CN101082051A - Recombined foot-and-mouth disease virus polypeptide fragment and and uses thereof - Google Patents

Abstract

The present invention reveals one kind of recombinant foot-and-mouth disease virus polypeptide fragment and its gene products. The recombinant foot-and-mouth disease virus polypeptide fragment includes two or more single-chain oligonucleotide segments and one or more flexible amino acid segments. Each two of the single-chain oligonucleotide segments are connected through one flexible amino acid segment, and at least one of the single-chain oligonucleotide segments contains the kernel segment RGD of the foot-and-mouth disease virus gene.

Description

Recombined foot-and-mouth disease virus polypeptide fragment and application thereof

Technical field

The invention relates to a kind of recombined foot-and-mouth disease virus polypeptide fragment, particularly about a kind of recombined foot-and-mouth disease virus polypeptide fragment that can be widely used in fields such as immunoassay, vaccine safely and effectively.

Background technology

Foot and mouth disease is a kind of transmissible disease of infecting both domestic animals and human, can infect in domestic animals to all cloven-hoofed animals.Its etiology is the Aptho virus (Apthovirus) in the Picornavirus, comprises seven kinds of serotypes: O type, A type, C type, SAT1 type, SAT2 type, SAT3 type and Asia 1 type.Be Pan Asia O type at Britain's popular at present, toxicity is very strong, and this disease shows as febris acuta in animal, bubble occurs on the normal sensation in the mouth hoof of animal thereupon.Foot and mouth disease is very big to the hazardness of animal husbandry development.This disease on the cloven-hoofed zoogenetic infections such as pig, ox, sheep, production performance descends, and serious meeting causes death, and the mortality ratio of young animal is higher.Foot and mouth disease can be given human by animal infection, and children's sense then serious myocarditis can take place, and is important zoonosis.

Along with being gradually improved of international trade system, foot and mouth disease has become one of animality disease of restriction living animal and animals products trade.One routine foot and mouth disease takes place in a state or area, with the trade barrier that cause immediately forming at this country related products.Early 1990s, European Union determines foot and mouth disease is taked " containing strategy " after having passed through cost effectiveness analysis, promptly livestock is not carried out at this sick preventive vaccination.But, will forbid that immediately the country that epidemic situation takes place exports any living animal or animals products in case break out epidemic situation.Forbid simultaneously the migration of cloven-hoofed animal in this state, and immediately to animal in infected animals, the infected area and had any animal that contacts to butcher and burn with infected animals.

The animal trade of long distance can cause the propagation of foot and mouth disease virus, and near the increase of the Pest-or disease-free area animal density in epidemic-stricken area also can cause the outburst of this area's epidemic situation.In addition, foot and mouth disease virus also can be borrowed moving of wind, people, animal or contaminated vehicle and be propagated.The main route of transmission of inter-State foot and mouth disease is legal or living animal, meat product and the milk preparation of contraband of import infection.The food that the passenger takes back from external epidemic-stricken area also can be propagated this disease.Foot and mouth disease virus can survive considerable time in green meat goods, false add worker meat product, smoked meat goods, the meat product of not curing animal and the halfway milk preparation of pasteurization.And infected animal just can secrete virion before the clinical symptom performance.Therefore, the harm of foot and mouth disease has certain disguise, is badly in need of a kind of means effectively and instrument and detects animal timely and accurately and whether suffer from foot and mouth disease.

In addition, foot and mouth disease is a kind of transmissible disease of infecting both domestic animals and human, for fear of working the mischief to society, country does not allow to produce in batches the related products of foot and mouth disease totivirus, therefore, totivirus foot and mouth disease detection kit can not be applied to common livestock industry aquaculture, has incured loss through delay the discovery and the diagnosis of epidemic situation.In addition, the making of totivirus foot and mouth disease detection kit and preservation all need to carry out strict isolation, therefore involve great expense.

The Protein Detection of a kind of foot and mouth disease virus (FMDV) recombinant gene expression that is disclosed for No. 03100579 as the Chinese patent livestock FMDV antibody technique of falling ill.Wherein, utilize the extracorporeal recombination clone or synthesize FMD vaccine gene (synthetic unit price, multivalence polypeptide vaccine gene, subunit vaccine gene and nonstructural protein gene etc.), it is efficiently expressed in certain expression vector system, and generation has immunogenic albumen.Through immunoblotting assay, its expressing protein product can be discerned by the FMDV positive serum, shows that this albumen has immunologic competence, thereby available this recombinant protein is as having the antigen that immunogenic antigen replaces traditional use virus transfection cell preparation.

