CN103467599A - Bactrian camel source C-strain E2 VHH and application - Google Patents

Abstract

The invention discloses bactrian camel source C-strain E2 VHH and application. The bactrian camel source C-strain E2 VHH and the application aim at solving the problem that production and preparation of high-purity high-appetency classical swine fever virus antibodies can not be obtained in the existing classical swine fever virus antigen diagnostic reagent research and development process. The bactrian camel source C-strain E2 VHH are characterized by being provided with a nucleotide sequence: SEQIDNO.1, SEQIDNO.2 or SEQIDNO.3. The invention further provides the application of the bactrian camel source C-strain E2 VHH in preparing a classical swine fever virus antigen testing diagnostic reagent. The bactrian camel source C-strain E2 VHH and the application have the advantages that the VHH coded sequence of C-strain antigen protein E2 is obtained for the first time through a Bactrian camel, ELISA experimental results show that the recombination VHH expressed in E.coli have the same antigen binding capacity as classical swine fever virus polyclone antiserum, and therefore the recombination VHH can replace the classical swine fever virus polyclone antiserum to be used for researching the classical swine fever virus antigen diagnostic reagent.

Description

The anti-hog cholera lapinised virus vaccine strain in two-humped camel source E2 antigen heavy chain antibody VHH and purposes

Technical field

The invention belongs to the vaccine field, be specifically related to the anti-hog cholera lapinised virus vaccine strain in a kind of two-humped camel source E2 antigen heavy chain antibody VHH.

Background technology

Swine fever claims again pig suddenly or classic swine fever (Classical swine fever, CSF), and its cause of disease is Pestivirus suis (CSF virus, CSFV).Swine fever is one of Important Infectious Diseases of pig, and its pathological characters is little vascular wall degeneration, causes multiple hemorrhage, the necrosis of internal organs and infarct.China is the big country of raising pigs, preventive vaccination hog cholera lapinised virus vaccine (C-strain) is the effective means of prevention and control swine fever, however, swine fever is still one of the most serious transmissible disease of pig industry harm, generation at China's prevention and control swine fever, have important economic implications and social effect.In pig body after that infect at CSFV or C-strain vaccine immunity, the specificity antivirus neutralizing antibody is the topmost antiviral immunity reaction of body, CSFV genome encoding ground E2 albumen is the main antigen protein that body produces the swine fever virus resistant serum antibody, is the most important candidate albumen of development recombinant vaccine and antigen, antibody diagnosis method.

1993, at first Hamers etc. find to exist a kind of natural disappearance light chain in the serum such as camel and shark, the IgG2 of heavy chain and the antibody of IgG3 type are only arranged, its antigen binding site only is comprised of the variable region of heavy chain single domain, therefore also referred to as single domain heavy chain antibody (variable domain of the heavy chain of HCAbs, VHH).Studies confirm that in a large number, lacked light chain and still kept with the identical biological function of common IgG antibody with CH1 constant region VHH antibody, and show the new biological function that common IgG antibody and mono-clonal do not possess.VHH antibody has the complementary land (CDR3) of longer antigen aminoacid sequence, not only can be combined with the epitope of antigenic surface, can also deeply have the antigen inside of crack sunk structure, as the avtive spot of enzyme, and virus and host Cell binding site etc.; Because it is molecular weight little (monoclonal antibody 1/10), so the tissue penetration sexuality is better than conventional IgG antibody, research finds that it even can stride across hemato encephalic barrier, arrives the position that common IgG antibody molecule can't contact, and gives full play to the biological function of VHH antibody; VHH antibody FR2 district exists some to be different from the hydrophilic amino acid of conventional IgG antibody, make VHH antibody there is satisfactory stability in the aqueous solution, can realize antibody large-scale production and purifying, the high purity recombinant antibodies that obtain that good water solubility, stability are strong, biological activity and antibody structure approaches natural antibody more; The avidity that VHH is combined with antigen reaches the nmole level, suitable with single-chain antibody avidity, and this antibody is as a kind of genetic engineering antibody of miniaturization in Study on etiology, and hypersensitivity diagnostic antigen method field has broad application prospects.

