CN103467599B - 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

Bactrian camel source anti-hog cholera lapinised virus vaccine strain E2 antigen heavy chain antibody VHH and purposes

Technical field

The invention belongs to vaccines arts, be specifically related to a kind of Bactrian camel source anti-hog cholera lapinised virus vaccine strain E2 antigen heavy chain antibody VHH.

Background technology

Also known as pig suddenly or classic swine fever (Classical swine fever, CSF), its cause of disease is Pestivirus suis (CSF virus, CSFV) to swine fever.Swine fever is one of Important Infectious Diseases of pig, and its pathological characters is the sex change of thin vessels wall, causes internal organs multiple hemorrhages, necrosis and infarct.China is big country of raising pigs, preventive vaccination hog cholera lapinised virus vaccine (C-strain) is the most effective means of prevention and control swine fever, however, swine fever is still one of pig industry harm transmissible disease the most serious, in the generation of China's prevention and corntrol swine fever, there is important economic implications and social effect.CSFV infect or in pig body after C-strain vaccine immunity, 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 swine fever virus resistant serum antibody, is the most important candidate albumen of development recombinant vaccine and antigen, antibody diagnosis method.

1993, first Hamers etc. find in the serum such as camel and shark, there is a kind of natural deletions light chain, only there is the antibody of IgG2 and the IgG3 type of heavy chain, its antigen binding site is only made up of 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).Large quantity research confirms, has lacked light chain and has still kept with the identical biological function of common IgG antibody with CH1 constant region VHH antibody, and shows the new biological function that common IgG antibody and mono-clonal do not possess.VHH antibody has complementary land (CDR3) aminoacid sequence of longer antigen, not only can be combined with the epitope of antigenic surface, and the antigen can also deeply with crack sunk structure is inner, as the avtive spot of enzyme, and virus and host cellular binding sites etc.; Because it is molecular weight little (1/10 of monoclonal antibody), so 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 cannot contact, gives full play to the biological function of VHH antibody; There is the hydrophilic amino acid that some are different from conventional IgG antibody in VHH antibody FR2 district, VHH antibody is made to have satisfactory stability in aqueous, antibody large-scale production and purifying can be realized, obtain that good water solubility, stability are strong, biological activity and antibody structure be more close to the high purity recombinant antibodies of natural antibody; The avidity that VHH is combined with antigen reaches 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 object of this invention is to provide a kind of Bactrian camel source anti-hog cholera lapinised virus vaccine strain E2 antigen heavy chain antibody VHH, to solve the problem of production and preparation of swine fever virus resistant antibody that cannot obtain high purity, high-affinity existed in existing Pestivirus suis antigen diagnose reagent R&D process.

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

Technical solution of the present invention is as follows: a kind of Bactrian camel source anti-hog cholera lapinised virus vaccine strain 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, containing described nucleotide sequence.

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

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

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

Accompanying drawing explanation

Fig. 1 is VHH antibody amino acid signature analysis figure;

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. 2,2:VHH-E2-1 supernatant; 3:VHH-E2-2 inclusion body, 4:VHH-E2-2 supernatant; 5 VHH-E2-3 inclusion bodys; M: protein standard marker (from top to bottom

Molecular weight is followed successively by: 75,65,40,35,14 kDa); 6:VHH-E2-3 supernatant; 7 and 8: empty vector control; 9: do not add IPTG control group; In figure, arrow instruction is object 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 are separated and cDNA synthesis

1. materials and methods

1.1 materials-experimental animal, antigen, adjuvant

The female young two-humped camel selecting for two ages peak year about one year old as immunity receptor animal, (raise scattered on the Gobi desert near the Jinchang City of Gansu throughout the year, mainly cut camel hair and sell by these two-humped camels.Poultry main only in the winter time careless withered, summer Extreme drought and the lambing of mother camel time recall supplementary forage grass, water and salt, never inoculated any vaccine, be the suitable acceptor animal 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 affinity chromatography method purification of soluble E2 fusion rotein (GST-tag) as the antigen of immune two-humped camel, the E2 antigen of screening specificity single domain heavy chain antibody (VHH).Utilize SDS-PAGE electrophoresis determination purity of protein, by uv-absorbing standard measure.

1.2 method

Emulsification is fully mixed complete with 206 adjuvants and E2-GST antigen equal-volume, respectively at the neck of two-humped camel, two hind leg and dorsal sc multi-point injection, first immunisation dosage is 300 μ g/ time (2mL), 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 E2 antigen immune, in body, anti-E2 protein specific antibody titre extends along with immunization time and rises gradually, after the 4th (when 35 days) immunity, antibody horizontal reaches highest level, after the 5th immunity, antibody titers is without significantly improving, according to serum antibody growth and decline feature, the 6th immunity gathers jugular vein peripheral blood in latter 5 days.

