CN109810190A - It can be used for detecting the monoclonal antibody and kit of PPR virus - Google Patents

It can be used for detecting the monoclonal antibody and kit of PPR virus Download PDF

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CN109810190A
CN109810190A CN201910275169.0A CN201910275169A CN109810190A CN 109810190 A CN109810190 A CN 109810190A CN 201910275169 A CN201910275169 A CN 201910275169A CN 109810190 A CN109810190 A CN 109810190A
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马宏伟
金燕萍
徐宏科
李彤
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Suzhou Institute of Nano Tech and Nano Bionics of CAS
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    • G01N2333/115—Paramyxoviridae, e.g. parainfluenza virus
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Abstract

The present invention relates to the monoclonal antibodies and kit that can be used for detecting PPR virus.Monoclonal antibody of the invention can specifically bind PPR virus.Peste des petits ruminants diagnostic kit of the invention includes substrate and the monoclonal antibody of the invention that is fixed in the substrate, be can be used in the biological sample of test object biological source with the presence or absence of PPR virus.

Description

It can be used for detecting the monoclonal antibody and kit of PPR virus
Technical field
The invention belongs to biological detection and technical field of medical detection, and in particular to one kind can be used for detecting peste des petits ruminants The monoclonal antibody and kit of virus.
Background technique
Peste des petits ruminants (peste des petits ruminants, PPR) is commonly called as sheep pest also known as false rinderpest (pseudorinderpest), the compound disease (stomatitis- of pneumoenteritis (pneumoenteritis), stomatitis pneumoenteritis Pneumoenteritis complex), it is a kind of acute viral infectious disease as caused by PPR virus, it is main to feel Small ruminant is contaminated, characterized by fever, stomatitis, diarrhea, pneumonia.
Nineteen forty-two, this disease occurred in the Ivory Coast for the first time, and thereafter, African Senegal, Ghana, Togo, Benin etc. have this Disease is reported, this disease also has occurred in the sheep and goat of Nigeria, and cause heavy losses.Some countries in Asia also report This disease of road, according to World Organization for Animal Health (OIE) 1993 " world animal health " reports, the goat of Bangladesh had this The disease of similar rinderpest has occurred in disease generation, India De Labang and some areas sheep of horse Harrar Shi Telabang, it is last true It examines as peste des petits ruminants, hereafter, safe Mir's ladd nation is also infected report.1993, Israel's first time report had small Epizootic disease is ruminated, source of infection is unknown, and to prevent this disease from propagating, Israel is vaccinated with the sheep and goat of its northern territory Rinderpest vaccines.1992, this disease-specific antibody was had found in the sheep and goat of Jordan, 1993, there are 11 farms to occur Clinical case, more than 100 sheep and goat death.1993, Saudi Arabia found 133 cases for the first time.In July, 2007, The epidemic disease is passed to China Tibet region for the first time.
PPR virus category paramyxovirus section Morbillivirus.There is similar physical chemistry with rinderpest virus and is immunized Learn characteristic.Virus is in pleomorphism, usually coarse spherical shape.Virion is big compared with rinderpest virus, and nucleocapsid is spiral hollow stem Shape simultaneously has characteristic subunit, there is cyst membrane.Virus can be in tire sheep renal, testicular cell, the Vero cell of tire sheep and newborn sheep Upper proliferation, and cytopathy (CPE) is generated, form plasomidum.
The small ruminants such as this disease main infection goat, sheep, U.S. white-tailed deer are popular in African western, middle part and Asia The some areas in continent.In epidemic-stricken area, this disease is that fragmentary generation can happening and prevelence when susceptible animal increases.This disease mainly passes through Immediate contagion, the secretion and excreta of ill domestic animal are the infections sources, and the sick sheep for facing the type of examining in Asia is especially dangerous.It is small to ruminate beast Epidemic disease incubation period is 4-5 days, longest 21 days.Natural occurrence is detected in goat and sheep.Goat morbidity is serious, and sheep also occasionally has seriously Case occurs.The lip of some rehabilitation goats forms aphtha sample lesion.Infection animal clinical symptom is similar to rinderpest infected cattle.It is acute Type body temperature can rise to 41 DEG C, and continue 3~5 days.Infection animal is had the fidgets, and dorsal body setae is unglazed, and mouth and nose are dry, appetite stimulator. Mucus purulent rhinorrhea is flowed, foul gas is breathed out.At first 4 days of fever, mucous membrane of mouth was congested, cheek mucous membrane progressive popularity damage Evil leads to polysialia, gangrenosum acne lesion then occurs, starts mucous membrane of mouth and small coarse red superficial necrotic lesion occurs, with After become pink, infection site includes lower lip, lower gums etc..The visible necrotic lesion of several cases involves tooth pad, palate, cheek And its nipple, tongue etc..There is band blood watery diarrhea in later period, and serious dehydration is thin, therewith temperature decline.There is cough, exhale It inhales abnormal.Disease incidence is up to 100%, and when seriously breaking out, the death rate 100%, in slight occur, the death rate is no more than 50%.Growing animal morbidity severe morbidity and dead all very high, a kind of disease delimited for China.
Because this virus has special affinity to gastrointestinal lymphoid cell and epithelial cell, therefore feature venereal disease can be caused Become.Generally occur acidophilia cytoplasmic inclusions and multinucleate giant cell in infection cell.In lymphoid tissue, peste des petits ruminants disease Poison can cause lymphocyte downright bad.Spleen, tonsillotome, lymph node cells are destroyed.The multicore of the cytoplasmic inclusions containing acidophilia is huge Cell occurs, rarely intranuclear inclusion.In digestive system, it is bad that virus causes the epithelial cell in Ma Erjishi layer depth portion to occur Extremely, infection cell generates karyopycnosis and karyorrhexis, and it is big and small to form the multicore containing acidophilia cytoplasmic inclusions in stratum germinativum epidermidis Born of the same parents.
Currently, effective ways there is no to treat peste des petits ruminants, vaccine inoculation, epidemic situation can only be taken to slaughter and periodically after occurring The method of sero monitoring is controlled.
Currently, peste des petits ruminants is mainly to be examined by the PPR virus antibody in detection biological sample It is disconnected.World Organization for Animal Health recommends the PPR virus antibody detection method used mainly to have virus neutralization tests (VNT) and enzyme-linked immunosorbent assay (ELISA).Wherein, the testing result of virus neutralization tests method is accurate, and being that detection is small ruminates beast The goldstandard of epidemic disease poison, but this method detection time is long and is unsuitable for detecting great amount of samples.In contrast, enzyme-linked immunosorbent assay Specificity and sensibility are higher, detection time is shorter than virus neutralization tests and therefore the suitable a large amount of samples of detection are widely used In the detection of PPR virus antibody.