Summary of the invention

The purpose of this invention is to provide the anti-hog cholera lapinised virus vaccine strain in a kind of two-humped camel source E2 antigen heavy chain antibody VHH, to solve the problem of the production and preparation that has the swine fever virus resistant antibody that can't obtain high purity, high-affinity existed in Pestivirus suis antigen diagnose reagent R&D process now.

Another object of the present invention is to provide the purposes of the anti-hog cholera lapinised virus vaccine strain in a kind of two-humped camel source E2 antigen heavy chain antibody VHH.

Technical solution of the present invention is as follows: the anti-hog cholera lapinised virus vaccine strain in a kind of two-humped camel source E2 antigen heavy chain antibody VHH is characterized in that described antibody has nucleotide sequence: SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

Further, the invention provides a kind of recombinant expression vector, contain described nucleotide sequence.

Further, the invention provides a kind of host cell, described host cell is the prokaryotic cell prokaryocyte that contains described nucleotide sequence.Described host cell is the prokaryotic cell prokaryocyte that contains described expression vector.

Further, the invention provides the anti-hog cholera lapinised virus vaccine strain in a kind of two-humped camel source E2 antigen heavy chain antibody VHH and detect the application in the Pestivirus suis antigen diagnose reagent in preparation.

Invention characteristics of the present invention are to obtain first the VHH antibody coding sequence that utilizes the two-humped camel screening to obtain swine fever virus resistant rabbitization attenuated vaccine strain antigen protein E2; The ELISA experimental result shows, e.colithe restructuring VHH antibody of middle expression has identical antigen binding capacity with the Pestivirus suis polyclonal antiserum, can replace the research of Pestivirus suis polyclonal antibody for the Pestivirus suis antigen diagnose reagent.

The accompanying drawing explanation

Fig. 1 is VHH antibody amino acid characteristics analysis chart;

Fig. 2 is that SDS-PAGE analyzes anti-CSFV E2 VHH antibody and exists e.coliin expression figure;

1:VHH-E2-1 inclusion body in Fig. 2, the 2:VHH-E2-1 supernatant; The 3:VHH-E2-2 inclusion body, the 4:VHH-E2-2 supernatant; 5 VHH-E2-3 inclusion bodys; M: the protein standard molecular weight (from top to bottom

Molecular weight is followed successively by: 75,65,40,35, and 14 kDa); The 6:VHH-E2-3 supernatant; 7 and 8: the empty carrier contrast; 9: do not add the IPTG control group; In figure, the arrow indication is the purpose band;

Fig. 3 is that Western blotting analyzes VHH-E2 albumen figure.

Embodiment

The following examples can further illustrate the present invention, but do not limit the present invention in any way.

Embodiment 1

One, two-humped camel immunity, peripheral blood lymphocyte separate and cDNA synthesizes

1. materials and methods

1.1 material-experimental animal, antigen, adjuvant

Select two ages peak year female young two-humped camel about one year old as the immunity receptor animal, (these two-humped camels are raised scattered near the Gobi desert Jinchang City of Gansu throughout the year, are mainly to cut camel hair to sell.Poultry main only in the winter time careless withered, summer Extreme drought and recall during female hunchbacked lambing and supplement forage grass, water and salt, never inoculated any vaccine, be the suitable receptor of preparation VHH antibody.), adjuvant 206 adjuvants, antigen is E2-GST antigen.

The preparation of hog cholera lapinised virus vaccine strain (CSFV) E2 antigen adopts existing method: utilize the antigen of affinity chromatography method purification of soluble E2 fusion rotein (GST-tag) as immune two-humped camel, the E2 antigen of screening specificity single domain heavy chain antibody (VHH).Utilize the SDS-PAGE electrophoresis to determine purity of protein, by the uv-absorbing standard measure.