But, need the participation of Virus Sample in the making processes of No. 03100579 recombination that technology obtained that patent disclosed, and Virus Sample obtain both formality complexity, easily the development personnel are caused infection again, thereby cause the manufacturing cost height, be unsuitable for extensive popularization; In addition, the recombination of its acquisition spatially distributes simply, can not fully form the stereoscopic three-dimensional structure, thereby causes its antigenicity lower.

Therefore, provide a kind of means safely and effectively and instrument, detect animal timely and accurately and whether suffer from the problem that the foot and mouth disease disease becomes the solution of industry urgent need.

Summary of the invention

The technical problem to be solved in the present invention provides a kind of can apply to the terminal user, recombined foot-and-mouth disease virus polypeptide fragment and by gene prod that this recombined foot-and-mouth disease virus polypeptide fragment obtained safely and effectively.

A kind of technical scheme of the present invention is: a kind of recombined foot-and-mouth disease virus polypeptide fragment is provided, it comprises two or two above oligonucleotide single-chain fragments and flexible amino acid fragment more than or, connect by a flexible amino acid fragment between per two oligonucleotide single-chain fragments, and comprise the core fragment of foot-and-mouth disease virus gene at least therein in oligonucleotide single-chain fragmentRGD, wherein:

The oligonucleotide single-chain fragment is selected from one of them of following slice groups:

ISISEIKGVIVHKIETILF；

VYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KKKIITITRITIITTID；

SKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KYGDTSTNNVRGDLQVLAQKAERTL；

GSSKYGDTSTNNVRGDLQVLAQKAERTL；

RHKQKIVAPAKQ；

TNNVRGDLQVLAQKAERTLP；

TNNVRGDLQVLAQKAERT；

Flexible amino acid fragment is selected from one of them of following slice groups:

K；

PG；

QFELEFMVPSR；

CC；

PS。

As a kind of embodiment, recombined foot-and-mouth disease virus polypeptide fragment comprises two oligonucleotide single-chain fragments and a flexible amino acid fragment, and recombined foot-and-mouth disease virus polypeptide fragment is selected one of them in the following array mode for use:

ISISEIKGVIVHKIETILF-K-VYNGS?SKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KKKIITIRIITIITTID-K-SKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

TNNVRGDLQVLAQKAERTLP-CC-TNNVRGDLQVLAQKAERTLP。

As a kind of preferred implementation, recombined foot-and-mouth disease virus polypeptide fragment comprises three oligonucleotide single-chain fragments and two flexible amino acid fragments, and recombined foot-and-mouth disease virus polypeptide fragment is selected one of them in the following array mode for use:

GSSKYGDTSTNNVRGDLQVLAQKAERTL-PG-RHKQKIVAPAKQ-QFELEFMVPSR-GSSKYGDTSTNNVRGDLQVLAQKAERTL；

TNNVRGDLQVLAQKAERT-CC-RHKQKIVAPAKQ-PS-TNNVRGDLQVLAQKAERT。

Another kind of technical scheme of the present invention is: the multiple gene prod of being made by recombined foot-and-mouth disease virus polypeptide fragment of the present invention is provided.Such as: recombined foot-and-mouth disease virus polypeptide fragment of the present invention can be used to make biochip, gene chip, DIFA or DICA or ELISA test kit, test strip or vaccine.Wherein, at least one probe of biochip adopts recombined foot-and-mouth disease virus polypeptide fragment of the present invention; At least one probe of gene chip has adopted the dna fragmentation that is transformed by recombined foot-and-mouth disease virus polypeptide fragment of the present invention; The part of test kit or test strip has adopted recombined foot-and-mouth disease virus polypeptide fragment of the present invention; Vaccine has adopted recombined foot-and-mouth disease virus polypeptide fragment of the present invention.

Selectively, the two ends of recombined foot-and-mouth disease virus polypeptide fragment of the present invention can be suitable according to actual needs prolong or shorten, recombined foot-and-mouth disease virus polypeptide fragment of the present invention can comprise the oligonucleotide single-chain fragment more than four or four, and adopting the flexible amino acid fragment more than three or three to connect, wherein same oligonucleotide single-chain fragment can repeatedly repeat in a recombined foot-and-mouth disease virus polypeptide fragment.

