CN100412088C - Enzyme-linked immunosorbent test detecting reagent kit for non-structure protein of pig and cow foot and mouth disease virus - Google Patents
Enzyme-linked immunosorbent test detecting reagent kit for non-structure protein of pig and cow foot and mouth disease virus Download PDFInfo
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Abstract
The present invention relates to the cloning and the expression of a foot-and-mouth disease virus non-structural protein 3AB in an escherichia coli expression system, and the application of an expression product thereof at the aspects of foot-and-mouth disease vaccine injections and the differential diagnosis of infected animals. The differential diagnosis of an infection antibody and an immune antibody can be specifically performed on foot-and-mouth disease susceptible animals, such as pigs, cattle, etc., with high efficiency by utilizing a detecting reagent kit of the present invention. The reagent kit of the present invention has the advantages of high specificity, convenient use and low price.
Description
Technical field
The present invention relates to biological technical field, relate more specifically to clone and the expression of foot and mouth disease virus Nonstructural Protein 3AB in escherichia expression system, and the application of the test kit of expression product and other related reagent composition aspect the differential diagnosis of foot and mouth disease notes seedling and infection animal.
Background technology
(foot-and-mouth disease FMD) is domestic animal and wild deadly infectious disease artiodactylous to foot and mouth disease.In case break out then bamboo telegraph, can cause large-scale susceptible animal infection morbidity, cause enormous economic loss.Since Burrows (1966) and Van Bekkum (1966) etc. isolates foot and mouth disease virus from esophagus-pharyngeal secretory product (O-P liquid) of clinical health ox since, the recessive band poison of foot and mouth disease rehabilitation animal, the problem that may become the potential epidemic disease source of foot and mouth disease outburst has caused the great attention of countries in the world, and has carried out big quantity research in succession.In view of the anti-major measure of making foot and mouth disease of China is to carry out immunization, regularly annotate seedling 2-3 time to susceptible droves such as ox, sheep, pig are annual.Therefore, set up and to distinguish the diagnostic method that infects live virus (morbidity or injection attenuated vaccine) and injection inactivated vaccine animal accurately and fast and detecting and the generation of anti-system FMD, the aspects such as raiseeing quarantine of living are all very necessary and press for.In recent years, fragments such as Nonstructural Protein (NSP-) 2C of state scholar counterpart fever aphthous viruses such as English, U.S., Holland, 3A, 3B, 3ABC have been done a large amount of careful researchs, set up enzyme linked immunosorbent assay (ELISA), be applicable to differential diagnosis infection and vaccine injected animal.
The DNA recombinant technology is adopted in this research, at expression in escherichia coli foot and mouth disease Nonstructural Protein 3AB, and with this recombinant protein as detecting antigen and positive control serum preparation antigen, preparation ELISA detection kit, be used for pig/ox foot and mouth disease Nonstructural Protein detection of antibodies, judge by detected result whether tested animal infects foot and mouth disease virus.Now finished trial production and popularization in up to ten thousand parts, application result proves that test kit is effectively, can be used for the differential diagnosis that foot and mouth disease is annotated seedling and infection animal.
The test kit that this project is developed is used to the notes seedling and the differential diagnosis of infecting antibody of foot and mouth disease susceptible animal pig/ox, provides technical guarantee, the loss that minimizing even subduction livestock industry production bring because of foot and mouth disease for China prevents system and the final foot and mouth disease of eliminating.Simultaneously, the use of this technology will promote the international status of China's livestock industry production and converted products comprehensively, quickens the progress that China's livestock industry product moves towards the world market.
In addition, the test kit that this project is developed is expected to substitute the import like product, for country saves a large amount of foreign exchanges, has independent intellectual property right in case the more important thing is us, and the foot and mouth disease diagnosis quarantine technology of China is reached world-class levels.
Summary of the invention
Purpose of the present invention just provides a kind of new technology and the related kit that are used for differential diagnosis foot and mouth disease notes seedling and infection animal.
Description of drawings
Fig. 1 has shown the double digestion result of 3AB gene RT-PCR amplification and recombinant vectors pET3AB.Wherein each swimming lane is: the RT-PCR amplified production of 3AB (swimming lane 4), and with the pET3AB (swimming lane 2) of BamH I and Hind III digestion, DNA standard substance DL15000 (swimming lane 1, Takara company), DNA standard substance DL2000 (swimming lane 3, Takara company).