1.2 method

Fully mix emulsification with 206 adjuvants and E2-GST antigen equal-volume complete, respectively at the neck of two-humped camel, the subcutaneous multi-point injection of two hind leg and back, first immunisation dosage is 300 μ g/ time (2mL), the 14th day, the 21st day, the 35th day, the 49th day and the 54th day booster immunization, immunizing dose is 200 μ g/ time (2mL).Gather venous blood before each immunity, separation of serum, with indirect ELISA test determination serum antibody titer.Two-humped camel is with after the E2 antigen immune, in body, anti-E2 protein specific antibody titre rises gradually along with immune time lengthening, after the 4th (35 days time) immunity, antibody horizontal reaches highest level, after the 5th immunity, antibody titers is without significantly improving, according to serum antibody growth and decline characteristics, the 6th immunity gathers the jugular vein peripheral blood in latter 5 days.

Gathering respectively two peak each 200mL of laboratory animal peripheric venous blood mixes as sample, then with 3.8% Sodium Citrate, as antithrombotics, with sample, by the volume ratio of 1:9, mix, add lymphocyte separation medium to separate peripheral blood lymphocyte (PBMC), collect and 2 * 10 altogether ⁹individual lymphocyte, lymphocyte is according to 1 * 10 ⁸individual/pipe packing, be kept in liquid nitrogen.Extract cell total rna with Trizol reagent, the first chain cDNA is synthesized in reverse transcription, using that it adopts primer (in Table 1-express the primer of the VHH antibody sequence of anti-E2) of amplification VHH as template, carry out 3 and take turns pcr amplification and obtain the specificity VHH fragment of big or small about 450bp for building phage display library.

Two, the structure in swine fever VHH immunity library

1. prepare the elementary storehouse of VHH phage antibody

Get the VHH antibody fragment of 6 μ g bacterium purifying and the phage vector pHEN4 of 28 μ g and use respectively restriction enzyme pstIthe and restriction endonuclease notIafter digestion, at 22 ℃ of temperature, use T ₄the DNA ligase connection of spending the night is carried out deactivation to enzyme at 65 ℃ of temperature, after deactivation 10min, cooling with ice bath, connect product utilization Biorad electroporation electricity be transformed into intestinal bacteria ( e.coli) the super competent cell of TG1, after transforming, bacterium liquid is 37 ^oat the C temperature, rotating speed concussion with 160rpm/min is cultivated 1 hour, recover resistance, be coated on (the glucose 100 μ g that the nutrient solution on culture plate is concentration 2%, 100 μ g/ml penbritins) on 2 * YT culture plate after concentrated, the bacterium liquid of simultaneously getting after the bacterium liquid 10 μ L after concentrated is diluted to 10 ⁴individual bacterium colony, coated plate then, incubated overnight at 37 ℃ of temperature, connect and transform in a large number through three times, and obtaining storage capacity is 1.29 * 10 ⁸camel immunity single domain antibody storehouse.Mono-clonal bacterium colony on culture plate is washed with the LB liquid nutrient medium, and adding therein concentration is 10% glycerine, is kept at-80 after packing ^oin the C refrigerator, preparation becomes the elementary storehouse of VHH phage antibody.

2. set up the VHH immune antibody library

Add helper phage M13K07 rescue library in the elementary storehouse of VHH phage antibody, obtain the phage antibody library titre and reach respectively 1.2 * 10 after 3 batches of rescues ⁸, 4.0 * 10 ⁸, 8.0 * 10 ⁸storage capacity (VHH immune antibody library storage capacity and diversity table after table 2-tri-time rescue), sequencing primer is: L1: 5'-TggAATTgTgAgCggATAACAATT-3'; S6: 5'-gTAAATgAATTTTCTgTATgAgg-3'.