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

Two, the structure of swine fever VHH non-immune libraries

1. prepare the elementary storehouse of VHH phage antibody

The phage vector pHEN4 of the VHH antibody fragment and 28 μ g of getting 6 μ g bacterium purifying uses restriction enzyme respectively pstIand restriction endonuclease notIafter digestion, at 22 DEG C of temperature, use T ₄dNA ligase spends the night connection, carries out deactivation, after deactivation 10min, be cooled with an ice bath at 65 DEG C of temperature to enzyme, 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 C temperature, with the rotating speed of 160rpm/min concussion cultivation 1 hour, recover resistance, be coated on (nutrient solution on culture plate is glucose 100 μ g, the 100 μ g/ml penbritins of concentration 2%) on 2 × YT culture plate after concentrated, the bacterium liquid simultaneously got after the bacterium liquid 10 μ L after concentrating is diluted to 10 ⁴individual bacterium colony, then coated plate, incubated overnight at 37 DEG C of temperature, connect in a large number through three times and transform, obtaining storage capacity is 1.29 × 10 ⁸camel immunity single domain antibody storehouse.Washed by mono-clonal bacterium colony LB liquid nutrient medium on culture plate, adding concentration is wherein 10% glycerine, is kept at-80 after packing ^oin C refrigerator, preparation becomes the elementary storehouse of VHH phage antibody.

2. set up VHH immune antibody library

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

Each batch of sequencing result is shown, obtain being unique sequence after sequence utilizes DNAstar, BLAST comparative analysis determines that the specificity clone selecting in 3 crowdes to measure is the heavy chain antibody VHH sequence deriving from camel and belong to, and illustrates that structure immune antibody library diversity is better.

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

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

1. bag quilt: by target molecule by CSFV E 2 Antigen: it is even that carbonate bag is buffered liquid=1:4 dilution mixture, 50ul/ hole, and 4 degree of bags are spent the night, and 1 × PBS washes 3 times;

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

3., in conjunction with phage: PBS washes 3 times, get 5ul phage+45ul 1.0% M-PBS and mix 50ul/ hole, under 37 DEG C of conditions, leave standstill 1.0 h;

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

5. two resist: 0.1%PBST washes 3 times, press 1:3000 dilute HRP-goat anti-rabbit IgG pAb 50ul/ hole, 37 DEG C of standing 1.0h with 1.0% M-PBS;

6. develop the color: 0.1%PBST washes 4 times, gets 0.2M/l Na ₂the each 4.5ml of HPO4+0.1M/L citric acid adds a small amount of OPD, 60ul H ₂o ₂mixing 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) screen

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

1.1 bag quilts: CSFVE2 pH 9.6 carbonate coating buffer is diluted to 15 ug/ml, and 4 DEG C are spent the night;

1.2 close: add 2% MPBS, 100 uL/ holes, 37 DEG C of incubation 1.0h;

1.3 combine: phage is with final concentration 0.5% MPBS by 10 times of dilutions, and all the other add 0.5% MPBS, and 37 DEG C of temperature educate 1.0 h;

1.4 primary antibodies: the anti-M13 of rabbit;

1.5 2 resist: HRP-goat-anti rabbit;

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

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

1.1 bag quilts: CSFV E2 pH 9.6 coating buffer is diluted to 10 ug/ml, and 4 DEG C are spent the night;

1.2 close: 2% MPBS, 37 DEG C of 1.0h;

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

1.4 primary antibodies: the anti-M13 of rabbit;

1.5 2 resist: 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, 19 and 24 yin and yang attribute differences are the most remarkable, again carry out third round mono-clonal ELISA and screen E2 specificity VHH antibody.

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

1.1 bag quilts: CSFV E2 pH9.6 coating buffer is diluted to 10 ug/ml, spends the night at 4 DEG C of temperature;

1.2. close: 2% MPBS, 37 DEG C of 1.0h;

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

1.4 primary antibodies: the anti-M13 of rabbit;

1.5 2 resist: HRP-goat-anti rabbit;

1.6 colour developing.

Third round the selection result in table 6-direct coated E2 antigen polyclone screening mono-clonal phage ELISA, wherein mono-clonal the 3,9,17,19with 24yin and yang attribute difference is the most remarkable, wherein 17,19 and 24 still show and to have stable and specific combination with E2 antigen after third round screening, illustrate still to obtain specific E2 VHH antibody from immune library under taking turns screening and high eluting power condition 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 clone separately, and the results are shown in Table the order-checking of 7-specificity VHH DNA sequence dna and analyze, the sequence recorded is shown in sequence table.