But in the presence of for PPR virus antibody, simultaneously nontarget organism suffers from peste des petits ruminants in biological sample Positive evidence, this is the intrinsic deficiency that the diagnostic method of peste des petits ruminants is diagnosed by detection antibody.Thus, it is desirable to have directly Detect the ELISA method of the PPR virus in biological sample.
But ELISA method is both needed to using monoclonal antibody, and just it has been observed that PPR virus is a kind of cyst membrane Virus, the monoclonal antibody that can be used for ELISA method detection PPR virus are not readily available.
Summary of the invention
In view of above-mentioned problems of the prior art, the purpose of the present invention is to provide one kind can be specific and small anti- The monoclonal antibody that hay epizootic disease virus combines, and the specificity of the monoclonal antibody is utilized and high small of sensibility ruminates beast Epidemic disease diagnostic kit.
Inventor has made intensive studies to solve above-mentioned technical problem, and house is immunized with purified PPR virus Rabbit is prepared for rabbit-anti PPR virus monoclonal antibody (code name: NMS-1-2H3L2), the rabbit-anti PPR virus list Clonal antibody can specifically combine PPR virus, and can be used in preparing and ruminate beast based on the small of ELISA principle Epidemic disease diagnostic kit.
That is, the present invention includes:
A kind of monoclonal antibody can be used for detecting PPR virus, it includes three complementary determining region of heavy chain (CDR-H1, CDR-H2 and CDR-H3) and three complementary determining region of light chain (CDR-L1, CDR-L2 and CDR-L3), in which:
(a) amino acid sequence of CDR-H1 is as shown in SEQ ID NO:1；
(b) amino acid sequence of CDR-H2 is as shown in SEQ ID NO:2；
(c) amino acid sequence of CDR-H3 is as shown in SEQ ID NO:3；
(d) amino acid sequence of CDR-L1 is as shown in SEQ ID NO:4；
(e) amino acid sequence of CDR-L2 is as shown in SEQ ID NO:5；And
(f) amino acid sequence of CDR-L3 is as shown in SEQ ID NO:6；
Wherein,
SEQ ID NO:1:GIDLNSAT (one-letter symbol of amino acid, similarly hereinafter)
SEQ ID NO:2:IVSGSTN
SEQ ID NO:3:ARYAGTIAYQWYFNI
SEQ ID NO:4:ESISSI
SEQ ID NO:5:RTS
SEQ ID NO:6:QQGVSNSNVDNI；
Or include three complementary determining region of heavy chain (CDR-H4, CDR-H5 and CDR-H6) and three complementary determining region of light chain (CDR-L4, CDR-L5 and CDR-L6), in which:
(g) amino acid sequence of CDR-H4 is as shown in SEQ ID NO:9；
(h) amino acid sequence of CDR-H5 is as shown in SEQ ID NO:10；
(i) amino acid sequence of CDR-H6 is as shown in SEQ ID NO:11；
(j) amino acid sequence of CDR-L4 is as shown in SEQ ID NO:12；
(k) amino acid sequence of CDR-L5 is as shown in SEQ ID NO:13；And
(l) amino acid sequence of CDR-L6 is as shown in SEQ ID NO:14；
Wherein,
SEQ ID NO:9:GIDLGGYA
SEQ ID NO:10:IDTTDS
SEQ ID NO:11:ARYAGDGGGGYFFDY
SEQ ID NO:12:QSVYNNNC
SEQ ID NO:13:GAS
SEQ ID NO:14:VGAYIGSNYA；
Or include three complementary determining region of heavy chain (CDR-H7, CDR-H8 and CDR-H9) and three complementary determining region of light chain (CDR-L7, CDR-L8 and CDR-L9), in which:
(m) amino acid sequence of CDR-H7 is as shown in SEQ ID NO:17；
(n) amino acid sequence of CDR-H8 is as shown in SEQ ID NO:18；
(o) amino acid sequence of CDR-H9 is as shown in SEQ ID NO:19；
(p) amino acid sequence of CDR-L7 is as shown in SEQ ID NO:10；
(q) amino acid sequence of CDR-L8 is as shown in SEQ ID NO:21；And
(r) amino acid sequence of CDR-L9 is as shown in SEQ ID NO:22；
Wherein,
SEQ ID NO:17:GIDLGGYA
SEQ ID NO:18:IDTTDS
SEQ ID NO:19:ARYAGDGGGGYFFDY
SEQ ID NO:10:QSIYNN
SEQ ID NO:21:EAS
SEQ ID NO:22:QQGYSITNVDNT.
