C of effect 1h, takes out and uses PBST board-washing 3 times, pats dry；The monoclonal antibody PEDV-McAB2 for taking HRP to mark makees
For secondary antibody, 1000 times of dilutions are done, 100 holes μ L/ are placed in 37 DEG C of effect 1h, take out and use PBST board-washing 3 times, pat dry；TMB (four is added
Methyl biphenyl amine) 50 hole μ L/ of substrate solution, 37 DEG C of reaction 10min；The 2M sulfuric acid for being eventually adding 50 holes μ L/ terminates reaction, uses enzyme
It marks instrument and detects OD450nm.Result judgement standard: with the OD of positive control450nmThe OD of value and negative control450nmValue is used as standard, root
According to the OD of sample450nmValue comes the yin and yang attribute of judgement sample, i.e. positive control OD450nmValue-negative control OD450nmValue > 0.5 is then tried
Test establishment, positive control OD450nmValue/negative control OD450nmValue >=2.1, measuring samples OD450nmIt is the positive when value >=0.3,
It otherwise is feminine gender.
According to 3.1 mesogen strain of embodiment and doubling dilution, the matter of double antibodies sandwich Sandwich-ELISA-McAB1/2 is carried out
Quantity research carries out sensitivity, sensibility, specificity and repetitive test, the results showed that (1) sensitivity test: visualizingre agent box
Monitoring lower-cut be 103.0TCID50/ml；(2) sensitivity assays: kit is under HN1301 plants and CV777 plants of PEDV of detection
Limit is 103.0TCID50/ml；(3) specific assay: visualizingre agent box detects transmissible gastro-enteritis virus (magnificent strain) virus
Liquid, porcine rotavirus (OSU plants) virus liquid, pig parvoviral (WH-1 plants) virus liquid, swine fever virus (C plants) virus liquid, pig are pseudo-
Hydrophobin (K-61 plants of Bartha) virus liquid, and the specific sample of 6 parts of negative fecal specimens is feminine gender；(4) it repeats
Property test: the coefficient of variation between visualizingre agent box batch batch is respectively less than 10%.
Clinical application: preparing 10 parts of clinical samples therein to embodiment 3.1 according to detection method and carry out clinical detection,
It the results are shown in Table 5.
Table 5RT-PCR and double antibodies sandwich Sandwich-ELISA-McAB1/2 testing result compare
As shown in Table 5: the testing result and RT-PCR testing result of double antibodies sandwich Sandwich-ELISA-McAB1/2 is complete
Meet entirely, sensitivity reaches PCR magnitude, shows that 2 plants of monoclonal antibodies can be used as the former material of double crush syndrome kit
Material, for detecting Porcine epidemic diarrhea virus antigen.
The competition Competitive-ELISA-McAB1/2/1+2 of 3.4 kits
The preparation of ELISA2: purified PEDV HN1301 plants (10 is taken7.5TCID50/ ml) viral antigen coating buffer
It is coated with after (carbonate buffer solution of pH9.6) dilution 1:10000,100 holes μ L/, while doing negative control and (being buffered with PBS
Liquid replacement), (inactivation after purification HN1301 plant of PEDV, content is 10 to positive control before inactivating4.0TCID50/ ml), 4 DEG C are overnight,
It takes out and uses PBST board-washing 3 times, pat dry；It is added with 5% skimmed milk power with 100 holes μ L/, 37 DEG C are closed 1 hour, board-washing 3 times, are clapped
It is dry；PEDV CV777 plant (10 is bred and purified according to embodiment 1.17.0TCID50/ ml), with dilution according to 1:10V/V
Ratio carry out gradient dilution, and added by 100 holes μ L/, while by each measuring samples according to the dilution proportion 3 of 1:10V/V