Fig. 2 has shown rNS-3AB fusion rotein IPTG abduction delivering SDS-PAGE electrophoresis (12%), target protein rNS-3AB purification result SDS-PAGE electrophoresis (12%), and immunoblotting reaction result.Wherein each swimming lane is: PET3AB does not induce (swimming lane 1), and PET3AB IPTG induces ( swimming lane 2,3,4,6), molecular weight of albumen standard ( swimming lane 5,7, Biolabs), PET3ABC expression product purification result ( swimming lane 8,9,10), Western trace result (swimming lane 12), one anti-be the bovine serum that FMDV infects, two anti-be albumin A/G (Pierce company) that coupling has horseradish peroxidase, the protein step standard substance that dyes in advance (swimming lane 11, Gibco BRL company).
Embodiment
The inventor is through going deep into and extensive studies for many years, successfully made up the expression vector of expressing foot and mouth disease virus Nonstructural Protein 3AB, this carrier is behind transformed into escherichia coli, can obtain expressing the proteic recombinant strains of 3AB, and the target protein rNS-3AB that expresses does not influence the normal breeding of bacterium to the host cell toxicological harmless.To the target protein rNS-3AB that expresses after sex change, purifying, renaturation are handled, the final product that obtains is as detecting antigen, set up indirect enzyme-linked immunosorbent assay (I-ELISA) foot and mouth disease infection and vaccine injected animal are carried out the differential diagnosis test, existed test results shows, the rNS-3AB albumen that the present invention obtained can be used as the detection antigen that foot and mouth disease is annotated effective, the high specific of seedling and infection animal differential diagnosis.Finished the present invention on this basis.
Detection kit of the present invention can make a differential diagnosis to foot and mouth disease susceptible animal pig, ox etc.This test kit high specificity, easy to use, cheap.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:ColdSpring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Foot and mouth disease virus Nonstructural Protein 3AB expresses and purifying
1. the amplification of foot and mouth disease virus Nonstructural Protein 3AB gene order
Give birth to worker's test kit with Shanghai and extract total RNA from the pig of cell cultures, hostis pecoris, operation steps is undertaken by the test kit specification sheets.The total RNA that obtains with extraction is a template, carries out reverse transcription PCR, and the reverse transcription test kit is available from the precious biotechnology in Dalian company limited, ProductName: Reverse Transcriptase XL (AMV).The synthetic primer of cDNA article one chain is Oligo (dT), target cDNA's is synthetic, it is synthetic that primer is given birth to the worker by Shanghai, primer sequence: 5 ' end primer: 5 '-GGATCCATCTCAATTCCTTCTC-3 ', 3 ' end primer: 5 '-AAGCTTCTCAGTGACAATCAGG-3 ' introduces BamH I and Hind III restriction enzyme site at 5 ' end and 3 ' end respectively during the design primer.Pcr amplification carries out according to a conventional method, 1%Agarose gel electrophoresis, observations.The PCR product is served the sea and is given birth to the order-checking of worker biotech company simultaneously.
As a result, be template with the foot-and-mouth disease virus genome RNA, the RT-PCR amplification has obtained 3AB dna sequence dna (Fig. 1), and the about 0.66kb of sequence length is consistent with bibliographical information length.
The structure of 2pET3AB expression plasmid
2.1T-3AB construction of recombinant plasmid
The 3AB pcr amplified fragment is connected with the T carrier after 1% low melting point Agrose gel-purified.The T carrier is given birth to worker's biotechnology Services Co., Ltd available from Shanghai, ProductName: T-Vector PCR Product ClongingKit, ligase enzyme are Dalian precious biotechnology company limited product, ProductName: DNA Ligation Kit Ver.2.Recombinant plasmid T-3AB transforms DH5 α host bacterium.Extracting plasmid (the centrifugal plasmid extraction test kit of worker's UNIQ-10 post is given birth in Shanghai), BamH I/Hind III double digestion are identified, check order simultaneously (giving birth to worker's order-checking portion by Shanghai finishes).
As a result, the T-3AB plasmid that obtains having recombinated, BamH I, Hind III double digestion are identified and are confirmed to comprise the 3AB fragment in the recombinant vectors.