Above-mentioned monoclonal antibody, it includes heavy chains and light chain, wherein
The amino acid sequence such as SEQ of heavy chain comprising three complementary determining region of heavy chain (CDR-H1, CDR-H2 and CDR-H3) Shown in ID NO:7；
The amino acid sequence such as SEQ of light chain comprising three complementary determining region of light chain (CDR-L1, CDR-L2 and CDR-L3) Shown in ID NO:8；
Wherein,
SEQ ID NO:7:
CQSLEESGGRLVTPGGSLTLTCTVSGIDLNSATVGWVRQAPGKGLEWIGDIVSGSTNTDYANWASGRFT ISKTSSTTVDLKMTSLTTEDTATYFCARYAGTIAYQWYFNIWGPGTLVTVSSGQPKAPSVFPLAPCCGDTPSSTVTL GCLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSK PTCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVV STLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVE WEKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
SEQ ID NO:8:
DVVMTQTPASVEVAVGGTVTIKCQASESISSILAWYQQKPGQRPNLLMYRTSTLASGVSSRFKGSGSGT DFTLTISGVQCDDAATYYCQQGVSNSNVDNIFGGGTEVVVTGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDV TVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
The amino acid sequence such as SEQ of heavy chain comprising three complementary determining region of heavy chain (CDR-H4, CDR-H5 and CDR-H6) Shown in ID NO:15；
The amino acid sequence such as SEQ of light chain comprising three complementary determining region of light chain (CDR-L4, CDR-L5 and CDR-L6) Shown in ID NO:16；
Wherein,
SEQ ID NO:15:
CQSLEESGGRLVTPGGSLTLTCTVSGIDLGGYAVGWVRQAPGKGLEYIGIIDTTDSTYYASWAKGRFTS SKTSSTTVDLKMTSLTTEDTATYFCARYAGDGGGGYFFDYWGSGTLVTITSGQPKAPSVFPLAPCCGDTPSSTVTLG CLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKP TCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVS TLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEW EKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
SEQ ID NO:16:
AAVLTQTPSPISAAVGGTVTINCQSSQSVYNNNCLSWYQQKPGQPPKFLIYGASTLASGVPSRFKGSGS GTEFTLTISDVQCADAATYYCVGAYIGSNYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFPDV TVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC
The amino acid sequence such as SEQ of heavy chain comprising three complementary determining region of heavy chain (CDR-H7, CDR-H8 and CDR-H9) Shown in ID NO:23；
The amino acid sequence such as SEQ of light chain comprising three complementary determining region of light chain (CDR-L7, CDR-L8 and CDR-L9) Shown in ID NO:24；
Wherein,
SEQ ID NO:23:
CQSLEESGGRLVTPGGSLTLTCTVSGIDLGGYAVGWVRQAPGKGLEYIGIIDTTDSTYYASWAKGRFTS SKTSSTTVDLKMTSLTTEDTATYFCARYAGDGGGGYFFDYWGSGTLVTITSGQPKAPSVFPLAPCCGDTPSSTVTLG CLVKGYLPEPVTVTWNSGTLTNGVRTFPSVRQSSGLYSLSSVVSVTSSSQPVTCNVAHPATNTKVDKTVAPSTCSKP TCPPPELLGGPSVFIFPPKPKDTLMISRTPEVTCVVVDVSQDDPEVQFTWYINNEQVRTARPPLREQQFNSTIRVVS TLPIAHQDWLRGKEFKCKVHNKALPAPIEKTISKARGQPLEPKVYTMGPPREELSSRSVSLTCMINGFYPSDISVEW EKNGKAEDNYKTTPAVLDSDGSYFLYSKLSVPTSEWQRGDVFTCSVMHEALHNHYTQKSISRSPGK
SEQ ID NO:24:
AAVLTQTPSPISAAVGGTVTINCQSSQSVYNNNCLSWYQQKPGQPPKFLIYGASTLASGVPSRFKGSG SGTEFTLTISDVQCADAATYYCVGAYIGSNYAFGGGTEVVVKGDPVAPTVLIFPPAADQVATGTVTIVCVANKYFP DVTVTWEVDGTTQTTGIENSKTPQNSADCTYNLSSTLTLTSTQYNSHKEYTCKVTQGTTSVVQSFNRGDC。
A kind of detection device comprising substrate and the said monoclonal antibody being fixed in the substrate.
Above-mentioned detection device is used in the biological sample of test object biological source with the presence or absence of peste des petits ruminants disease Poison.
Above-mentioned detection device, wherein the object organisms are goat or sheep.
Above-mentioned detection device, wherein the biological sample is whole blood, blood plasma or serum.
Whether a kind of peste des petits ruminants diagnostic kit is used in the biological sample by test object biological source deposit In PPR virus, diagnose whether object organisms suffer from peste des petits ruminants.
Above-mentioned peste des petits ruminants diagnostic kit, wherein the object organisms are goat or sheep.
Above-mentioned peste des petits ruminants diagnostic kit, wherein the biological sample is whole blood, blood plasma or serum.
The specific embodiment of invention
Embodiment 1 is used as the purifying of the PPR virus of antigen
1.1 purifying concentration systems: desk-top membrane filtration system, AKTA Pure Protein purification chromatographic system, high speed refrigerated centrifuge Machine, superspeed refrigerated centrifuge, 500K hollow fiber column, 100kd ultra-filtration centrifuge tube.
1.2 main agents: 0.02mol/L PBS, 0.5N NaOH solution sephrose 6FF chromatography media, sucrose.
The preparation of 1.3 antigens and clarification
Under aseptic condition, 2000mL PPR virus antigen, 4 DEG C, 5000rpm centrifugation 30min are taken.Precipitating is abandoned, is taken Supernatant is for being concentrated by ultrafiltration.
1.4 concentrating and purifying
1.4.1,500K hollow fiber column and pipeline, then the hollow fiber column with 2%NaOH lye wash cycles 500K are installed And soaked overnight, then to neutrality and water flux is surveyed with residual lye in purified water washing hollow-fiber column.It is flat with sterile PBS liquid Weigh hollow fiber column.
1.4.2 the feed liquor of sterile connection hollow fiber column and concentration is carried out after returning liquid, when processing keeps pump revolution to exist Between 10%~15%.Sample detection when every batch of sample distinguishes cycles of concentration to 6 times, with sterile PBS 6 times of concentrates of continuous filter wash 4 times, liquid is gone out with filter wash and calculates filter wash number (i.e. filter wash, which goes out when liquid volume is 5 times 2 times of concentrate, terminates filter wash), is taken respectively Sample at least five after each filter wash.
1.4.3 gel filtration chromatography
It takes 24mL PPR that liquid is concentrated by ultrafiltration, carries out loading according to 5mL/min volume flow rate, use 0.02mol/L PBS (pH =7.4, Cond=18.0ms/cm) it is eluted, it is collected according to 280 ultraviolet absorption peak of UV.
1.4.4 100KD ultrafiltration membrane is concentrated
Using Merck-Mi Libo 100KD ultra-filtration centrifuge tube, 5 times of concentrations are carried out to 1# absorption peak.
1.4.5 sucrose density gradient centrifugation
1mL sample is added in discontinuous sucrose density gradient (15%-80%) w/v solution, 35000rpm/min, 4 DEG C It is centrifuged 3h, every 0.5mL collects 1 pipe, totally 23 pipe, number consecutively 1-23.Every pipe takes 70uL to carry out SDS-PAGE detection.
1.5 detection
No. 1-23 is managed, every pipe takes 70uL to carry out the detection of SDS-PAGE protein electrophoresis.Testing result shows 11-14 Protein concentration highest in number pipe.Therefore, merge the sample in 11-14 pipe, as immune antigen.
2 antigen of embodiment is immune, B cell is screened and the preparation of monoclonal antibody
Biotechnology Co., Ltd is insulted in antigen immunizing rabbit, B cell screening and the preparation of monoclonal antibody commission Hangzhou hundred It completes.With the immune use antigen immunizing rabbit prepared in above-described embodiment 1, screened from the peripheral blood for completing the rabbit after being immunized The single B cell of anti-PPR virus monoclonal antibody is secreted, and obtains the coding core of the monoclonal antibody by sequencing Nucleotide sequence (so as to know the amino acid sequence of the monoclonal antibody, referring to SEQ ID NO:1-8).By by the coding Nucleotide sequence is cloned into expression vector appropriate, and the expression vector is imported suitable host cell and carries out expression and pure Change, to obtain monoclonal antibody NMS-1-2H3L2.