It is a；By the monoclonal antibody diluted of embodiment 1 after purification to 100 μ g/mL, added by 100 holes μ L/, it is anti-in 37 DEG C
It answers 30 minutes, washs 3 times, pat dry, while setting blank control；The sheep anti mouse secondary antibody of HRP label presses 1:1 × 10 with dilution4V/V
It is diluted, then 100 holes μ L/ are added, and 37 DEG C are reacted 30 minutes, wash 3 times, pat dry；TMB (tetramethyl benzidine) bottom is added
50 hole μ L/ of object solution is added, and 37 DEG C are reacted 10 minutes；The 2M sulfuric acid for being eventually adding 50 holes μ L/ terminates reaction, is detected with microplate reader
OD450nm。
Wherein, when monoclonal antibody is monoclonal antibody PEDV-McAB1 as Competitive-ELISA-McAB1,
When monoclonal antibody is monoclonal antibody PEDV-McAB2 as Competitive-ELISA-McAB2, work as monoclonal antibody
As Competitive-ELISA-McAB1+ when being mixed with monoclonal antibody using 1:1V/V for monoclonal antibody PEDV-McAB1
2.Then, according to CV777 plants of gradient dilutions of PEDV through Competitive-ELISA-McAB1, Competitive-ELISA-
OD after McAB2, Competitive-ELISA-McAB1+2 detection450nmIt is bent that value and the concentration of gradient dilution draw competition test
Line, and the OD detected according to measuring samples450nmAs a result the PEDV content contained by it is calculated.
According to 3.1 mesogen strain of embodiment and doubling dilution, the Competitive-ELISA-McAB1 that is at war with,
The quality research of Competitive-ELISA-McAB2, Competitive-ELISA-McAB1+2 kit, progress sensitivity,
Sensibility, specificity and repetitive test, the results showed that (1) sensitivity test: the Monitoring lower-cut of visualizingre agent box is
103.0TCID50/ml；(2) sensitivity assays: 3 kits are to HN1301 plants and CV777 plants of PEDV of Monitoring lower-cut
103.0TCID50/ml；(3) specific assay: 3 kit detection transmissible gastro-enteritis virus (magnificent strain) virus liquids of display,
Porcine rotavirus (OSU plants) virus liquid, pig parvoviral (WH-1 plants) virus liquid, swine fever virus (C plants) virus liquid, pseudorabies
Virus (K-61 plants of Bartha) virus liquid, and the specific sample of 6 parts of negative fecal specimens is feminine gender；(4) repeatability examination
Test: batch interior coefficient of variation is respectively less than 10% between display 3 kits batch.
Clinical research: embodiment 3.1 is prepared into 10 parts of clinical samples therein according to detection method and carries out clinical detection, meter
The results are shown in Table 6 for calculation:
6 competitive ELISA of table is compared with RT-PCR detection clinical sample result
As the result is shown: Competitive-ELISA-McAB1/2/1+2 testing result matches with RT-PCR testing result.
4 monoclonal antibody of embodiment identifies connection seedling
By the pig fluidity diarrhea of Harbin Wei Ke biotechnology development company production, transmissible gastroenteritis of swine bigeminal live vaccine
Vaccine is diluted to 10 according to vaccine operation instructions with DMEM culture solution5.0TCID50/ml。
Vaccine after taking isometric dilution respectively with monoclonal antibody PEDV-McAB1 (neutralization titer is greater than 1:512), anti-
PEDV hyper-immune serum (preparing according to Chinese patent CN103705918A, neutralization titer is greater than 1:512) is placed in 37 DEG C of neutralizations
1h.The first 5 times of dilutions of the virus liquid after neutralizing are taken, then after 10 times of doubling dilutions, 96 orifice plates of inoculation measure malicious valence.After statistics neutralizes
Cytopathy variable orifice, calculate malicious valence.It the results are shown in Table 7.