Each batch of sequencing result shown, obtain after sequence is utilized DNAstar being unique sequence, the BLAST comparative analysis determines that the specificity clone of Selective determination in 3 batches is the heavy chain antibody VHH sequence that derives from the camel genus, illustrates that structure immune antibody library diversity is better.

three, the VHH antibody screening of the anti-E2 of specificity

(1) adopt the 3 VHH antibody of taking turns in Biopanning technology screening immune antibody library with the E2 specific binding, the basic fundamental route is as follows:

1. coated: by target molecule by CSFV E 2 Antigen: carbonate be coated with damping fluid=1:4 dilution and mixes, the 50ul/ hole, 4 spend to be coated with and spend the night, 1 * PBS washes 3 times;

2. sealing: PBS washes 3 times, uses 2% M-PBS, 300 ul/ holes, 37 ℃ of 1h;

3. in conjunction with phage: PBS washes 3 times, gets 5ul phage+45ul 1.0% M-PBS and mixes the 50ul/ hole, standing 1.0 h under 37 ℃ of conditions;

4. primary antibodie: 0.1%PBST washes 3 times, with 1.0% M-PBS, presses 1:3000 dilution Rabbit anti-M13 pAb 50ul/, 37 ℃ of standing 1.0h;

5. two is anti-: 0.1%PBST washes 3 times, with 1.0% M-PBS, presses 1:3000 dilution HRP-goat anti-rabbit IgG pAb 50ul/ hole, 37 ℃ of standing 1.0h;

6. colour developing: 0.1%PBST washes 4 times, gets 0.2M/l Na ₂hPO4+each 4.5ml of 0.1M/L citric acid adds a small amount of OPD, 60ul H ₂o ₂mix the 50ul/ hole, 2M sulfuric acid stops 25ul/ hole, OD ₄₉₀nm detects.

Three-wheel is eluriated and specifically be the results are shown in Table 3.

(2) screening

1. screening for the first time: direct coated E2 antigen polyclone ELISA screening specificity clone, the basic fundamental route is as follows:

1.1 coated: CSFVE2 is diluted to 15 ug/ml with pH 9.6 carbonate coating buffers, and 4 ℃ are spent the night;

1.2 sealing: add 2% MPBS, 100 uL/ holes, 37 ℃ of incubation 1.0h;

1.3 in conjunction with: phage is with final concentration 0.5% MPBS by 10 times of dilutions, and all the other add 0.5% MPBS, and 37 ℃ of temperature are educated 1.0 h;

1.4 primary antibodie: the anti-M13 of rabbit;

1.5 two is anti-: HRP-goat-anti rabbit;

1.6 colour developing: the OPD colour developing the results are shown in Table 4-direct coated E2 antigen polyclone ELISA.

2. programmed screening: direct coated E2 antigen polyclone ELISA screening specificity clone, the basic fundamental route is as follows:

1.1 coated: CSFV E2 is diluted to 10 ug/ml with pH 9.6 coating buffers, and 4 ℃ are spent the night;

1.2 sealing: 2% MPBS, 37 ℃ of 1.0h;

1.3 in conjunction with: phage is with final concentration 0.5% MPBS by 10 times of dilutions, and all the other holes add 0.5% MPBS, 37 ℃ of 1.0h;

1.4 primary antibodie: the anti-M13 of rabbit;

1.5 two is anti-: HRP-goat-anti rabbit, colour developing.

The selection result is in Table 5-direct coated E2 antigen polyclone screening mono-clonal phage ELISA, and wherein mono-clonal the 17th, and 19 and 24 yin and yang attribute differences are the most remarkable, again carries out third round mono-clonal ELISA screening E2 specificity VHH antibody.

screening for the third time: direct coated E2 antigen polyclone ELISA screening specificity clone,the basic fundamental route is as follows:

1.1 coated: as CSFV E2 to be diluted to 10 ug/ml with the pH9.6 coating buffer, to spend the night at 4 ℃ of temperature;

1.2. sealing: 2% MPBS, 37 ℃ of 1.0h;

1.3 in conjunction with: phage is with final concentration 0.5% MPBS by 10 times of dilutions, and all the other holes add 0.5% MPBS, 37 ℃ of 1.0 h;

1.4 primary antibodie: the anti-M13 of rabbit;

1.5 two is anti-: HRP-goat-anti rabbit;

1.6 colour developing.