The preparation and application of 3 peste des petits ruminants diagnostic kit of embodiment (detection device)
Using conventional method on conventional ELISA solid phase carrier point sample said monoclonal antibody NMS-1-2H3L2 Solution, in addition one PB point of point sample is prepared for detection device (diagnostic kit) as negative Quality Control point.
Detecting step (antibody sandwich)
(1), with purified water by 20 × concentrated cleaning solutions (TBST:0.4M Tris-HCl, 2.74MNaCl, 2%Tween20, PH7.2 ± 0.2) it is diluted by 1:20, obtain cleaning solution.It, will about 200 with liquid-transfering gun to make to detect device surface complete wetting μ L cleaning solution is added to detection device surface, and impregnates detection device certain time.
It (2), will with Sample dilution (0.05MPBS, 1%BSA, 0.2%PVP, 0.5%Tween20, pH7.2 ± 0.2) Test serum sample is diluted according to 1:50.
(3), for having discarded the detection device of cleaning solution, in the state that surface is completely wet, after drawing 200 μ L dilution Serum sample be added to detection device on.
(4), it will test device in constant-temperature table with 150 revs/min to be incubated for 30 minutes, temperature is room temperature.
(5), serum sample is discarded, the surface of detection device is cleaned with cleaning solution.
(6), after cleaning, the molten of the monoclonal antibody NMS-1-2H3L2 of the HRP label of 200 μ L is added on detection device Liquid will test device in constant-temperature table with 150 revs/min and be incubated for 30 minutes, and temperature is room temperature.
(7), enzyme labelled antibody solution is discarded, device surface is will test with cleaning solution and cleans up.
(8), after the completion of cleaning, luminous substrate liquid (Thermo, the Prod# of 20 μ L are uniformly sprawled on detection device surface 37074)。
(9), chemiluminescence imaging will be carried out to detection device with gel imager, and determines result.
(10), result judgement: for every a serum, the monoclonal antibody NMS-1- on the detection device is counted respectively Whether 2H3L2 has response (that is, signal-to-noise ratio (SNR) is more than or equal to 2), and is determined.That is,
When detecting that monoclonal antibody NMS-1-2H3L2 has response, it is determined as the PPR virus positive；Conversely, It is determined as feminine gender.
Wherein, signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point Signal value refers to the chemiluminescence intensity value for the polypeptide point that software is read, and negative control point signal value refers to the feminine gender that software is read The chemiluminescence intensity value of control point.
For 800 parts of goats and sheep serum sample from China regions, be respectively adopted above-mentioned diagnostic kit with And viral nucleic acid amplification is detected, testing result is as follows.
1 800 portions of goats of table and sheep serum pattern detection result
Sensibility=493/560*100%=88%
Specificity=224/240=93%
As known from the above, the sensibility and specificity of the diagnostic kit (and is using nucleic acid amplification 85% or more Method validation is not the verifying of other contrast agents cassette methods), fully meet the requirement of disease diagnosis.
Embodiment 4 is used as the purifying of the PPR virus of antigen
1.1 purifying concentration systems: desk-top membrane filtration system, AKTA Pure Protein purification chromatographic system, high speed refrigerated centrifuge Machine, superspeed refrigerated centrifuge, 500K hollow fiber column, 100kd ultra-filtration centrifuge tube.
1.2 main agents: 0.02mol/L PBS, 0.5N NaOH solution sephrose 6FF chromatography media, sucrose.
The preparation of 1.3 antigens and clarification
Under aseptic condition, 2000mL PPR virus antigen, 4 DEG C, 5000rpm centrifugation 30min are taken.Precipitating is abandoned, is taken Supernatant is for being concentrated by ultrafiltration.
1.4 concentrating and purifying
1.4.1,500K hollow fiber column and pipeline, then the hollow fiber column with 2%NaOH lye wash cycles 500K are installed And soaked overnight, then to neutrality and water flux is surveyed with residual lye in purified water washing hollow-fiber column.It is flat with sterile PBS liquid Weigh hollow fiber column.
1.4.2 the feed liquor of sterile connection hollow fiber column and concentration is carried out after returning liquid, when processing keeps pump revolution to exist Between 10%~15%.Sample detection when every batch of sample distinguishes cycles of concentration to 6 times, with sterile PBS 6 times of concentrates of continuous filter wash 4 times, liquid is gone out with filter wash and calculates filter wash number (i.e. filter wash, which goes out when liquid volume is 5 times 2 times of concentrate, terminates filter wash), is taken respectively Sample at least five after each filter wash.
1.4.3 gel filtration chromatography
It takes 24mL PPR that liquid is concentrated by ultrafiltration, carries out loading according to 5mL/min volume flow rate, use 0.02mol/L PBS (pH =7.4, Cond=18.0ms/cm) it is eluted, it is collected according to 280 ultraviolet absorption peak of UV.
1.4.4 100KD ultrafiltration membrane is concentrated
Using Merck-Mi Libo 100KD ultra-filtration centrifuge tube, 5 times of concentrations are carried out to 1# absorption peak.
1.4.5 sucrose density gradient centrifugation
1mL sample is added in discontinuous sucrose density gradient (15%-80%) w/v solution, 35000rpm/min, 4 DEG C It is centrifuged 3h, every 0.5mL collects 1 pipe, totally 23 pipe, number consecutively 1-23.Every pipe takes 70uL to carry out SDS-PAGE detection.
1.5 detection
No. 1-23 is managed, every pipe takes 70uL to carry out the detection of SDS-PAGE protein electrophoresis.Testing result shows 11-14 Protein concentration highest in number pipe.Therefore, merge the sample in 11-14 pipe, as immune antigen.
5 antigen of embodiment is immune, B cell is screened and the preparation of monoclonal antibody
Biotechnology Co., Ltd is insulted in antigen immunizing rabbit, B cell screening and the preparation of monoclonal antibody commission Hangzhou hundred It completes.With the immune use antigen immunizing rabbit prepared in above-described embodiment 4, screened from the peripheral blood for completing the rabbit after being immunized The single B cell of anti-PPR virus monoclonal antibody is secreted, and obtains the coding core of the monoclonal antibody by sequencing Nucleotide sequence (so as to know the amino acid sequence of the monoclonal antibody, referring to SEQ ID NO:9-16).By by the volume Code nucleotide sequence is cloned into expression vector appropriate, and the expression vector is imported suitable host cell and carries out expression and pure Change, to obtain monoclonal antibody NMS-1-17H6L3.