2.2pET3AB the structure of expression plasmid
Carrier pET32a and host bacterium BL21 (DE3) plysS are all available from Novagen company.Reorganization T-3AB plasmid and carrier pET32a use BamH I, Hind III double digestion respectively, enzyme is cut product 3AB fragment and carrier pET32a enzyme and is cut big fragment and connect after 1% low melting point Agrose gel-purified, obtain recombinant plasmid pET3AB, transform host bacterium BL21 (DE3) plysS, coating contains the LB flat board of acillin, obtains recombinant clone bacterium colony PET3AB.From the reorganization bacterium colony, extract plasmid, BamH I/Hind III double digestion, the 1%Agrose gel electrophoresis is identified, simultaneously, is checked order by Shanghai Shen You Bioisystech Co., Ltd.
The plasmid enzyme restriction result as shown in Figure 1.Sequencing result is carried out Computer Analysis, show reorganization 3ABC sequence and bibliographical information sequence coincidence rate greater than 90%, it is correct that on position is read frame.
3. the expression of target protein 3AB
Clone's bacterium colony is cultivated in the LB fermented liquid that contains 100 μ g/mL penbritins, when treating that the OD value reaches 0.5 left and right sides, adds IPTG and induces, and IPTG concentration is 1mmol/L, induction time 3-4h, and sample SDS-PAGE electrophoresis observation result takes a morsel behind the receipts bacterium.
Reorganization bacterium abduction delivering product determines that through the 12%SDS-PAGE electrophoresis fusion protein molecule amount of expression is about 45KD (Fig. 2).This target protein is named as rNS-3AB.Its aminoacid sequence and nucleotide sequence are as shown in Figure 3.
4. the Western trace of expressing protein 3AB is identified
Carry out with reference to method described in " molecular cloning ".The electrotransfer condition is 100V electrophoresis 1 hour, and confining liquid is 5% skim-milk, 100mmol/L Tris-HCl (pH7.5), 0.9%NaCl, 0.1%Tween 20, one anti-bovine serums for the foot and mouth disease virus infection, two anti-are horseradish peroxidase-labeled albumin A/G, and substrate is DAB.
The Western trace detects and to show, expressed proteins can with foot and mouth disease virus infected animals serum generation immune response (Fig. 2).
5. the purifying of expression product
Fermented liquid is through 5000rpm, and 4 ℃, 15 minutes centrifugal, remove supernatant, precipitate with 1/20 fermentation volume NTA0 damping fluid (20mmol/L Tris.HCl, 100mmol/L NaCl, 10% glycerine) suspend, ultrasonication, 13500rpm, 4 ℃, 20 minutes centrifugal, remove supernatant, precipitation is dissolved with GuNTA0 (NTA0 that contains the 6M Guanidinium hydrochloride) solution, 4 ℃ of sex change of spending the night.With Ni
2+The NTA post is connected with FPLC (high pressure liquid chromatography (HPLC)) system, with 2 to 3 times of volume NTA0 balance columns, last sample, washing post to foreign protein peak value with NTA0 is 0 o'clock, in elutriant, add GuNTA500 (GuNTA0 that contains the 500mmol/L imidazoles), making GuNTA500 is 20-30% in the ratio of elutriant, and wash-out target protein rNS-3AB collects elutriant when elution peak occurring on the supervisory computer screen.The elutriant renaturation of under 4 ℃ of conditions, dialysing.Getting sample SDS-PAGE detects.
Expression product rNS-3AB is through Ni
2+Behind the NTA column purification (Fig. 2), as antigen, the result proves through the indirect ELISA test experience with purified product, and this albumen can be used as detection antigen, is used for the notes seedling of foot and mouth disease susceptible animal and the differential diagnosis of infection animal.
The foundation of pig, hostis pecoris Nonstructural Protein antibody ELISA detection method
The foundation of 1 detection method
1.1 detection method
In the present embodiment, the recombinant expressed Nonstructural Protein rNS-3AB of the foot and mouth disease virus (FMDV) that obtains in the Application Example 1 differentiates foot and mouth disease pig/cattle infected animal and immune animal, and this method is indirect enzyme-linked immunosorbent assay (I-ELISA).
1.2 detect antigenic working concentration
The purifying antigen albumen rNS-3AB concentration of determining test kit elisa plate bag quilt is 0.1 μ g/ hole.
