CN102746398B - Single-chain antibody ZL1 against chicken-derived Newcastle disease virus P protein, preparation method and application thereof - Google Patents
Single-chain antibody ZL1 against chicken-derived Newcastle disease virus P protein, preparation method and application thereof Download PDFInfo
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- CN102746398B CN102746398B CN 201210228688 CN201210228688A CN102746398B CN 102746398 B CN102746398 B CN 102746398B CN 201210228688 CN201210228688 CN 201210228688 CN 201210228688 A CN201210228688 A CN 201210228688A CN 102746398 B CN102746398 B CN 102746398B
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Abstract
The invention relates to preparation of a genetically engineered single-chain antibody ZL1 against chicken-derived Newcastle disease virus P protein by using molecular biological means. The single-chain antibody ZL1 at least comprises a light chain variable region having an amino acid sequence as represented by SEQ ID No. 1, a heavy chain variable region having an amino acid sequence as represented by SEQ ID No. 2 and intermediate connecting peptide located between the light chain variable region and the heavy chain variable region. According results of ELISA experiments, the prepared genetically engineered single-chain antibody ZL1 against Newcastle disease virus can specifically bind to prokaryotically expressed Newcastle disease virus P protein, which improves that the single-chain antibody has activity in binding to Newcastle disease virus P protein and can be used for prevention and/or treatment of Newcastle disease.
Description
Technical field
The preparation method who the present invention relates to a kind of phage single chain antibody ZL1 of anti-newcastle disease virus, the expression vector of expressing this single-chain antibody and host cell, this single-chain antibody with and uses thereof, described single-chain antibody can be combined activity with newcastle disease virus P protein-specific.
Background technology
Newcastle disease is that the bird that causes of Avian pneumo-encephalitis virus is acute, the transmissible disease of height contact and lethality, to the harm of China and even world's aviculture and serious, being defined as the category-A transmissible disease by World Organization for Animal Health, also is one of important diseases of China's poultry export quarantine.For viral infectious, still there is not the specific treatment medicine at present.Genetic engineering antibodies such as single-chain antibody with its unique antivirus action and advantage that can the large-scale engineering preparation, have shown the potentiality of huge research and development antiviral, are subjected to this field and pay much attention to.
Summary of the invention
The purpose of this invention is to provide the phage single chain antibody ZL1 of a kind of chicken source property anti-newcastle disease virus P albumen, this single-chain antibody can be combined with newcastle disease virus P protein-specific, can be used for preventing and/or treating of newcastle disease.
The single-chain antibody ZL1 of a kind of chicken source property anti-newcastle disease virus P albumen, it has the variable region of light chain of aminoacid sequence shown in SEQ ID No.1 at least, shown in SEQ ID No.2 the variable region of heavy chain of aminoacid sequence and the middle connection peptides between variable region of light chain and variable region of heavy chain, described in the middle of connection peptides be (Gly4Ser) 3.
Above-mentioned single-chain antibody has the aminoacid sequence shown in SEQ ID No.3.
Another object of the present invention provides the gene of a kind of single-chain antibody ZL1 of the described chicken source property anti-newcastle disease virus P albumen of encoding, and it has the nucleotide sequence shown in the SEQ ID No.4.
In order easily single-chain antibody to be detected purifying and further operation, can on the basis of above-mentioned sequence, further design restriction enzyme site and recognition sequence, preferably further contain restriction enzyme site NotI, NcoI and recognition sequence.Comprising NcoI:CCATGG NotI:GCGGCCGC.
A further object of the present invention provides a kind of expression carrier that contains the above-mentioned single-chain antibody of encoding.Above-mentioned expression vector is prokaryotic expression carrier, is preferably the pOPE101-XP carrier.
A further object of the present invention provides a kind of method for preparing the single-chain antibody (ZL1) of above-mentioned chicken source property anti-newcastle disease virus P albumen, may further comprise the steps:
(1) adopt RT-PCR directly from the chicken spleen RNA of ND GA/VA vaccine immunity, to amplify heavy chain variable region gene and the chain variable region gene of antibody coding gene;
(2) utilize the SOE-PCR method that linker is linked to each other with the VL gene with the VH gene and make up chicken source property single-chain antibody encoding gene;
(3) the chicken source property single-chain antibody encoding gene of step (2) is cloned among the prokaryotic expression carrier pOPE101-XP construction recombination plasmid;
(4) the prokaryotic expression carrier pOPE101-XP with step (3) is transformed into E.coli JM109 competent cell (Beijing Quanshijin Biotechnology Co., Ltd), cultivates, expresses single-chain antibody;
(5) indirect ELISA that the single-chain antibody of step (4) expression is set up as envelope antigen in order to the Avian pneumo-encephalitis virus P albumen of prokaryotic expression screens, and positive colony is the single-chain antibody of anti-new castle disease virus P albumen;
(6) purification procedures (5) gained positive colony cultured products namely obtains the single-chain antibody of described chicken source property anti-newcastle disease virus.(carrying out according to the Shanghai deep bio tech ltd purification column specification sheets operation of anticipating)
Need to prove, more than the experiment material that adopts of each step be the standard material that regular company obtains, method therefor is the described method of standard reagent box product description (seeing corresponding embodiment), the intermediates that each step obtains and last finished product all can repeat to obtain through the test of many times proof, and its biological property keeps stable and consistent.Illustrate that intermediates and finished product that each testing sequence of the present invention is related all can accurately obtain according to the method for sending out that the present invention states.