The preparation and application of 6 peste des petits ruminants diagnostic kit of embodiment (detection device)
Using conventional method on conventional ELISA solid phase carrier point sample said monoclonal antibody NMS-1-17H6L3 Solution, in addition one PB point of point sample is prepared for detection device (diagnostic kit) as negative Quality Control point.
Detecting step (antibody sandwich)
(1), with purified water by 20 × concentrated cleaning solutions (TBST:0.4M Tris-HCl, 2.74MNaCl, 2%Tween20, PH7.2 ± 0.2) it is diluted by 1:20, obtain cleaning solution.It, will about 200 with liquid-transfering gun to make to detect device surface complete wetting μ L cleaning solution is added to detection device surface, and impregnates detection device certain time.
It (2), will with Sample dilution (0.05MPBS, 1%BSA, 0.2%PVP, 0.5%Tween20, pH7.2 ± 0.2) Test serum sample is diluted according to 1:50.
(3), for having discarded the detection device of cleaning solution, in the state that surface is completely wet, after drawing 200 μ L dilution Serum sample be added to detection device on.
(4), it will test device in constant-temperature table with 150 revs/min to be incubated for 30 minutes, temperature is room temperature.
(5), serum sample is discarded, the surface of detection device is cleaned with cleaning solution.
(6), after cleaning, the molten of the monoclonal antibody NMS-1-17H6L3 of the HRP label of 200 μ L is added on detection device Liquid will test device in constant-temperature table with 150 revs/min and be incubated for 30 minutes, and temperature is room temperature.
(7), enzyme labelled antibody solution is discarded, device surface is will test with cleaning solution and cleans up.
(8), after the completion of cleaning, luminous substrate liquid (Thermo, the Prod# of 20 μ L are uniformly sprawled on detection device surface 37074)。
(9), chemiluminescence imaging will be carried out to detection device with gel imager, and determines result.
(10), result judgement: for every a serum, the monoclonal antibody NMS-1- on the detection device is counted respectively Whether 17H6L3 has response (that is, signal-to-noise ratio (SNR) is more than or equal to 2), and is determined.That is,
When detecting that monoclonal antibody NMS-1-17H6L3 has response, it is determined as the PPR virus positive；Conversely, It is determined as feminine gender.
Wherein, signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point Signal value refers to the chemiluminescence intensity value for the polypeptide point that software is read, and negative control point signal value refers to the feminine gender that software is read The chemiluminescence intensity value of control point.
For 800 parts of goats and sheep serum sample from China regions, be respectively adopted above-mentioned diagnostic kit with And viral nucleic acid amplification is detected, testing result is as follows.
2 800 portions of goats of table and sheep serum pattern detection result
Sensibility=505/560*100%=90%
Specificity=226/240=94%
As known from the above, the sensibility and specificity of the diagnostic kit (and is using nucleic acid amplification 85% or more Method validation is not the verifying of other contrast agents cassette methods), fully meet the requirement of disease diagnosis.
Embodiment 7 is used as the purifying of the PPR virus of antigen
1.1 purifying concentration systems: desk-top membrane filtration system, AKTA Pure Protein purification chromatographic system, high speed refrigerated centrifuge Machine, superspeed refrigerated centrifuge, 500K hollow fiber column, 100kd ultra-filtration centrifuge tube.
1.2 main agents: 0.02mol/L PBS, 0.5N NaOH solution sephrose 6FF chromatography media, sucrose.
The preparation of 1.3 antigens and clarification
Under aseptic condition, 2000mL PPR virus antigen, 4 DEG C, 5000rpm centrifugation 30min are taken.Precipitating is abandoned, is taken Supernatant is for being concentrated by ultrafiltration.
1.4 concentrating and purifying
1.4.1,500K hollow fiber column and pipeline, then the hollow fiber column with 2%NaOH lye wash cycles 500K are installed And soaked overnight, then to neutrality and water flux is surveyed with residual lye in purified water washing hollow-fiber column.It is flat with sterile PBS liquid Weigh hollow fiber column.
1.4.2 the feed liquor of sterile connection hollow fiber column and concentration is carried out after returning liquid, when processing keeps pump revolution to exist Between 10%~15%.Sample detection when every batch of sample distinguishes cycles of concentration to 6 times, with sterile PBS 6 times of concentrates of continuous filter wash 4 times, liquid is gone out with filter wash and calculates filter wash number (i.e. filter wash, which goes out when liquid volume is 5 times 2 times of concentrate, terminates filter wash), is taken respectively Sample at least five after each filter wash.
1.4.3 gel filtration chromatography
It takes 24mL PPR that liquid is concentrated by ultrafiltration, carries out loading according to 5mL/min volume flow rate, use 0.02mol/L PBS (pH =7.4, Cond=18.0ms/cm) it is eluted, it is collected according to 280 ultraviolet absorption peak of UV.
1.4.4 100KD ultrafiltration membrane is concentrated
Using Merck-Mi Libo 100KD ultra-filtration centrifuge tube, 5 times of concentrations are carried out to 1# absorption peak.
1.4.5 sucrose density gradient centrifugation
1mL sample is added in discontinuous sucrose density gradient (15%-80%) w/v solution, 35000rpm/min, 4 DEG C It is centrifuged 3h, every 0.5mL collects 1 pipe, totally 23 pipe, number consecutively 1-23.Every pipe takes 70uL to carry out SDS-PAGE detection.
1.5 detection
No. 1-23 is managed, every pipe takes 70uL to carry out the detection of SDS-PAGE protein electrophoresis.Testing result shows 11-14 Protein concentration highest in number pipe.Therefore, merge the sample in 11-14 pipe, as immune antigen.
8 antigen of embodiment is immune, B cell is screened and the preparation of monoclonal antibody
Biotechnology Co., Ltd is insulted in antigen immunizing rabbit, B cell screening and the preparation of monoclonal antibody commission Hangzhou hundred It completes.With the immune use antigen immunizing rabbit prepared in above-described embodiment 7, screened from the peripheral blood for completing the rabbit after being immunized The single B cell of anti-PPR virus monoclonal antibody is secreted, and obtains the coding core of the monoclonal antibody by sequencing Nucleotide sequence (so as to know the amino acid sequence of the monoclonal antibody, referring to SEQ ID NO:17-24).By by the volume Code nucleotide sequence is cloned into expression vector appropriate, and the expression vector is imported suitable host cell and carries out expression and pure Change, to obtain monoclonal antibody NMS-1-21H1L3.