Five, VHH sequence signature is analyzed

From the symbolic characteristic that the amino acid such as Fig. 1: Val37Phe, Gly44Glu, Leu45Arg, and Gly47Trp are composition heavy chain VHH antibody, illustrate that 3 VHH clones of screening acquisition are the heavy chain antibodies from two-humped camel.Relatively find, hypervariable region amino acid significant difference between the CDR3 of 3 the different VHH sequences combined with E2 antigen-specific, the CDR3 length of VHH-E2-1 has 18 amino acid, all the other two clone CDR3 districts are made up of 20 amino acid, illustrating that 3 VHH antibody are from 3 different epitopes from E2 antigen, is independently 3 antibody sequences.By finding the amino acid length analysis of different genera CDR3 district, 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 antibodies disappearance light chain, but by extending 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 expression vector p-SMK(80 μ g/ml, kantlex, Kan ^r) on, positive restructuring data is transformed into ecolibL21-CodonPlus (DE3) (34 μ g/ml, paraxin, Cm ^r) by state cell, picking mono-clonal causes in 5 ml LB liquid nutrient mediums, 37 DEG C, 220rpm incubated overnight.Next day is inoculated into the 100ml LB substratum of new configuration according to 2% ratio, cultivates OD ₆₀₀value is to 0.6, and add the IPTG induction of final concentration 0.1mM, 20 DEG C, 220rpm induces 20h, centrifugal receipts bacterium, and SDS-PAGE analyzes the target stripe (Fig. 2) of size about 39 kDa, 1:VHH-E2-1 inclusion body in Fig. 2,2:VHH-E2-1 supernatant; 3:VHH-E2-2 inclusion body, 4:VHH-E2-2 supernatant; 5 VHH-E2-3 inclusion bodys; M: protein standard marker (from top to bottom molecular weight is followed successively by: 75,65,40,35,14 kDa); 6:VHH-E2-3 supernatant; 7 and 8: empty vector control; 9: do not add 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 of ultrasonic treatment induction, analyzes and shows that VHH albumen is expressed with soluble form, with affinitive layer purification VHH recombinant protein.

seven, double-antibody sandwich elisa mensuration VHH tests with restructuring E2 albumen and 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 plate, rabbit anti-swine fever polyclone 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 compares discovery, VHH antibody is a little less than the anti-CSFV hyper-immune serum of rabbit, may be because VHH antibody is only made up of in conjunction with epi-position the antigen of 1 single anti-CSFV E2, and the antibody not only containing anti-E2 antigen in CSFV hyper-immune serum, also include the antibody of anti-E0 albumen, therefore under similarity condition, polyclonal antiserum can 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 bag quilt: being buffered (pH 9.6) liquid by mixed 3 strain VHH antibody dilution to content with 0.05 M carbonate bag is 15.5 μ g/ml.In enzyme plate reacting hole, add 100 μ l/ holes, 4 DEG C are spent the night.Next day, discard solution in hole, with 1 × PBST lavation buffer solution, 200 μ l/ holes, wash plate 4 times.Add 200 μ l/ hole confining liquid (1 × PBST, 5% skim-milk) 37 DEG C and close 1 hour, with 1 × PBST lavation buffer solution, 200 μ l/ holes, wash plate 4 times.

2. add antigen: all samples does holes (see table 9-Pestivirus suis antigen double-antibodies sandwich ELISA operation signal), with 1 × PBS, measuring samples is done 1:2 dilution, 100 μ l/ holes have been wrapped in the reacting hole of quilt in above-mentioned, 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 DEG C act on 2 hours, with 1 × PBST lavation buffer solution, 200 μ l/ holes, wash plate 4 times.

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

4. rabbit anti-pig enzyme labelled antibody: anti-for horseradish peroxidase-labeled rabbit pig enzyme labelled antibody is done 1:100 dilution, 100 μ l/ holes add in each reacting hole, 37 DEG C act on 1 hour, repeat to wash plate step.

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

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

7. result judges: read result in microplate reader in 450nm place, all samples gets the mean value decision content as a result of holes.After blank control wells zeroing, (all samples hole OD value-blank well OD value) is as the OD value of the actual detection of various kinds sample wells.

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

(2) result judges: testing sample OD ₄₅₀>=negative control OD ₄₅₀2.1 times that are worth, be judged to be the positive.For negative sample, polymerase chain reaction (PCR) can be passed through and again detect.

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

The 73 increment product [5] of 1984 to 2006 that the applicant preserves are detected with double-antibody sandwich elisa, detected result following (table 10-double-antibody sandwich elisa and PCR two kinds of methods detect swine fever antigen result table).

In table 10,73 increment product are defined as CSFV nucleic acid for positive after being and being detected by PCR method.As the double-antibodies sandwich ELISA of capture antigen, VHH antibody detects that wherein 61 increment product are that virus antigen is positive, positive rate is 83.56%.It is positive that double-antibodies sandwich ELISA detects Pestivirus suis antigen, can confirm that really there is CSFV in sample infects, contributing to censorship pig farm takes anti-epidemic measure to process the pig that falls ill in time, take urgent vaccination ways to avoid by virus infection to compromised swinery, thus at utmost reduce financial loss.

Sequence table

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

<120> Bactrian camel source anti-hog cholera lapinised virus vaccine strain 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. a Bactrian camel source anti-hog cholera lapinised virus vaccine strain E2 antigen heavy chain antibody VHH, it is characterized in that, the nucleotides sequence of encoding said antibody VHH is classified as: 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 according to claim 1.

3. a host cell, is characterized in that described host cell is the prokaryotic cell prokaryocyte containing nucleotide sequence according to claim 1.

4. host cell as claimed in claim 3, is characterized in that described host cell is the prokaryotic cell prokaryocyte containing expression vector according to claim 2.

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