1.3 the eliminating of nonspecific reaction
The foot and mouth disease Nonstructural Protein rNS-3AB that utilizes gene recombination technology to obtain still has a spot of carrier proteins and host's mycoprotein in the purified expression product.And intestinal bacteria antibody is prevalent in the animal serum, therefore, utilizes this expression product as diagnostic antigen, will inevitably cause nonspecific reaction.In order to get rid of these nonspecific reactions, select blocker for use and make shaker test.Test of many times result relatively back determines:
(1) with 3% gelatin and 5% skim-milk blocking antigen bag by the hole.
(2) the following diluted of tested serum sample: 0.01mol/L PBST contains 5% skim-milk in (pH7.2～7.4), the intestinal bacteria split product of cultivating in 0.1%12 hour.
1.4 tested serum weaker concn
Tested serum dilutes by 1: 40 with above-mentioned diluent.
2 detected results
This test-results is with the OD value representation of ELISA experiment.
2.1 negative control
Use no foot and mouth disease to infect the normal pig of antibody or bovine serum as the test kit negative control sera.
2.2 positive control
With rNS-3AB as immunogen, immune new zealand rabbit, the hyper-immune serum of preparation is as the test kit positive control serum.
2.3 judging criterion
Calculate tested serum S/P value by following formula, and determine whether tested serum is positive.
S/P＝(X-N)/(P-N)
X: tested serum OD
450Value; N: negative serum arithmetical av; P: positive serum arithmetical av.
2.4 normal bovine serum sample
Not morbidity, not vaccinated pig, bovine serum antibody, 200 parts of serum S/P values are all less than 0.10.
2.5FMDV infection serum sample
Test pig, ox infect the back 1 thoughtful 12 months through manually attacking poison, collection serum, and this serum is used as positive control serum.Detected result shows that the S/P value of 73 parts of tested serum all＞0.25.
2.6 pig, bovine serum behind the inoculation FMD inactivated vaccine
Part tested serum S/P value is all less than 0.25 surplus in the of 2000.
The result shows, test kit provided by the present invention can be used for foot and mouth disease is annotated the differential diagnosis of seedling and infection animal.This method and VIAA-AGID (VIA-sepharose immunodiffusion) compare, and specificity and susceptibility are significantly improved.
Pig, hostis pecoris Nonstructural Protein antibody ELISA detection kit are formed
(1) elisa plate: reorganization rNS-3AB by elisa plate, seals (confining liquid: PBS, pH7.2 contain 3% gelatin, 5% skim-milk) with confining liquid with 0.1 μ g/ hole bag;
(2) sample diluting liquid: 12mL (PBS, pH7.2 wherein contain 1% gelatin, 5% skim-milk, 0.1% intestinal bacteria extract);
(3) enzyme conjugates: 200 μ L (albumin A/G preserves liquid with the PBS that contains 50% glycerine and dilutes with certain proportion); Enzyme conjugates diluent: 12mL (PBS PBS, pH7.2);
(4) TMB A liquid: 6mL (TMB is dissolved in the 0.1mol/L citrate buffer solution, final concentration 0.2mg/mL, pH5.0);
(5) TMB B liquid: 6mL (hydrogen peroxide urine is dissolved in the 0.1mol/L citrate buffer solution, final concentration 0.2%, pH5.0);
(6) terminator: 12mL (2M H
2SO
4).
Pig, hostis pecoris Nonstructural Protein antibody ELISA detection kit detection method
(1) take out antigen coated microplate in the test kit, and sample diluting liquid, 37 ℃ of sample diluting liquids were hatched 3～5 minutes.
(2) add 100 μ L sample diluting liquids in every hole, respectively add 5 μ L negative control seras in A1, B1 two holes, respectively add 5 μ L positive control serums in C1, D1 two holes, all the other each holes add test sample, the sample in every hole, 5 μ L/ holes.
(3) hatch 60 minutes (± 1 minute) for rearmounted 37 ℃ with shrouding film shrouding.
(4) washing working fluid preparation: measure on demand, washings is done 25 times of dilutions with deionized water or distilled water.
(5) with washing working fluid washing antigen coated microplate, every hole 200～300 μ L wash 6 times, pat dry.