7 bigeminal live vaccine of table TGEV poison valence after the neutralization of PEDV antibody
As the result is shown: above-mentioned data show, bigeminal live vaccine using in hyper-immune serum and after PEDV, the loss of TGEV poison valence compared with
It is more, and use in monoclonal antibody PEDV-McAB1 and after PEDV, TGEV poison valence is measured, closer to the theoretical value for matching seedling.Cause
This monoclonal antibody PEDV-McAB1 can be used for the measurement of viral level in bigeminy or multi-joint live vaccine.
The effect assessment of 5 monoclonal antibody PEDV-McAB1 of embodiment prevention and/or treatment virus infection
The pre- preventing virus infection effect assessment of 5.1 monoclonal antibody PEDV-McAB1
The double-negative 2-3 age in days piglet 15 of PEDV, PoRV, TGEV antigen, antibody is screened, is randomly divided into 3 groups, first group
The monoclonal antibody PEDV-McAB12mL prepared in embodiment 1 is taken orally respectively with second group, and third group takes orally PBS buffer solution
2mL, 24 hours first group and third groups take orally PEDV prevalence strain HN1301 small intestine poison 2mL (containing 1000 minimum hairs after taking orally
Sick dosage), second group of oral CV777 small intestine poison (containing 1000 minimum dosage of falling ill), the daily clinical symptoms for observing piglet, even
Continuous observation 14 days by clinical incidence, occurring degree, the death rate and uses PCR method to detect the virus monitor in anus swab
Toxin expelling number of days come evaluate monoclonal antibody PEDV-McAB1 prevention diarrhea virus infection effect, the results are shown in Table 8.
Table 8 takes the test result of prevention PEDV infection after monoclonal antibody PEDV-McAB1
5.2 monoclonal antibody PEDV-McAB1 treat virus infection effect assessment
The double-negative 2-3 age in days piglet 20 of PEDV, PoRV, TGEV antigen, antibody is screened, is randomly divided into 4 groups, first group
It is taken orally respectively PEDV prevalence strain HN1301 small intestine poison 2mL (containing 1000 minimum dosage of falling ill) with second group, third group and the
Four groups of oral CV777 small intestines poison (containing 1000 minimum dosage of falling ill), it is oral after 12 hours first group and third components Kou Fu not
Monoclonal antibody PEDV-McAB1-2mL, second group and the 4th group oral PBS prepared by embodiment 1 observes facing for piglet daily
Bed symptom, is observed continuously 14 days, by clinical incidence, occurring degree, the death rate and PCR method is used to detect in anus swab
Virus monitors toxin expelling number of days, to evaluate the effect of monoclonal antibody PEDV-McAB1 treatment Porcine epidemic diarrhea virus infection
Fruit the results are shown in Table 9.
The test result of monoclonal antibody PEDV-McAB1 treatment is taken within 12 hours after after table 9PEDV infection
The result shows that monoclonal antibody PEDV-McAB1 can reduce the clinical condition as caused by Porcine epidemic diarrhea virus
Shape reduces the death rate and reduces toxin expelling number of days, there is prevention and/or therapeutic effect well.
The preparation of 6 genetic engineering antibody of embodiment
According to Li Yue etc., (clone of Li Yue .A type influenza virus single-chain antibody gene and antiviral activity study the agriculture of the Xinjiang
Sparetime university learn master thesis, 2014) establish single-chain antibody preparation operating method, respectively use monoclonal antibody PEDV-
The variable region sequences of McAB1 and monoclonal antibody PEDV-McAB2 prepare corresponding single-chain antibody 1 and single-chain antibody 2, with list
The heavy chain variable region of clonal antibody PEDV-McAB1 and the light chain variable region of monoclonal antibody PEDV-McAB2 prepare single-chain antibody
3, it is prepared with the heavy chain variable region of monoclonal antibody PEDV-McAB2 and the light chain variable region of monoclonal antibody PEDV-McAB1 single
Chain antibody 4.