Certainly, recombined foot-and-mouth disease virus polypeptide fragment can also not comprise flexible amino acid fragment, only is the oligonucleotide single-chain fragment, such as:

VYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KYGDTSTNNVRGDLQVLAQKAERTL。

Recombined foot-and-mouth disease virus polypeptide fragment of the present invention can change into dna fragmentation as required, and directly carries out types of applications with dna fragmentation.

The invention has the beneficial effects as follows: adopt the mode of synthetic to form recombination, avoided in development or use and direct contact of virus, effectively prevented viral diffusion, and guaranteed the personnel's that develop safety; Adopt flexible amino acid fragment to be connected between the oligonucleotide single-chain fragment, help recombined foot-and-mouth disease virus polypeptide fragment and form 3-D solid structure, thereby strengthened the antigenicity of recombined foot-and-mouth disease virus gene; The gene prod security of making based on recombined foot-and-mouth disease virus polypeptide fragment of the present invention is good, be convenient to preserve, easy to operate, price is low, effective; Because the virus-free infectivity of recombined foot-and-mouth disease virus polypeptide fragment of the present invention, thus the gene prod of making thus can produce in batches and promote the use of to the terminal user, thereby can find epidemic situation the very first time and in time epidemic situation is controlled at bud.

Below in conjunction with drawings and Examples, further specify the present invention, but the present invention is not limited to these embodiment, any on essence spirit of the present invention improvement or substitute, still belong to scope required for protection in claims of the present invention.

Description of drawings

Fig. 1 is the synoptic diagram of foot and mouth disease detection kit of the present invention.

Fig. 2 is the cross-sectional schematic of foot and mouth disease detection kit of the present invention.

Fig. 3 is the positive synoptic diagram of foot and mouth disease detection kit of the present invention.

Fig. 4 is the negative status synoptic diagram of foot and mouth disease detection kit of the present invention.

Fig. 5 is the disarmed state synoptic diagram of foot and mouth disease detection kit of the present invention.

Embodiment

Embodiment 1

The invention provides a kind of recombined foot-and-mouth disease virus polypeptide fragment, be used to make the dangerous gene prod of virus-free diffusion, the making method of recombined foot-and-mouth disease virus polypeptide fragment is as follows:

Bioactive peptide (HXT) gene synthetic organization plan

According to the immune-active peptides aminoacid sequence of foot and mouth disease virus major antigen site A, utilize DNASTAR software to carry out the antigenic activity analysis.Adopt the codon of intestinal bacteria preference, by the base sequence of Computer Design bioactive peptide (HXT5) gene, and design EcoRI restriction enzyme site.But seven kinds of selection schemes are as follows, and wherein, the italicized item in the sequence is flexible amino acid, indicate underscore and partly are the core fragment of foot-and-mouth disease virus gene.

Scheme one:

ISISEIKGVIVHKIETILF-K-VYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

Scheme two:

KKKIITITRIITIITTID-K-SKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

Scheme three:

VYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

Scheme four:

KYGDTSTNNVRGDLQVLAQKAERTL；

Scheme five:

GSSKYGDTSTNNVRGDLQVLAQKAERTL-PG-RHKQKIVAPAKQ-QFELEFMVPSR-GSSKYGDTSTNNVRGDLQVLAQKAERTL；

Scheme six:

TNNVRGDLQVLAQKAERTLP-CC-TNNVRGDLQVLAQKAERTLP；

Scheme seven:

TNNVRGDLQVLAQKAERT-CC-RHKQKIVAPAKQ-PS-TNNVRGDLQVLAQKAERT。

Gene synthesizes underlying condition

Adopt full gene synthesis method, it is synthetic that 242 bases of whole gene are divided into 6 oligonucleotide fragments, in external all fragment phosphorylations will be except that gene two terminal.The single-chain fragment of salvage after the mixing annealing, adds dna ligase, obtains required full polypeptide gene fragment.Finish by biotech company, and standby through the PAGE purifying.