(6) enzyme conjugates working fluid preparation: with the enzyme conjugates diluent enzyme conjugates is done 100 times of dilutions, now with the current.
(7) every hole adds 100 μ L enzyme conjugates working fluids, hatches 30 minutes (± 1 minute) for rearmounted 37 ℃ with shrouding film shrouding.
(8) repeating step 1.9.5.
(9) tmb substrate working fluid preparation:, now with the current with tmb substrate A liquid and tmb substrate B liquid balanced mix.
(10) every hole adds 100 μ L tmb substrate working fluids, hatches 15 minutes for 37 ℃.
(11) every hole adds 100 μ L stop buffers, and the mixing that vibrates gently.
(12) in 15 minutes, be determined at OD value under the 450nm wavelength with elisa reading instrument.
All documents that the present invention mentions are all quoted as a reference in this application, are just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Dna sequence dna and the aminoacid sequence of recombinant protein rNS-3AB are as follows:
----→ TrxTagTM Trx
atg?agc?gat?aaa?att?att?cac?ctg?act?gac?gac?agt?ttt?gac?acg?gat 48
Met?Ser?Asp?Lys?Ile?Ile?His?Leu?Thr?Asp?Asp?Ser?Phe?Asp?Thr?Asp
1 5 10 15
gta?ctc?aaa?gcg?gac?ggg?gcg?atc?ctc?gtc?gat?ttc?tgg?gca?gag?tgg 96
Val?Leu?Lys?Ala?Asp?Gly?Ala?Ile?Leu?Val?Asp?Phe?Trp?Ala?Glu?Trp
20 25 30
tgc?ggt?ccg?tgc?aaa?atg?atc?gcc?ccg?att?ctg?gat?gaa?atc?gct?gac 144
Cys?Gly?Pro?Cys?Lys?Met?Ile?Ala?Pro?Ile?Leu?Asp?Glu?Ile?Ala?Asp
35 40 45
gaa?tat?cag?ggc?aaa?ctg?acc?gtt?gca?aaa?ctg?aac?atc?gat?caa?aac 192
Glu?Tyr?Gln?Gly?Lys?Leu?Thr?Val?Ala?Lys?Leu?Asn?Ile?Asp?Gln?Asn
50 55 60
cct?ggc?act?gcg?ccg?aaa?tat?ggc?atc?cgt?ggt?atc?ccg?act?ctg?ctg 240
Pro?Gly?Thr?Ala?Pro?Lys?Tyr?Gly?Ile?Arg?Gly?Ile?Pro?Thr?Leu?Leu
65 70 75 80
ctg?ttc?aaa?aac?ggt?gaa?gtg?gcg?gca?acc?aaa?gtg?ggt?gca?ctg?tct 288
Leu?Phe?Lys?Asn?Gly?Glu?Val?Ala?Ala?Thr?Lys?Val?Gly?Ala?Leu?Ser
85 90 95
aaa?ggt?cag?ttg?aaa?gag?ttc?ctc?gac?gct?aac?ctg?gcc?ggt?tct?ggt 336
Lys?Gly?Gln?Leu?Lys?Glu?Phe?Leu?Asp?Ala?Asn?Leu?Ala?Gly?Ser?Gly
100 105 110
----→ HisTag----→ zymoplasm
tct?ggc?cat?atg?cac?cat?cat?cat?cat?cat?tct?tct?ggt?ctg?gtg?cca 384
Ser?Gly?His?Met?His?His?His?His?His?His?Ser?Ser?Gly?Leu?Val?Pro
115 120 125
cgc?ggt?tct?ggt?atg?aaa?gaa?acc?gct?gct?gct?aaa?ttc?gaa?cgc?cag 432
Arg?Gly?Ser?Gly?Met?Lys?Glu?Thr?Ala?Ala?Ala?Lys?Phe?Glu?Arg?Gln
130 135 140
----→ enteropeptidase