Example 1.1 prepares HN1301 plants of PEDV inoculating cells, until 30% lesion occurs in cell, with 80% acetone 4
DEG C fixed 30min, is washed 3 times with PH7.4PBS, 5min/ times, is separately added into single-chain antibody 1, single-chain antibody 2,3 and of single-chain antibody
Single-chain antibody 4 is placed in 37 DEG C of effect 1h, is washed 3 times with pH7.4PBS, 5min/ times, the rabbit anti-mouse igg that FITC label is added is placed in
37 DEG C of effect 1h are washed 3 times with pH7.4PBS, 5min/ times, are placed in fluorescence microscopy under the microscope.
According to embodiment 1.3.5 operating method, single-chain antibody 1, single-chain antibody 2, single-chain antibody 3 and single-chain antibody 4 are measured
Neutralization titer.
As the result is shown: single-chain antibody 1, single-chain antibody 2, single-chain antibody 3 and single-chain antibody 4 can and pig epidemic diarrhea
Specific reaction occurs for virus.The neutralization titer of single-chain antibody 1 and single-chain antibody 3 is 1:64, and single-chain antibody 2 and single-chain antibody
4 without neutralization titer.
The above results show that SEQ ID No.1, SEQ ID No.3, SEQ ID No.5 and SEQ ID No.7 can be used for pig
The preparation of epidemic diarrhea virus genetic engineering antibody.
It describes the preferred embodiment of the present invention comprehensively above, but various alternatives and modifications can be carried out to them.Therefore,
Above description be reference should not be made to determine the scope of the present invention, but should refer to the appended claims and its whole equivalents to determine
Determine the scope of the present invention.Any feature, (being whether preferred) can be with any other feature (being whether preferred) phases
In conjunction with.Claims of the present invention is understood not to have the limitation of method+function, unless leading in a certain claim
It crosses term " ... method " and clearly enumerates such limitation.
Claims (20)
1. a kind of mouse monoclonal antibody PEDV-McAB1 for specifically binding Porcine epidemic diarrhea virus, wherein 1) weight chain variable
Region amino acid sequence is shown in SEQ ID No.2；2) chain variable region amino acid sequence is shown in SEQ ID No.4.
2. the antibody that one kind is made of heavy chain variable region described in claim 1 and light chain variable region；The antibody is monoclonal
Antibody, genetic engineering antibody；Wherein, the genetic engineering antibody includes single-chain antibody, chimeric mAb, reshaping monoclonal
Antibody, pig resource monoclonal antibody.
3. antibody according to claim 2, the antibody is monoclonal antibody PEDV-McAB1, the monoclonal antibody
The amino acid sequence of PEDV-McAB1 heavy chain variable region is shown in SEQ ID No.2, and the amino acid sequence of light chain variable region is SEQ
Shown in ID No.4.
4. a kind of mouse monoclonal antibody PEDV-McAB2 for specifically binding Porcine epidemic diarrhea virus, wherein 1) weight chain variable
Region amino acid sequence is shown in SEQ ID No.6；2) chain variable region amino acid sequence is shown in SEQ ID No.8.
5. one kind is made of light chain variable region in heavy chain variable region in variable region as claimed in claim 4 and the variable region
Antibody；The antibody is monoclonal antibody, genetic engineering antibody；Wherein, the genetic engineering antibody include single-chain antibody, it is embedding
Close monoclonal antibody, reshaping monoclonal antibody, pig resource monoclonal antibody.
6. antibody according to claim 5, wherein the antibody is monoclonal antibody PEDV-McAB2, the monoclonal
The amino acid sequence of antibody PEDV-McAB2 heavy chain variable region is the amino acid sequence of light chain variable region shown in SEQ ID No.6
For shown in SEQ ID No.8.
7. a kind of pharmaceutical composition, wherein described pharmaceutical composition includes the antibody as claimed in claim 2 of immune amount, and
Pharmaceutically acceptable carrier.