The preparation and application of 9 peste des petits ruminants diagnostic kit of embodiment (detection device)
Using conventional method on conventional ELISA solid phase carrier point sample said monoclonal antibody NMS-1-21H1L3 Solution, in addition one PB point of point sample is prepared for detection device (diagnostic kit) as negative Quality Control point.
Detecting step (antibody sandwich)
(1), with purified water by 20 × concentrated cleaning solutions (TBST:0.4M Tris-HCl, 2.74MNaCl, 2%Tween20, PH7.2 ± 0.2) it is diluted by 1:20, obtain cleaning solution.It, will about 200 with liquid-transfering gun to make to detect device surface complete wetting μ L cleaning solution is added to detection device surface, and impregnates detection device certain time.
It (2), will with Sample dilution (0.05MPBS, 1%BSA, 0.2%PVP, 0.5%Tween20, pH7.2 ± 0.2) Test serum sample is diluted according to 1:50.
(3), for having discarded the detection device of cleaning solution, in the state that surface is completely wet, after drawing 200 μ L dilution Serum sample be added to detection device on.
(4), it will test device in constant-temperature table with 150 revs/min to be incubated for 30 minutes, temperature is room temperature.
(5), serum sample is discarded, the surface of detection device is cleaned with cleaning solution.
(6), after cleaning, the molten of the monoclonal antibody NMS-1-21H1L3 of the HRP label of 200 μ L is added on detection device Liquid will test device in constant-temperature table with 150 revs/min and be incubated for 30 minutes, and temperature is room temperature.
(7), enzyme labelled antibody solution is discarded, device surface is will test with cleaning solution and cleans up.
(8), after the completion of cleaning, luminous substrate liquid (Thermo, the Prod# of 20 μ L are uniformly sprawled on detection device surface 37074)。
(9), chemiluminescence imaging will be carried out to detection device with gel imager, and determines result.
(10), result judgement: for every a serum, the monoclonal antibody NMS-1- on the detection device is counted respectively Whether 21H1L3 has response (that is, signal-to-noise ratio (SNR) is more than or equal to 2), and is determined.That is,
When detecting that monoclonal antibody NMS-1-21H1L3 has response, it is determined as the PPR virus positive；Conversely, It is determined as feminine gender.
Wherein, signal-to-noise ratio=(polypeptide point signal value-negative control point signal value)/negative control point signal value.Polypeptide point Signal value refers to the chemiluminescence intensity value for the polypeptide point that software is read, and negative control point signal value refers to the feminine gender that software is read The chemiluminescence intensity value of control point.
For 800 parts of goats and sheep serum sample from China regions, be respectively adopted above-mentioned diagnostic kit with And viral nucleic acid amplification is detected, testing result is as follows.
The testing result of table 3 800 portions of goats and sheep serum sample
Sensibility=487/560*100%=87%
Specificity=221/240=92%
As known from the above, the sensibility and specificity of the diagnostic kit (and is using nucleic acid amplification 85% or more Method validation is not the verifying of other contrast agents cassette methods), fully meet the requirement of disease diagnosis.
The above is only presently preferred embodiments of the present invention, is not intended to limit the invention in any way, it is all according to the present invention Technical spirit any simple modification, change and equivalence change to the above embodiments, still fall within the technology of the present invention side In the protection scope of case.
Sequence table
<110>Suzhou Institute of Nano-tech. and Nano-bionics, Chinese Academy of Sciences
<120>it can be used for detecting the monoclonal antibody and kit of PPR virus
<141> 2019-04-08
<150> CN201810932611.8
<151> 2018-08-16
<150> CN201810932595.2
<151> 2018-08-16
<150> CN201810932679.6
<151> 2018-08-16
<160> 24
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Oryctolagus cuniculus
<400> 1
Gly Ile Asp Leu Asn Ser Ala Thr
1 5
<210> 2
<211> 7
<212> PRT
<213> Oryctolagus cuniculus
<400> 2
Ile Val Ser Gly Ser Thr Asn
1 5
<210> 3
<211> 15
<212> PRT
<213> Oryctolagus cuniculus
<400> 3
Ala Arg Tyr Ala Gly Thr Ile Ala Tyr Gln Trp Tyr Phe Asn Ile
1 5 10 15
<210> 4
<211> 6
<212> PRT
<213> Oryctolagus cuniculus
<400> 4
Glu Ser Ile Ser Ser Ile
1 5
<210> 5
<211> 3
<212> PRT
<213> Oryctolagus cuniculus
<400> 5
Arg Thr Ser
1
<210> 6
<211> 12
<212> PRT
<213> Oryctolagus cuniculus
<400> 6
Gln Gln Gly Val Ser Asn Ser Asn Val Asp Asn Ile
1 5 10
<210> 7
<211> 444
<212> PRT
<213> Oryctolagus cuniculus
<400> 7
Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Asn Ser Ala
20 25 30
Thr Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45
Gly Asp Ile Val Ser Gly Ser Thr Asn Thr Asp Tyr Ala Asn Trp Ala
50 55 60
Ser Gly Arg Phe Thr Ile Ser Lys Thr Ser Ser Thr Thr Val Asp Leu
65 70 75 80
Lys Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala
85 90 95
Arg Tyr Ala Gly Thr Ile Ala Tyr Gln Trp Tyr Phe Asn Ile Trp Gly
100 105 110
Pro Gly Thr Leu Val Thr Val Ser Ser Gly Gln Pro Lys Ala Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val
130 135 140
Thr Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val
145 150 155 160
Thr Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser
165 170 175
Val Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val
180 185 190
Thr Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr
195 200 205
Asn Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro
210 215 220
Thr Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe
225 230 235 240
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
245 250 255
Thr Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe
260 265 270
Thr Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu
275 280 285
Arg Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro
290 295 300
Ile Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val
305 310 315 320
His Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
325 330 335