cac?atg?gac?agc?cca?gat?ctg?ggt?acc?gac?gac?gac?gac?aag?gcc?atg 480
His?Met?Asp?Ser?Pro?Asp?Leu?Gly?Thr?Asp?Asp?Asp?Asp?Lys?Ala?Met
145 150 155 160
BamH?I----→3A
gct?gat?atc?gga?tcc?atc?tca?att?cct?tct?caa?aag?gct?gtg?ctg?tac 528
Ala?Asp?Ile?Gly?Ser?Ile?Ser?Ile?Pro?Ser?Gln?Lys?Ala?Val?Leu?Tyr
165 170 175
ttt?ctc?att?gag?aag?ggt?cag?cac?gaa?gca?gca?att?gaa?ttc?ttt?gag 576
Phe?Leu?Ile?Glu?Lys?Gly?Gln?His?Glu?Ala?Ala?Ile?Glu?Phe?Phe?Glu
180 185 190
ggg?atg?gtg?cat?gac?tcc?atc?aag?gag?gag?ctc?cgg?cct?ctc?atc?caa 624
Gly?Met?Val?His?Asp?Ser?Ile?Lys?Glu?Glu?Leu?Arg?Pro?Leu?Ile?Gln
195 200 205
cag?acc?tca?ttc?gtg?aag?cgc?gct?ttt?aag?cgc?ctg?aag?gaa?aac?ttt 672
Gln?Thr?Ser?Phe?Val?Lys?Arg?Ala?Phe?Lys?Arg?Leu?Lys?Glu?Asn?Phe
210 215 220
gag?ata?gtt?gcc?ctg?tgt?ttg?act?ctt?ttg?gca?aac?ata?gtg?atc?atg 720
Glu?Ile?Val?Ala?Leu?Cys?Leu?Thr?Leu?Leu?Ala?Asn?Ile?Val?Ile?Met
225 230 235 240
cta?cgc?gaa?gcg?cgc?aag?agg?cgc?cag?tca?gtg?gat?gac?tca?ctg?gat 768
Leu?Arg?Glu?Ala?Arg?Lys?Arg?Arg?Gln?Ser?Val?Asp?Asp?Ser?Leu?Asp
245 250 255
gac?gac?gcg?gct?ctt?gac?gat?gcg?gaa?aag?aac?cct?cta?gag?gcg?agt 816
Asp?Asp?Ala?Ala?Leu?Asp?Asp?Ala?Glu?Lys?Asn?Pro?Leu?Glu?Ala?Ser
260 265 270
ggc?gcc?agc?gcc?gtt?ggt?ttc?aga?gag?aga?tcc?ccc?atc?gag?caa?aag 864
Gly?Ala?Ser?Ala?Val?Gly?Phe?Arg?Glu?Arg?Ser?Pro?Ile?Glu?Gln?Lys
275 280 285
Thr?Cys?Asp?Asp?Val?Asn?Thr?Glu?Pro?Val?Val?Pro?Gly?Arg?Glu?Gln
290 295 300
----→3B
ccg?cga?gct?gaa?gga?ccc?tac?gcc?ggg?cca?ctc?gaa?cgt?cag?aaa?cct 960
Pro?Arg?Ala?Glu?Gly?Pro?Tyr?Ala?Gly?Pro?Leu?Glu?Arg?Gln?Lys?Pro
305 310 315 320
ctt?aaa?gtg?aaa?gcc?agg?ttg?cca?caa?caa?gag?gga?cct?tac?gcc?ggt 1008
Leu?Lys?Val?Lys?Ala?Arg?Leu?Pro?Gln?Gln?Glu?Gly?Pro?Tyr?Ala?Gly
325 330 335
ccc?atg?gag?cgg?cag?aaa?ccg?ctg?aaa?gtg?aaa?gca?aaa?gcc?ccc?gtc 1056
Pro?Met?Glu?Arg?Gln?Lys?Pro?Leu?Lys?Val?Lys?Ala?Lys?Ala?Pro?Val
340 345 350
gtg?aag?gaa?gga?ccc?tac?gag?ggg?ccg?gtg?aaa?aag?cct?gtc?gct?ttg 1104
Val?Lys?Glu?Gly?Pro?Tyr?Glu?Gly?Pro?Val?Lys?Lys?Pro?Val?Ala?Leu
355 360 365
Hind?III
aaa?gtg?aaa?gca?aag?aac?ttg?att?gtc?act?gag?aag?ctt?gtc?gag?aag 1152
Lys?Val?Lys?Ala?Lys?Asn?Leu?Ile?Val?Thr?Glu?Lys?Leu?Val?Glu?Lys
370 375 380
tac?tag
Tyr?*
385
Fig. 3. the nucleotide sequence of recombinant protein rNS-3AB and amino acid sequence
Claims (10)
1. an albumen is characterized in that, it comprises aminoacid sequence shown in Figure 3.