The third round the selection result is in Table 6-direct coated E2 antigen polyclone screening mono-clonal phage ELISA, and wherein mono-clonal the 3,9,17,19with 24yin and yang attribute difference is the most remarkable, wherein 17,19 and 24 still show with E2 antigen and have stable and specific combination after the third round screening, illustrate and are taking turns under screening and high eluting power condition and still can from immune storehouse, obtain specific E2 VHH antibody through 3.

four, VHH mono-clonal bacterium colony order-checking comparison

By 3,9,17,19 and 24 totally 5 clones carry out nucleotide sequencing and sequential analysis, found that 3 and 9,17 and 24 is tumor-necrosis factor glycoproteinss, be defined as identical clone; 19 is to clone separately, the results are shown in Table the order-checking of 7-specificity VHH DNA sequence dna and analyzes, and the sequence recorded is shown in sequence table.

Five, the VHH sequence signature is analyzed

From Fig. 1: Val37Phe, Gly44Glu, Leu45Arg, the amino acid such as and Gly47Trp are the symbolic characteristics that forms heavy chain VHH antibody, illustrate that 3 VHH clones that screening obtains are the heavy chain antibodies from two-humped camel.Relatively find, remarkable with the hypervariable region amino acid difference between the CDR3 of 3 of the combination of E2 antigen-specific different VHH sequences, the CDR3 length of VHH-E2-1 has 18 amino acid, all the other two clone CDR3 districts are comprised of 20 amino acid, illustrating that 3 VHH antibody are from 3 different epitopes from E2 antigen, is 3 antibody sequences independently.By the amino acid length analysis of different genera CDR3 district is found, heavy chain VHH antibody CDR3 district is longer than common IgG antibody CDR3 district ammonia, this feature of VHH antibody is the result of the advancing of the functional compensation of heavy chain antibody of disappearance light chain, although camel VHH antibody disappearance light chain, but by having extended the CDR3 district after natural evolution, therefore, VHH antibody still has perfect antigen binding capacity with common IgG antibody.

six, VHH antibody exists e.colimiddle expression, purifying and Western blotting

3 VHH fragments are cloned into respectively to expression vector p-SMK(80 μ g/ml, kantlex, Kan ^r) upper, positive restructuring data is transformed into ecolibL21-CodonPlus (DE3) (34 μ g/ml, paraxin, Cm ^r) be subject in the state cell, the picking mono-clonal causes in 5 ml LB liquid nutrient mediums, and 37 ℃, the 220rpm incubated overnight.Be inoculated into the 100ml LB substratum of new configuration next day according to 2% ratio, cultivate OD ₆₀₀value to 0.6, add the IPTG of final concentration 0.1mM to induce, and 20 ℃, 220rpm induces 20h, centrifugal receipts bacterium, and SDS-PAGE analyzes the approximately target stripe of 39 kDa (Fig. 2) of size, 1:VHH-E2-1 inclusion body in Fig. 2,2:VHH-E2-1 supernatant; The 3:VHH-E2-2 inclusion body, the 4:VHH-E2-2 supernatant; 5 VHH-E2-3 inclusion bodys; M: the protein standard molecular weight (from top to bottom molecular weight is followed successively by: 75,65,40,35, and 14 kDa); The 6:VHH-E2-3 supernatant; 7 and 8: the empty carrier contrast; 9: do not add the IPTG control group.

Western blotting experiment obtains specific band (Fig. 3), 1:VHH-E2-1 in Fig. 3; 2:VHH-E2-2; 3:VHH-E2-3.The thalline that ultrasonic treatment is induced, the analysis showed that VHH albumen expresses with soluble form, with affinitive layer purification VHH recombinant protein.

seven, double-antibody sandwich elisa is measured VHH and restructuring E2 albumen and the experiment of CSFV C-strain binding ability

Three kinds of VHH(15.5 μ g/ml, 100 μ l/ holes) respectively as capture antibody direct coated 96 hole enzyme plates, the anti-swine fever polyclone of rabbit hyper-immune serum (1:3000 dilution) is as positive control, (E2-GST label and concentrated C-strain cell toxicant are as middle antigen for purifying E2 recombinant protein, normal camel and rabbit serum are as negative control, the anti-CSFV hyper-immune serum of pig is as second antibody, TMB is as substrate, measure absorbancy OD450, the results are shown in Table 8-double-antibody sandwich and detect VHH and E2 recombinant protein and CSFV C-strain binding ability experiment (OD450).