8. pharmaceutical composition according to claim 7, wherein described pharmaceutical composition includes the monoclonal of immune amount
Antibody PEDV-McAB1 and pharmaceutically acceptable carrier.
9. pharmaceutical composition according to claim 7, wherein described pharmaceutical composition includes the claim 1 of immune amount
The single-chain antibody and pharmaceutically acceptable carrier of the heavy chain variable region preparation of the monoclonal antibody PEDV-McAB1.
10. pharmaceutical composition according to claim 7, wherein described pharmaceutical composition includes the claim 1 of immune amount
The single-chain antibody of heavy chain variable region and the light chain variable region preparation of the monoclonal antibody PEDV-McAB1, and can pharmaceutically connect
The carrier received.
11. pharmaceutical composition according to claim 7, wherein described pharmaceutical composition includes the Dan Ke of immune amount
The preparation of the light-chain variable sequence of the heavy chain variable region of grand antibody PEDV-McAB1 and the monoclonal antibody PEDV-McAB2
Single-chain antibody and pharmaceutically acceptable carrier；The amino acid of the heavy chain variable region of the monoclonal antibody PEDV-McAB1
Sequence is shown in SEQ ID No.2, and the amino acid sequence of the monoclonal antibody PEDV-McAB2 light chain variable region is SEQ ID
Shown in No.8.
12. a kind of kit, wherein the kit includes a effective amount of antibody as claimed in claim 2 and/or effective quantity
Claim 5 described in antibody, and for carrying out the inspection that is detected of antigen-antibody reaction to Porcine epidemic diarrhea virus
Test agent, negative control, positive control.
13. kit according to claim 12, wherein the antibody as claimed in claim 2 and claim 5 institute
The antibody stated is marked using enzyme, described to try for carrying out the detection that antigen-antibody reaction is detected to Porcine epidemic diarrhea virus
Agent is the substrate that color reaction is generated with the enzyme of the label.
14. kit according to claim 12, wherein the kit includes a effective amount of monoclonal antibody
PEDV-McAB1 and/or a effective amount of monoclonal antibody PEDV-McAB2, for being carried out to Porcine epidemic diarrhea virus
Detection reagent that antigen-antibody reaction is detected, negative control, positive control.
15. kit according to claim 12, wherein described for carrying out antigen-antibody to Porcine epidemic diarrhea virus
The detection reagent that reaction is detected is anti-for the ELIAS secondary antibody in conjunction with the antibody and with the enzyme generation color of the label
The substrate answered, the ELIAS secondary antibody include anti-, enzyme mark sheep anti mouse secondary antibody more than enzyme mark sheep anti mouse.
16. a kind of kit, wherein the kit includes monoclonal antibody PEDV- described in a effective amount of claim 3
Monoclonal antibody PEDV-McAB2 described in McAB1, a effective amount of claim 6, and for Porcine epidemic diarrhea virus into
The detection reagent that row antigen-antibody reaction is detected；
Wherein, the kit includes colloidal gold colloidal gold detection test paper strip, and the colloidal gold colloidal gold detection test paper strip includes bottom plate, the bottom plate
Have a first end and a second end, and successively have on the direction of second end along the first end filter paper, sample pad, gold-labelled pad,
Nitrocellulose filter and water absorption pad, the nitrocellulose filter are contacted with gold-labelled pad or are contacted with sample pad, gold-labelled pad so that anti-
The combination physical efficiency of the former and described monoclonal antibody PEDV-McAB2 is migrated to bottom plate second end on it；Contain in the gold-labelled pad
The monoclonal antibody PEDV-McAB2 of colloid gold label, on the position of the closely described bottom plate second end of the nitrocellulose filter
Including a detection line and a nature controlling line, immobilization has the monoclonal antibody PEDV-McAB1 in the detection line, described
Immobilization has sheep anti mouse secondary antibody or sheep anti mouse mostly anti-on nature controlling line.