Phage display

(1) plasmid, bacterial strain and phage

The integrated plasmid pR that is used for the T4 phage display, can be used for the reorganization of T4 phage SOC gene, modified SOC gene (3469～3969) 5 ' end has the part lysozyme gene e ' (3969～4219) of T4 phage, and 3 ' end has the gene denV ' (3319～3469) of T4 phage.Foreign gene can insert in its EcoRI site (3469), merges with SOC gene 3 ' end.This plasmid possesses beta-galactosidase gene (Ap) (4596～612) and aminoglycoside 3 ' phosphoric acid transferase gene (Kn) (1920～2732) simultaneously, can be used for the screening of penbritin and kantlex.The initial initial point of its dna replication dna (ORI) is positioned at 1326～1328.SOC gene 5 ' end primer (5 ' G AAT CAT ATGGCT AGT ACT CGC GGT 3 ') can be used for the screening of foreign gene direction of insertion.Other research materials also have the mutated phages φ T4-Z1 of T4 phage host strain E.coli E2, genetically engineered bacteria E.coli, wild type phage φ T4 and SOC genetically deficient, by this laboratory qualification and preservation.

(2) substratum, toolenzyme and main agents

Conventional bacteria culture medium is LB, and the penbritin that adds final concentration therein and be 100 μ g/ml is resistance substratum (LA).SC substratum and M9S substratum are prepared routinely.Ex^TMTaqDNA polysaccharase, T4 dna ligase, restriction enzyme Eco RI, alkaline phosphatase (CIAP) and DNA Marker are provided by TaKaRa company ,-20 ℃ of preservations.Plasmid takes out test kit for a short time and DNA purifying/recovery solution is U.S. Omega company product.Other reagent such as N,O-Diacetylmuramidase are homemade analytical reagent.Pcr amplification primer Pr.78 is synthetic by Shanghai Bo Ya company, adds the distilled water that DEPC handles, and dilution is 50 μ M, and packing postposition-20 ℃ preservation is standby.FMDV O type negative serum and positive serum are provided by Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences ,-20 ℃ of preservations.

(3) preparation of the amplification of HXT5 gene and pR plasmid

Take HXT5 (GSSKYGDTSTNNVRGDLQVLAQKAERTL-PG-RHKQKIVAPAKQ-QFELEFMVPSR-GSSKYGDTSTNNVRGDLQVLAQKAERTL) as the purpose fragment; Use the synthetic HXT5 of full gene, gene order is: GNWSNWSNAARTAYGGNGAYACNWSNACNAAYAAYGTNMGNGGNGAYYTNCARGTN YTNGCNCARAARGCNGARMGNACNYTNCCNGGNMGNCAYAARCARAARATHGTNGC NCCNGCNAARCARCARTTYGARYTNGARTTYATGGTNCCNWSNMGNGGNWSNWSNA ARTAYGGNGAYACNWSNACNAAYAAYGTNMGNGGNGAYYTNCARGTNYTNGCNCAR AARGCNGARMGNACNYTN. With the EcoRI enzyme HXT5 gene synthetic product being carried out enzyme cuts.Require to carry out electrophoresis and purifying/recovery HXT5 polypeptide fragment according to test kit.The streak culture E.coli DH5 α that contains the pR plasmid on the LA flat board, 37 ℃ of of overnight incubation.Picking list colony inoculation is in the LA nutrient solution, and 24h is cultivated 37 ℃ of of joltings of in (220r/min) .Reference reagent cassette method extracting pR plasmid.To pR plasmid enzyme restriction and dephosphorylation, in 0.2mL EP pipe, prepare 50 μ L reaction systems, electrophoresis purifying/recovery pR endonuclease bamhi with EcoRI.

(4) structure of integrated plasmid pR-HXT5 and evaluation

Get 0.2mL EP pipe, preparation T4 dna ligase system.Ordinary method prepares competence E.coli DH5 α recipient bacterium.Be connected product Transformed E .coli DH5 α recipient bacterium with HXT5 with pR.Carry out pcr amplification with 2 couples of primer HXT5-S2/HXT5-A2 and Pr.78/HXT5-A2 respectively, identify the transformant and the direction of insertion thereof that contain the HXT5 gene.Extracting recombinant plasmid pR-HXT5 carries out the EcoRI enzyme and cuts evaluation.PCR and enzyme are cut and are identified that correct plasmid and bacterium liquid send biotech company's evaluation of checking order.