2. a dna sequence dna is characterized in that, its coding comprises the albumen of aminoacid sequence shown in Figure 3.
3. dna sequence dna as claimed in claim 2 is characterized in that, it comprises the described nucleotide sequence of Fig. 3.
4. an expression vector is characterized in that, it contains the described dna sequence dna of claim 2.
5. a bioengineered strain is characterized in that, it contains the described expression vector of claim 4.
6. bioengineered strain as claimed in claim 5 is characterized in that it is intestinal bacteria.
7. one kind produces method of protein, it is characterized in that, comprises step:
(a) under conditions suitable for the expression, cultivate the described bioengineered strain of claim 6;
(b) isolate expressing protein from culture, described expressing protein comprises aminoacid sequence shown in Figure 3 in the specification sheets.
8. a test kit that detects foot and mouth disease is characterized in that, it contains the described albumen of claim 1.
9. test kit as claimed in claim 8 is characterized in that, it also contains positive control, negative control and other related reagent.
10. test kit as claimed in claim 8 is characterized in that, can be used for foot and mouth disease is annotated the differential diagnosis of seedling and infection animal pig or ox.
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| CNB200510070593XA CN100412088C (en) | 2005-05-08 | 2005-05-08 | Enzyme-linked immunosorbent test detecting reagent kit for non-structure protein of pig and cow foot and mouth disease virus |
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| CNB200510070593XA CN100412088C (en) | 2005-05-08 | 2005-05-08 | Enzyme-linked immunosorbent test detecting reagent kit for non-structure protein of pig and cow foot and mouth disease virus |
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| CN100412088C true CN100412088C (en) | 2008-08-20 |
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| CN102662062B (en) * | 2012-04-17 | 2015-05-27 | 中国农业科学院兰州兽医研究所 | Monoclonal antibody blocking enzyme-linked immunosorbent assay (ELISA) kit and method for detecting nonstructural protein (NSP) antibody of foot-and-mouth disease virus (FMDV) |
| CN102662063A (en) * | 2012-04-25 | 2012-09-12 | 中国农业科学院兰州兽医研究所 | Detection kit and method for non-structural protein antibody dot blots of foot and mouth disease viruses |
| CN102998460B (en) * | 2012-10-31 | 2014-12-10 | 广东海大畜牧兽医研究院有限公司 | Enzyme linked immunosorbent assay (ELISA) kit of foot-and-mouth disease virus antibody and preparation method thereof |
| CN109851675B (en) * | 2018-12-24 | 2020-09-01 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Foot-and-mouth disease diagnostic kit and foot-and-mouth disease diagnostic antigen used by same |
| CN109856396B (en) * | 2018-12-24 | 2022-02-08 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Enzyme linked immunosorbent assay kit for detecting foot and mouth disease virus infection antibody and application thereof |
| CN109851662B (en) * | 2018-12-24 | 2020-09-01 | 中国动物疫病预防控制中心（农业部屠宰技术中心） | Foot-and-mouth disease virus recombinant protein and related biological material and application thereof |
| CN109765366A (en) * | 2019-01-31 | 2019-05-17 | 中国农业科学院兰州兽医研究所 | A kind of kit and its detection method detecting foot and mouth disease virus 3AB antibody |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603831A (en) * | 2003-09-29 | 2005-04-06 | 中国农业科学院兰州兽医研究所 | Indirect ELISA process for discriminating livestock infected with foot-and-mouth disease virus and livestock immunologically vaccinated with foot-and-mouth disease vaccine utilizing nonstructural prot |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1603831A (en) * | 2003-09-29 | 2005-04-06 | 中国农业科学院兰州兽医研究所 | Indirect ELISA process for discriminating livestock infected with foot-and-mouth disease virus and livestock immunologically vaccinated with foot-and-mouth disease vaccine utilizing nonstructural prot |
Non-Patent Citations (1)
| Title |
|---|
| 应用口蹄疫非结构蛋白区分感染动物和注苗动物. 江鹏斐，谢庆阁.中国兽医科技，第29卷第12期. 1999 * |
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