Table 8 experimental result shows, VHH can be combined with restructuring E2 antigen and C-strain totivirus antigen-specific.OD ₄₅₀value is relatively found, VHH antibody is a little less than the anti-CSFV hyper-immune serum of rabbit, may be because VHH antibody only consists of in conjunction with epi-position the antigen of 1 single anti-CSFV E2, and not only contain the antibody of anti-E2 antigen in the CSFV hyper-immune serum, the antibody that also includes anti-E0 albumen, so under similarity condition, polyclonal antiserum can be more than VHH antibody in conjunction with the quantity of Pestivirus suis antigen.

eight, the development of Pestivirus suis antigen double-antibody sandwich elisa

1.VHH antibody is coated: be 15.5 μ g/ml with the coated buffering of 0.05 M carbonate (pH 9.6) liquid by mixed 3 strain VHH antibody dilution to content.Add 100 μ l/ holes in the enzyme plate reacting hole, 4 ℃ are spent the night.Next day, discard solution in hole, and with 1 * PBST lavation buffer solution, 200 μ l/ holes, wash plate 4 times.Add 37 ℃ of sealings of 200 μ l/ hole confining liquid (1 * PBST, 5% skim-milk) 1 hour, with 1 * PBST lavation buffer solution, 200 μ l/ holes, wash plate 4 times.

2. add antigen: all samples is done two holes (in Table 9-Pestivirus suis antigen double-antibodies sandwich ELISA operation signal), with 1 * PBS, sample to be checked is done to the 1:2 dilution, 100 μ l/ holes are in above-mentioned coated reacting hole, add blank (sample diluting liquid) simultaneously, negative control hole (health pig tissue suspension) and positive control (swine fever attenuated cell culture, 750 rabbit precursor reactant/mL), be positioned in wet box, 37 ℃ act on 2 hours, with 1 * PBST lavation buffer solution, 200 μ l/ holes, wash plate 4 times.

3. add polyclonal antibody: rabbit swine fever virus resistant antibody dilution, to 1:3000, is added in each reacting hole, 100 μ l/ holes, 37 ℃ act on 1 hour, repeat to wash the plate step.

4. the anti-pig enzyme labelled antibody of rabbit: the anti-pig enzyme labelled antibody of horseradish peroxidase-labeled rabbit is done to the 1:100 dilution, and 100 μ l/ holes add in each reacting hole, and 37 ℃ of effects 1 hour, repeat to wash the plate step.

5. substrate colour developing: add freshly prepared TMB substrate solution 100 μ l/ holes, 37 ℃ of lucifuge colour developing 12-15min.

6. termination reaction: add 1.5M H ₂sO ₄stop buffer 100 μ l/ holes.

7. result is judged: in microplate reader, in 450nm place reading result, the mean value that all samples is got two holes is decision content as a result of.Using after blank hole zeroing (all samples hole OD value-blank well OD value) as OD value of the actual detection of each sample well.

(1) experimental result is set up and is judged: if positive criteria OD ₄₅₀>=negative control OD ₄₅₀2.1 times of value, experimental result is set up.

(2) result is judged: testing sample OD ₄₅₀>=negative control OD ₄₅₀the value 2.1 times, be judged to be the positive.For negative sample, can again detect by polymerase chain reaction (PCR).

(9), Pestivirus suis antigen double-antibody sandwich elisa field sample detection

73 duplicate samples [5] of 1984 to 2006 that the applicant is preserved are detected with double-antibody sandwich elisa, detected result following (two kinds of methods of table 10-double-antibody sandwich elisa and PCR detect swine fever antigen table as a result).

In table 10, to be defined as CSFV nucleic acid after being and detecting by PCR method positive for 73 duplicate samples.It is the virus antigen positive that VHH antibody detects wherein 61 duplicate samples as the double-antibodies sandwich ELISA of capture antigen, and positive rate is 83.56%.It is positive that double-antibodies sandwich ELISA detects Pestivirus suis antigen, can confirm in sample really to exist CSFV to infect, contribute to the censorship pig farm to take in time anti-epidemic measure to be processed the pig that falls ill, to compromised swinery, take urgent vaccination ways to avoid by virus infection, thereby at utmost reduce financial loss.