17. a kind of kit, wherein the kit includes monoclonal antibody PEDV- described in a effective amount of claim 3
Monoclonal antibody PEDV-McAB2 described in McAB1, a effective amount of claim 6, and for Porcine epidemic diarrhea virus into
The detection reagent that row antigen-antibody reaction is detected；
Wherein, the kit includes buffer supply unit and enzyme immunochromatographydetecting detecting test strip；The buffer supply is single
Member is for supplying the enzyme immunochromatographydetecting detecting test strip for buffer；The enzyme immunochromatographydetecting detecting test strip includes nitric acid fibre
Plain film (1) is tieed up, the enzyme immunochromatographydetecting detecting test strip successively includes substrate supply area, sample supply area, detection in the longitudinal direction
Area；The substrate supply area includes substrate pad (3), is adsorbed with dry zymolyte, the substrate pad (3) and cellulose nitrate thereon
Plain film (1) contact, the zymolyte are dissolved in buffer and on nitrocellulose filter (1) to away from the buffer supply unit
Distal migration；The sample supply area includes enzyme mark pad (2), is adsorbed with the monoclonal antibody PEDV- of enzyme label thereon
McAB2, the zymolyte can generate chromogenic reaction, the enzyme mark pad with the enzyme marked on the monoclonal antibody PEDV-McAB2
(2) it is contacted with nitrocellulose filter (1), the monoclonal antibody PEDV-McAB2 is dissolved in buffer and in nitrocellulose filter
(1) to the distal migration away from the buffer supply unit on；And the detection zone immobilization has the monoclonal antibody
PEDV-McAB1；
Wherein, the buffer supply unit is gentle including expansion fluid cushion (5), substrate buffer liquid bath (8), substrate buffer solution (9)
Fliud flushing button, the substrate buffer solution (9) are placed in substrate buffer liquid bath (8), and the buffer button is located at substrate buffer
The top of liquid bath (8), pressing can immerse the expansion fluid cushion (5) in buffer (9)；The detection zone include detection line (6),
Nature controlling line (7), wherein the nature controlling line (7) compared with detection line (6) further from the sample supply area, on the detection line (6)
Immobilization has the monoclonal antibody PEDV-McAB1, and immobilization has sheep anti mouse secondary antibody or sheep anti mouse on the nature controlling line (7)
It is mostly anti-, and the monoclonal antibody PEDV-McAB2 for the enzyme label being adsorbed on enzyme mark pad (2) is for being fixed on detection line (6)
On the monoclonal antibody PEDV-McAB1 for be excessive；Nitrocellulose filter (1) full section is adhered to the branch
Support on object (10), supporter (10) connects the buffer supply unit, substrate supply area, sample supply area, detection zone and
Water absorption pad (4), the water absorption pad (4) is in the distalmost end away from the buffer supply unit；And test sample (11) addition
Position is the position of the enzyme mark pad (2).
18. a kind of kit, wherein the kit includes monoclonal antibody PEDV-McAB1 described in coating claim 3
Elisa plate, is used for pig epidemic diarrhea the reaction solution containing monoclonal antibody PEDV-McAB2 described in enzyme label claim 6
Virus carries out detection reagent, the negative control, positive control that antigen-antibody reaction is detected.
19. kit according to claim 18, wherein the kit includes being coated with the monoclonal antibody PEDV-
The elisa plate of McAB1, the reaction solution that the monoclonal antibody PEDV-McAB2 is marked containing enzyme, cleaning solution, dilution, substrate are aobvious
Color liquid, terminate liquid, negative control, positive control；Wherein, the cleaning solution is phosphate buffer, and the dilution is containing ox
Sero-abluminous solution, the substrate developing solution are tetramethyl benzidine TMB developing solution, and the terminate liquid is the dense of 2mol/l
H2SO4Solution, the negative control are phosphate buffer, and the positive control is comprising inactivating pig epidemic after purification
The solution of diarrhea virus.