Arg Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg
340 345 350
Glu Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly
355 360 365
Phe Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala
370 375 380
Glu Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser
385 390 395 400
Tyr Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg
405 410 415
Gly Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His
420 425 430
Tyr Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
435 440
<210> 8
<211> 214
<212> PRT
<213> Oryctolagus cuniculus
<400> 8
Asp Val Val Met Thr Gln Thr Pro Ala Ser Val Glu Val Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Lys Cys Gln Ala Ser Glu Ser Ile Ser Ser Ile
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Arg Pro Asn Leu Leu Met
35 40 45
Tyr Arg Thr Ser Thr Leu Ala Ser Gly Val Ser Ser Arg Phe Lys Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Gly Val Gln Cys
65 70 75 80
Asp Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Gly Val Ser Asn Ser Asn
85 90 95
Val Asp Asn Ile Phe Gly Gly Gly Thr Glu Val Val Val Thr Gly Asp
100 105 110
Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val
115 120 125
Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro
130 135 140
Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly
145 150 155 160
Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn
165 170 175
Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys
180 185 190
Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser
195 200 205
Phe Asn Arg Gly Asp Cys
210
<210> 9
<211> 8
<212> PRT
<213> Oryctolagus cuniculus
<400> 9
Gly Ile Asp Leu Gly Gly Tyr Ala
1 5
<210> 10
<211> 6
<212> PRT
<213> Oryctolagus cuniculus
<400> 10
Ile Asp Thr Thr Asp Ser
1 5
<210> 11
<211> 15
<212> PRT
<213> Oryctolagus cuniculus
<400> 11
Ala Arg Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr
1 5 10 15
<210> 12
<211> 8
<212> PRT
<213> Oryctolagus cuniculus
<400> 12
Gln Ser Val Tyr Asn Asn Asn Cys
1 5
<210> 13
<211> 3
<212> PRT
<213> Oryctolagus cuniculus
<400> 13
Gly Ala Ser
1
<210> 14
<211> 10
<212> PRT
<213> Oryctolagus cuniculus
<400> 14
Val Gly Ala Tyr Ile Gly Ser Asn Tyr Ala
1 5 10
<210> 15
<211> 443
<212> PRT
<213> Oryctolagus cuniculus
<400> 15
Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Gly Gly Tyr
20 25 30
Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Ile Ile Asp Thr Thr Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ser Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys
65 70 75 80
Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
85 90 95
Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr Trp Gly Ser
100 105 110
Gly Thr Leu Val Thr Ile Thr Ser Gly Gln Pro Lys Ala Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val
165 170 175
Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr
180 185 190
Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn
195 200 205
Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr
210 215 220
Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255
Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr
260 265 270
Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg
275 280 285
Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
290 295 300
Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
305 310 315 320
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg
325 330 335
Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu
340 345 350
Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe
355 360 365
Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
370 375 380
Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr
385 390 395 400
Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
405 410 415
Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430
Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
435 440
<210> 16
<211> 214
<212> PRT
<213> Oryctolagus cuniculus
<400> 16
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Ile Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn
20 25 30
Asn Cys Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Phe
35 40 45
Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Val
65 70 75 80
Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Val Gly Ala Tyr Ile Gly
85 90 95
Ser Asn Tyr Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp
100 105 110
Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val
115 120 125
Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro
130 135 140
Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly
145 150 155 160
Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn
165 170 175
Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys
180 185 190
Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser
195 200 205
Phe Asn Arg Gly Asp Cys
210
<210> 17
<211> 8
<212> PRT
<213> Oryctolagus cuniculus
<400> 17
Gly Ile Asp Leu Gly Gly Tyr Ala
1 5
<210> 18
<211> 6
<212> PRT
<213> Oryctolagus cuniculus
<400> 18
Ile Asp Thr Thr Asp Asn
1 5
<210> 19
<211> 15
<212> PRT
<213> Oryctolagus cuniculus
<400> 19
Ala Arg Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr
1 5 10 15
<210> 20
<211> 6
<212> PRT
<213> Oryctolagus cuniculus
<400> 20
Gln Ser Ile Tyr Asn Asn
1 5
<210> 21
<211> 3
<212> PRT
<213> Oryctolagus cuniculus
<400> 21
Glu Ala Ser
1
<210> 22
<211> 12
<212> PRT
<213> Oryctolagus cuniculus
<400> 22
Gln Gln Gly Tyr Ser Ile Thr Asn Val Asp Asn Thr
1 5 10
<210> 23
<211> 443
<212> PRT
<213> Oryctolagus cuniculus
<400> 23
Cys Gln Ser Leu Glu Glu Ser Gly Gly Arg Leu Val Thr Pro Gly Gly
1 5 10 15
Ser Leu Thr Leu Thr Cys Thr Val Ser Gly Ile Asp Leu Gly Gly Tyr
20 25 30
Ala Val Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile
35 40 45
Gly Ile Ile Asp Thr Thr Asp Ser Thr Tyr Tyr Ala Ser Trp Ala Lys
50 55 60
Gly Arg Phe Thr Ser Ser Lys Thr Ser Ser Thr Thr Val Asp Leu Lys
65 70 75 80
Met Thr Ser Leu Thr Thr Glu Asp Thr Ala Thr Tyr Phe Cys Ala Arg
85 90 95
Tyr Ala Gly Asp Gly Gly Gly Gly Tyr Phe Phe Asp Tyr Trp Gly Ser
100 105 110
Gly Thr Leu Val Thr Ile Thr Ser Gly Gln Pro Lys Ala Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Cys Cys Gly Asp Thr Pro Ser Ser Thr Val Thr
130 135 140
Leu Gly Cys Leu Val Lys Gly Tyr Leu Pro Glu Pro Val Thr Val Thr
145 150 155 160
Trp Asn Ser Gly Thr Leu Thr Asn Gly Val Arg Thr Phe Pro Ser Val