(5) preparation of T4 phage transformant and PCR identify

With the pR-HXT5 plasmid Transformed E .coli E2 competence bacterium of identifying.And identify positive transformant with PCR method.Obtain phage phi T4-Z1 clone, M9S nutrient solution (adding the 0.2mg/mL N,O-Diacetylmuramidase) enlarged culturing with plum blossom spot method.The N,O-Diacetylmuramidase dependence test is qualified, counts plaque quantity then, converts out the number (titre) of every mL results liquid pnagus medius.Picking contains single bacterium colony of E.coli E2 of pR-3C, inoculation LA nutrient solution 1mL, 3～5h is cultivated in 37 ℃ of joltings (220r/min), to OD600 be 0.3～0.5.Inoculate the dependent phage phi T4-Z1 of N,O-Diacetylmuramidase results liquid 10 μ L (bacterial count/phage number about 1: 2) therein, put room temperature (25～30 ℃) and continue the jolting overnight incubation.Phage phi T4-Z1 phage (clone) enlarged culturing that picking is grown on the SC flat board of lysozyme not.The phage phi T4-Z1 that these N,O-Diacetylmuramidase dependency characteristics change is considered to successful integration, and the phage phi T4-3C of formation is as further identifying.

(6) bacteriolyze patch test and Western-blot identify

Carry out the bacteriolyze patch test of phage phi T4-HXT5 with plum blossom spot method, compare with wild-type T4 phage and mutated phages φ T4-Z1 simultaneously.Carrying out SDS-PAGE and Western blotting according to a conventional method analyzes.With the anti-FMDV polyclonal antibody of cavy is one anti-, and goat-anti cavy IgG-HRP is two anti-, the DAB colour developing.

Embodiment 2

In the present embodiment, recombined foot-and-mouth disease virus polypeptide fragment is used to make the genes involved product of foot and mouth disease virus quarantine.

Traditional Animal Quarantine is to cultivate cause of disease or measure antibody, as use neutralization test, enzyme and connect immunosorbent and measure (Enzyme-Linked Immunosorbnent Assay, abbreviation ELISA), spot immune binding analysis (dot immunobinding assay is called for short DIBA), agar diffusion test and complement fixation test (CFT) etc. fast.At present, some inspection and quarantine new technologies have obtained applying, and as PCR, immunoblotting, recombinant antigen, monoclonal antibody and synthetic peptide etc., have become the important supplement of traditional inspection and quarantine technology, but these inspection and quarantine new technologies need expensive testing installation and test conditions.Therefore, routine diagnostic method still is being extensive use of in Animal Quarantine, and new Protocols in Molecular Biology has then enlarged the scope that detects diagnosis, for inspection and quarantine provides strong new tool.

At present, most widely used general and easily mass-produced is based on test kit or the test strip that ELISA principle or DIBA principle are made.

It is a kind of immunoenzyme technics that grows up after immunofluorescence and radioimmunoassay technique that enzyme connects immunosorbent mensuration (ELISA).This technology has developed very rapidly since coming out the beginning of the seventies, has been widely used in biology and medical science applied many fields at present.ELISA is based on immunological response, the very high experimental technique of a kind of susceptibility that the specific reaction of antigen, antibody and enzyme are combined to the efficient catalytic effect of substrate.Owing to carry out in the hole that is reflected at a kind of solid phase carrier-polystyrene microtiter plates of antigen, antibody, after a kind of reagent of every adding is hatched, can remove unnecessary free reactant by washing, thus warranty test result's specificity and stability.In actual applications, by different designs, concrete method steps can have multiple, such as: be used to detect antibody indirect method, be used to detect antigenic double antibody sandwich method and be used to detect small molecules antigen or haptenic antigenic competition method or the like.Commonly used is ELISA double antibody sandwich method and ELISA indirect method.

Recombined foot-and-mouth disease virus polypeptide fragment of the present invention can be used for the antigen or the antibody of ELISA test kit.

Be applied to the diagnostic industry based on clinical and non-clinical field along with immunoassay is increasingly extensive, immunoassay develops to both direction: a class is full-automatic immunoassay; Another kind of for being the tachysynthesis analysis of carrier with the nitrocellulose membrane.The former needs expensive full-automatic instrument to reach and the strict supporting all ingredients box of instrument, can only use in medical treatment and inspection center at present, though also can faster provide the result, but still need certain hour, be not suitable for area, more can not allow the terminal user to detect voluntarily easily away from medical treatment and inspection center.Therefore on the basis of enzyme immunoassay, mainly be that the fast diagnosis method of carrier grows up rapidly and widely with the nitrocellulose filter.Here it is quick spot immune binding analysis (DIBA).