20. a kind of kit, wherein the kit includes the elisa plate for being coated with Porcine epidemic diarrhea virus antigen, containing described
Reaction solution, cleaning solution, dilution, substrate developing solution, terminate liquid, the negative control, positive control of monoclonal antibody；Wherein, contain
The reaction solution of the monoclonal antibody is containing the reaction solution of monoclonal antibody PEDV-McAB1 described in claim 3 or to contain right
It is required that the reaction solution of the 6 monoclonal antibody PEDV-McAB2 or containing the monoclonal antibody PEDV-McAB1 and the Dan Ke
The mixed reaction solution of grand antibody PEDV-McAB2；Wherein, the cleaning solution is phosphate buffer, and the dilution is containing ox blood
The solution of pure albumen, the substrate developing solution are tetramethyl benzidine TMB developing solution, and the terminate liquid is the dense of 2mol/l
H2SO4Solution, the negative control are phosphate buffer, and the positive control is comprising inactivating pig epidemic after purification
The solution of diarrhea virus.
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Families Citing this family (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105891491A (en) * | 2016-04-11 | 2016-08-24 | 洛阳普莱柯万泰生物技术有限公司 | Kit and application thereof |
| CN105906712B (en) * | 2016-06-28 | 2019-05-24 | 河南农业大学 | Porcine epidemic diarrhea resisting virus pig source single-chain antibody and preparation method thereof |
| CN106146658B (en) * | 2016-07-25 | 2019-10-01 | 黑龙江八一农垦大学 | A kind of preparation method recombinating Porcine epidemic diarrhea virus antibody |
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| CN110967480A (en) * | 2018-09-28 | 2020-04-07 | 洛阳普泰生物技术有限公司 | Preparation and application of pig epidemic diarrhea virus IgA antibody ELISA kit |
| CN111665362B (en) * | 2019-03-07 | 2023-10-17 | 北京中科基因技术有限公司 | Method for preparing anti-porcine virus antibody immunochromatographic test strip containing quantum dot label, prepared test strip and application |
| CN110187097B (en) * | 2019-05-28 | 2021-09-14 | 中国农业科学院哈尔滨兽医研究所 | Fluorescent quantitative detection test strip for porcine epidemic diarrhea virus and preparation method and application thereof |
| CN114057868B (en) * | 2020-07-31 | 2023-08-15 | 洛阳普泰生物技术有限公司 | Swine delta coronavirus antibody, kit containing swine delta coronavirus antibody and application |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339192A (en) * | 2008-08-28 | 2009-01-07 | 河南省农业科学院 | Test paper for one-step detection for pig virus diarrhoea disease pathogen |
| CN104062430A (en) * | 2014-06-30 | 2014-09-24 | 洛阳普莱柯万泰生物技术有限公司 | Kit box used for detecting influenza virus in sample and detection method and application thereof |
-
2015
- 2015-12-17 CN CN201510948571.2A patent/CN105461805B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101339192A (en) * | 2008-08-28 | 2009-01-07 | 河南省农业科学院 | Test paper for one-step detection for pig virus diarrhoea disease pathogen |
| CN104062430A (en) * | 2014-06-30 | 2014-09-24 | 洛阳普莱柯万泰生物技术有限公司 | Kit box used for detecting influenza virus in sample and detection method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| 免疫检测用的胶体金制备工艺的优化研究;李红梅等;《工艺技术》;20091231;第30卷(第12期);第289-291页 |
| 抗猪流行性腹泻病毒单克隆抗体的研制及其特性鉴定;孙智锋等;《中国兽医学报》;19980331;第18卷(第2期);第131-134页 |
| 猪流行性腹泻病毒单克隆抗体的研制;张丽燕;《中国优秀硕士学位论文全文数据库 农业科技辑》;20130715(第7期);D050-228 |
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