Sequence table

<110 > Lanzhou Veterinary Inst., Chinese Acedemy of Agaricultural Sciences

<120 > the anti-hog cholera lapinised virus vaccine strain in two-humped camel source E2 antigen heavy chain antibody VHH and purposes

<130> Muyldermans S, Atarhouch T, Saldanha J, Barbosa JA, Hamers R.

Sequence and structure of VH domain from naturally occurring

camel heavy chain immunoglobulins lacking light chains.

1994,7(9):1129-35.

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 372

<212> DNA

<213> Bactrian camel

<220>

<221> gene

<222> (1)..(372)

<400> 1

caggtgcagc tgttggagtc tgggggaggc ttggtacagc ctggggggtc cctgcgtctc 60

tcctgtgcag cctccggata tagctttatc gataagacta tggcctgggt ccgccaggct 120

ccagggaagg gtctagagtg ggtatcagcc attcggatgc ataacggtag cacatactac 180

gcagactccg tgaagggccg gttcaccatc tcccgtgaca attccaagaa cacgctgtat 240

ctgcaaatga acagcctgcg tgccgaggat accgcggtat attattgcgc gcgactgtcg 300

cgtaggattc aggctaagga taaggcgaag ttgaagtatt ggggtcaggg aaccctggtc 360

accgtctcga gc 372

<210> 2

<211> 378

<212> DNA

<213> Bactrian camel

<220>

<221> gene

<222> (1)..(378)

<400> 2

gatgtgcagc tggtggagtc tgggggaggc ttggtgcagg caggggggtc tctgagactc 60

tcctgtacag cctctggatt cacttttgat gattatgcca tggcctggtt ccgccagcct 120

ccaggaaagg agcgcgaggg ggtctcatgt attagctcga gtggtggtag cacatggtat 180

gcagactccg ttaagggccg attcaccgtc tccagagaca acgccaagaa cacgctgtat 240

ctgcaaatga acagtctgaa acctgaggac acggccatgt attactgtgc ggcagactgg 300

gggcgactgt gtgtcgtcag aggtccacga aatgagtata agtactcggg ccaggggacc 360

caggtcactg tctcctca 378

<210> 3

<211> 378

<212> DNA

<213> Bactrian camel

<220>

<221> gene

<222> (1)..(378)

<400> 3

gatgtgcagc tagtggagtc tgggggaggc ttggtgcagg caggggggtc tctgagactc 60

tcctgtacag cctctggatt cacttttgat gattatgcca tggcctggtt ccgccagcct 120

ccaggaaagg agcgcgaggg ggtctcatgt attagctcga gtggtggtag cacatggtat 180

gcagactccg ttaagggccg attcaccgtc tccagagaca acgccaagaa cacgctgtat 240

ctgcaattga acagtctgaa acctgaggac acggccatgt attactgtgc ggcagactgg 300

gtgaaaatgt attttgtggt gggtccacga aatgagtata agtactcggg ccaggggacc 360

caggtcactg tctcctca 378

Claims (5)

1. the anti-hog cholera lapinised virus vaccine strain in a two-humped camel source E2 antigen heavy chain antibody VHH, is characterized in that described antibody has nucleotide sequence: SEQ ID NO.1, SEQ ID NO.2 or SEQ ID NO.3.

2. a recombinant expression vector, is characterized in that containing nucleotide sequence claimed in claim 1.

3. a host cell, is characterized in that described host cell is the prokaryotic cell prokaryocyte that contains nucleotide sequence claimed in claim 1.

4. host cell as claimed in claim 4, is characterized in that described host cell is the prokaryotic cell prokaryocyte that contains expression vector claimed in claim 2.

5. the anti-hog cholera lapinised virus vaccine strain in two-humped camel claimed in claim 1 source E2 antigen heavy chain antibody VHH detects the application in the Pestivirus suis antigen diagnose reagent in preparation.

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