DIBA mainly comprises two types, a kind of is that (dotimmunofiltrtion assay is analyzed in the spot immune diafiltration, be called for short DIFA), wherein, immune response is to be undertaken by vertically penetrating the nitrocellulose filter that is fixed with part (being recombined foot-and-mouth disease gene of the present invention in the present invention).Another kind is spot immune chromatographic analysis (dot immunochro-matography assay, be called for short DICA), and wherein, analysis principle is identical with DIFA, just reaction liquid flow be not straight to penetrate mobile, but the transverse flow of chromatography effect.Wherein, the marker that uses among the DIBA comprises the microballoon of colloidal metal (Radioactive colloidal gold or electroselenium), decentralized dyestuff and dye marker etc., widespread use now be Radioactive colloidal gold.Therefore, that DIFA is most widely used is colloidal gold immunity percolation analysis (GIFA), and that DICA is most widely used is colloidal gold immunochromatographimethod analysis (GICA).Because GICA is solid phase form, be easy to be made into test kit or test strip, and the product of making carry, preserve, all very convenient when using, therefore have bigger application value.

The principle of GICA is that reaction carriers is a millipore filtration, wraps by known antigen or antibody on film earlier, adds sample to be checked again, and antigen antibody reaction takes place on film, through the Radioactive colloidal gold binding substances demonstration detected result of mark.The advantage of using this method is that (1) is sensitive and accurate, is subjected to external influence little, but the assay prolonged preservation; (2) rapidly quick, can draw detected result in the several minutes; (3) safe ready without any need for plant and instrument, is convenient to apply in grass-roots unit in the testing process; (4) with low cost, only need a spot of reagent and sample.

Colloidal gold chromatographic (GICA) test strip was developed rapidly and is promoted in recent years, was to help the novel vitro diagnostic techniques that grass-roots unit applies.This test strip is used fixing known antigen of T film or antibody, absorption colloid gold label reagent on pad.Sample to be checked is permeated forward by sample pad, with the reaction of colloid gold label thing, to antibody or antigenic region, produces immunocomplex earlier, and be trapped within to detect and be with, but the visual inspection result.This method has been used for the detection and the diagnosis of multiple animal epidemic at present.

Recombined foot-and-mouth disease virus polypeptide fragment of the present invention can be used for above-mentioned various detection method, preferably is used to make GICA test kit or GICA test strip.

As Fig. 1 and shown in Figure 2, test kit comprises ahollow housing 10, the leading portion ofhousing 10 upper surfaces is offered an application ofsample window 12, aview port 14 is offered in the posterior segment ofhousing 10 upper surfaces, contain thenitrocellulose filter 20 of solid phase in thehousing 10, thisnitrocellulose filter 20 is provided with the solid phase colloid gold label and (marks hereinafter to be referred as gold between application ofsample window 12 andview port 14, figure does not show), thisnitrocellulose filter 20 is provided withtesting wire 27 andcontrol line 28 successively belowview port 12,testing wire 27 is coated with part (figure does not show), and part is recombined foot-and-mouth disease virus polypeptide fragment in the present invention.The rear end ofnitrocellulose filter 20 is provided with absorbent pad 30.

As shown in Figure 3; sample to be tested (pig, ox or sheep blood serum) is added drop-wise on thenitrocellulose filter 20 from application ofsample window 12; sample is to absorbent pad 30 direction chromatographic flow; after the approach gold cursor position; the gold mark is redissolved fully; form colloid gold label antibody-antigenic compound, it continues to flow toforward testing wire 27 places.If in the sample foot and mouth disease virus antibody is arranged, then colloid gold label antibody-antigenic compound will react with recombined foot-and-mouth disease virus polypeptide fragment, and be bridged on thetesting wire 27, when the golden scalar of bridge joint acquires a certain degree, the gold mark will develop the color for macroscopic level, be generally red-purple.Unnecessary gold mark continues swimming to controlline 28 positions, and at this moment, golden mark will be bridged on thecontrol line 28 and colour developing, generally also be red-purple.Thistesting wire 27 and the situation that controlline 28 all develops the color show that test result is positive, promptly have foot and mouth disease virus antibody in the sample.

As shown in Figure 4, if in the sample when not having foot and mouth disease virus antibody, the gold mark can not be bridged totesting wire 27, sotesting wire 27 do not develop the color, and controlline 28 will develop the color, and promptly there is not foot and mouth disease virus antibody in this negative result in the sample.