165 170 175
Arg Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Ser Val Thr
180 185 190
Ser Ser Ser Gln Pro Val Thr Cys Asn Val Ala His Pro Ala Thr Asn
195 200 205
Thr Lys Val Asp Lys Thr Val Ala Pro Ser Thr Cys Ser Lys Pro Thr
210 215 220
Cys Pro Pro Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe Pro
225 230 235 240
Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr
245 250 255
Cys Val Val Val Asp Val Ser Gln Asp Asp Pro Glu Val Gln Phe Thr
260 265 270
Trp Tyr Ile Asn Asn Glu Gln Val Arg Thr Ala Arg Pro Pro Leu Arg
275 280 285
Glu Gln Gln Phe Asn Ser Thr Ile Arg Val Val Ser Thr Leu Pro Ile
290 295 300
Ala His Gln Asp Trp Leu Arg Gly Lys Glu Phe Lys Cys Lys Val His
305 310 315 320
Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Arg
325 330 335
Gly Gln Pro Leu Glu Pro Lys Val Tyr Thr Met Gly Pro Pro Arg Glu
340 345 350
Glu Leu Ser Ser Arg Ser Val Ser Leu Thr Cys Met Ile Asn Gly Phe
355 360 365
Tyr Pro Ser Asp Ile Ser Val Glu Trp Glu Lys Asn Gly Lys Ala Glu
370 375 380
Asp Asn Tyr Lys Thr Thr Pro Ala Val Leu Asp Ser Asp Gly Ser Tyr
385 390 395 400
Phe Leu Tyr Ser Lys Leu Ser Val Pro Thr Ser Glu Trp Gln Arg Gly
405 410 415
Asp Val Phe Thr Cys Ser Val Met His Glu Ala Leu His Asn His Tyr
420 425 430
Thr Gln Lys Ser Ile Ser Arg Ser Pro Gly Lys
435 440
<210> 24
<211> 214
<212> PRT
<213> Oryctolagus cuniculus
<400> 24
Ala Ala Val Leu Thr Gln Thr Pro Ser Pro Ile Ser Ala Ala Val Gly
1 5 10 15
Gly Thr Val Thr Ile Asn Cys Gln Ser Ser Gln Ser Val Tyr Asn Asn
20 25 30
Asn Cys Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Phe
35 40 45
Leu Ile Tyr Gly Ala Ser Thr Leu Ala Ser Gly Val Pro Ser Arg Phe
50 55 60
Lys Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Asp Val
65 70 75 80
Gln Cys Ala Asp Ala Ala Thr Tyr Tyr Cys Val Gly Ala Tyr Ile Gly
85 90 95
Ser Asn Tyr Ala Phe Gly Gly Gly Thr Glu Val Val Val Lys Gly Asp
100 105 110
Pro Val Ala Pro Thr Val Leu Ile Phe Pro Pro Ala Ala Asp Gln Val
115 120 125
Ala Thr Gly Thr Val Thr Ile Val Cys Val Ala Asn Lys Tyr Phe Pro
130 135 140
Asp Val Thr Val Thr Trp Glu Val Asp Gly Thr Thr Gln Thr Thr Gly
145 150 155 160
Ile Glu Asn Ser Lys Thr Pro Gln Asn Ser Ala Asp Cys Thr Tyr Asn
165 170 175
Leu Ser Ser Thr Leu Thr Leu Thr Ser Thr Gln Tyr Asn Ser His Lys
180 185 190
Glu Tyr Thr Cys Lys Val Thr Gln Gly Thr Thr Ser Val Val Gln Ser
195 200 205
Phe Asn Arg Gly Asp Cys
210

Claims (9)

1. a kind of monoclonal antibody that can be used for detecting PPR virus, it includes three complementary determining region of heavy chain (CDR- H1, CDR-H2 and CDR-H3) and three complementary determining region of light chain (CDR-L1, CDR-L2 and CDR-L3), in which:
(a) amino acid sequence of CDR-H1 is as shown in SEQ ID NO:1；
(b) amino acid sequence of CDR-H2 is as shown in SEQ ID NO:2；
(c) amino acid sequence of CDR-H3 is as shown in SEQ ID NO:3；
(d) amino acid sequence of CDR-L1 is as shown in SEQ ID NO:4；
(e) amino acid sequence of CDR-L2 is as shown in SEQ ID NO:5；And
(f) amino acid sequence of CDR-L3 is as shown in SEQ ID NO:6；
Or include three complementary determining region of heavy chain (CDR-H4, CDR-H5 and CDR-H6) and three complementary determining region of light chain (CDR-L4, CDR-L5 and CDR-L6), in which:
(g) amino acid sequence of CDR-H4 is as shown in SEQ ID NO:9；
(h) amino acid sequence of CDR-H5 is as shown in SEQ ID NO:10；
(i) amino acid sequence of CDR-H6 is as shown in SEQ ID NO:11；
(j) amino acid sequence of CDR-L4 is as shown in SEQ ID NO:12；
(k) amino acid sequence of CDR-L5 is as shown in SEQ ID NO:13；And
(l) amino acid sequence of CDR-L6 is as shown in SEQ ID NO:14；
Or include three complementary determining region of heavy chain (CDR-H7, CDR-H8 and CDR-H9) and three complementary determining region of light chain (CDR-L7, CDR-L8 and CDR-L9), in which:
(m) amino acid sequence of CDR-H7 is as shown in SEQ ID NO:17；
(n) amino acid sequence of CDR-H8 is as shown in SEQ ID NO:18；
(o) amino acid sequence of CDR-H9 is as shown in SEQ ID NO:19；
(p) amino acid sequence of CDR-L7 is as shown in SEQ ID NO:10；
(q) amino acid sequence of CDR-L8 is as shown in SEQ ID NO:21；And
(r) amino acid sequence of CDR-L9 is as shown in SEQ ID NO:22.
2. monoclonal antibody according to claim 1, it includes heavy chains and light chain, wherein
The amino acid sequence of heavy chain comprising three complementary determining region of heavy chain (CDR-H1, CDR-H2 and CDR-H3) such as SEQ ID Shown in NO:7；
The amino acid sequence of light chain comprising three complementary determining region of light chain (CDR-L1, CDR-L2 and CDR-L3) such as SEQ ID Shown in NO:8；
The amino acid sequence of heavy chain comprising three complementary determining region of heavy chain (CDR-H4, CDR-H5 and CDR-H6) such as SEQ ID Shown in NO:15；
The amino acid sequence of light chain comprising three complementary determining region of light chain (CDR-L4, CDR-L5 and CDR-L6) such as SEQ ID Shown in NO:16；
The amino acid sequence of heavy chain comprising three complementary determining region of heavy chain (CDR-H7, CDR-H8 and CDR-H9) such as SEQ ID Shown in NO:23；
The amino acid sequence of light chain comprising three complementary determining region of light chain (CDR-L7, CDR-L8 and CDR-L9) such as SEQ ID Shown in NO:24.
3. a kind of detection device comprising substrate and the monoclonal of any of claims 1 or 2 being fixed in the substrate Antibody.
4. detection device according to claim 3 is used in the biological sample of test object biological source whether there is PPR virus.
5. detection device according to claim 4, wherein the object organisms are goat or sheep.
6. detection device according to claim 4, wherein the biological sample is whole blood, blood plasma or serum.
7. a kind of peste des petits ruminants diagnostic kit is used to whether there is in the biological sample by test object biological source Whether PPR virus, diagnosis object organisms suffer from peste des petits ruminants.
8. peste des petits ruminants diagnostic kit according to claim 7, wherein the object organisms are goat or sheep.
9. peste des petits ruminants diagnostic kit according to claim 7, wherein the biological sample be whole blood, blood plasma or Serum.
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