As shown in Figure 5, iftesting wire 27 does not all develop the color withcontrol line 28, show that then test kit lost efficacy.

Embodiment 3

As another embodiment of the present invention, recombined foot-and-mouth disease virus polypeptide fragment of the present invention can be used to prepare biochip, gene chip or vaccine, and wherein, at least one probe of biochip adopts recombined foot-and-mouth disease virus polypeptide fragment of the present invention; At least one probe of gene chip has adopted the dna fragmentation that is transformed by recombined foot-and-mouth disease virus polypeptide fragment of the present invention; The part of test kit or test strip has adopted recombined foot-and-mouth disease virus polypeptide fragment of the present invention; Vaccine has adopted recombined foot-and-mouth disease virus polypeptide fragment of the present invention.

Claims (10)

1, a kind of recombined foot-and-mouth disease virus polypeptide fragment, comprise two or two above oligonucleotide single-chain fragments and flexible amino acid fragment more than or, connect by a described flexible amino acid fragment between per two described oligonucleotide single-chain fragments, and comprise the core fragment of foot-and-mouth disease virus gene at least therein in described oligonucleotide single-chain fragmentRGD, wherein:

Described oligonucleotide single-chain fragment is selected from one of them of following slice groups:

ISISEIKGVIVHKIETILF；

VYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KKKIITITRIITIITTID；

SKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KYGDTSTNNVRGDLQVLAQKAERTL；

GSSKYGDTSTNNVRGDLQVLAQKAERTL；

RHKQKIVAPAKQ；

TNNVRGDLQVLAQKAERTLP；

TNNVRGDLQVLAQKAERT；

Described flexible amino acid fragment is selected from one of them of following slice groups:

K；

PG；

QFELEFMVPSR；

CC；

PS。

2, recombined foot-and-mouth disease virus polypeptide fragment as claimed in claim 1, it is characterized in that, described recombined foot-and-mouth disease virus polypeptide fragment comprises two described oligonucleotide single-chain fragments and a described flexible amino acid fragment, and described recombined foot-and-mouth disease virus polypeptide fragment is selected one of them in the following array mode for use:

ISISEIKGVIVHKIETILF-K-VYNGSSKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

KKKIITITRIITIITTID-K-SKYGDTSTNNVRGDLQVLAQKAERTLPTSFNYGAIK；

TNNVRGDLQVLAQKAERTLP-CC-TNNVRGDLQVLAQKAERTLP。

3, recombined foot-and-mouth disease virus polypeptide fragment as claimed in claim 1, it is characterized in that, described recombined foot-and-mouth disease virus polypeptide fragment comprises three described oligonucleotide single-chain fragments and two described flexible amino acid fragments, and described recombined foot-and-mouth disease virus polypeptide fragment is selected one of them in the following array mode for use:

GSSKYGDTSTNNVRGDLQVLAQKAERTL-PG-RHKQKIVAPAKQ-QFELEFMVPSR-GSSKYGDTSTNNVRGDLQVLAQKAERTL；

TNNVRGDLQVLAQKAERT-CC-RHKQKIVAPAKQ-PS-TNNVRGDLQVLAQKAERT。

4, a kind of biochip is characterized in that, at least one probe of described biochip has adopted the described recombined foot-and-mouth disease virus polypeptide fragment of one of claim 1～3.

5, a kind of gene chip is characterized in that, at least one probe of described gene chip has adopted the dna fragmentation that is transformed by the described recombined foot-and-mouth disease virus polypeptide fragment of one of claim 1～3.

6, a kind of test kit is characterized in that, the part of described test kit has adopted the described recombined foot-and-mouth disease virus polypeptide fragment of one of claim 1～3.

7, test kit as claimed in claim 6 is characterized in that, described test kit is DIFA test kit or DICA test kit or ELISA test kit.

8, test kit as claimed in claim 7 is characterized in that, described test kit is GIFA test kit or GICA test kit.

9, a kind of test strip is characterized in that, the part of described test strip has adopted the described recombined foot-and-mouth disease virus polypeptide fragment of one of claim 1～3.

10, a kind of aftosa vaccine is characterized in that, described vaccine has adopted the described recombined foot-and-mouth disease virus polypeptide fragment of one of claim 1～3.

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