The single-chain antibody that a further object of the present invention provides above-mentioned chicken source property anti-newcastle disease virus P albumen is for the preparation of the prevention of newcastle disease, the application in the medicine.
Know-why of the present invention is to adopt RT-PCR directly from the ND(newcastle disease) amplify variable region of heavy chain (VH) gene and variable region of light chain (VL) gene of antibody coding gene the chicken spleen RNA of GA/VA vaccine strain immunity.Utilize SOE-PCR(reorganization chain extension reaction) method is linker and VH gene structure chicken source property single-chain antibody (ScFv) gene that links to each other with the VL gene, and it is cloned among the prokaryotic expression carrier pOPE101-XP, construction recombination plasmid also changes escherichia coli expression over to, the positive colony of the anti-NDV single-chain antibody of indirect ELISA screening prokaryotic expression, carry out the Clustalw multisequencing after the order-checking relatively, prove that this single-chain antibody belongs to the single-chain antibody of chicken source property anti-newcastle disease virus P albumen.
The invention has the beneficial effects as follows: the genetic engineering antibody of anti-newcastle disease virus can be combined with newcastle disease virus P protein-specific, can be used in prevention and the treatment of newcastle disease.
Description of drawings:
Fig. 1 is the structure iron of the pOPE101-XP recombinant vectors of embodiment 1.
Fig. 2 is encoding gene and the corresponding amino acid sequence thereof of single-chain antibody ZL1.
Embodiment:
The preparation of the single-chain antibody of embodiment 1 chicken source property anti-newcastle disease virus
1: newcastle disease vaccine (Bio ND VG/GA is available from Cimmeria group) is pressed operation instruction immunity chicken (Luo Man hen, 4 monthly ages, body weight 1.5kg, think the professional cooperative society of sweet poultry farming available from Shanghai), when detecting serum antibody titer greater than 1:20000 with conventional (with reference to " fine works molecular biology experiment guides " such as F.M. Ao Sibai) ELISA method, the results chicken spleen, after homogenate was ground, (TRIZOL Reagent is available from TaKaRa company) extracted total RNA with the Trizol method.Total RNA with extraction is masterplate, adopts Oligo primer, according to the description of product operation steps of reverse transcription test kit (cDNA the 1st chain synthetic agent box is available from TaKaRa company), and synthetic the 1st chain cDNA.
2: the FR district design amplification antibody of the chicken antibody encoding gene variable region sequences of having announced according to GenBank (AJ298107.1) is light, the primer (table 1) of heavy chain, and wherein VH1F and VH1R are used for amplification VH district; VL1F and VL1R are used for amplification VL district; VH2R, VH2F are used for the VH gene and add restriction enzyme site and Linker sequence; VL2R, VL2F are used for the VL gene and add restriction enzyme site and Linker sequence.Wherein, VH2F, VL2R contain NotI and NcoI restriction enzyme site respectively; VH2R, VL2F contain complementary Linker sequence (restriction enzyme site and Linker sequence indicate with underscore) in table 1.Primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.
Primer and the amplified fragments size thereof of table 1 amplification antibody variable region
The amplification of 3:VH and VL gene
Be masterplate with cDNA, VH1F, VH1R are primer amplification VH gene; VL1F, VL1R are primer amplification VL gene.The PCR reaction system is 25 μ L:2 * PCR mix12.5 μ L, masterplate cDNA2 μ L, each 1 μ L of upstream and downstream primer (25 μ M), ddH
2O8.5 μ L.Amplification program is as follows: 95 ℃ of pre-sex change 3min; 94 ℃ of sex change 40s, 50 ℃ of annealing 40s, 72 ℃ are extended 1min, 30 circulations; Last 72 ℃ are extended 10min.1.5% agarose gel electrophoresis is identified product and goal gene (glue that provides according to AxyGEN company reclaims the specification sheets operation) is provided.
4: the acquisition of single-chain antibody encoding gene
Be masterplate with VH and VL gene respectively, VH2R, VL2F are variable region of heavy chain and the chain variable region gene that the primer PCR amplification has Linker, and the PCR condition is the same.Amplified production reclaims goal gene (VH and the VL gene that contain the Linker sequence) after 1% agarose gel electrophoresis is identified.VH and the VL gene that will contain the Linker sequence by reorganization chain extension reaction (SOE-PCR) are connected to the single-chain antibody encoding gene, and add NotI and NcoI restriction enzyme site.
5: the structure of single-chain antibody encoding gene prokaryotic expression plasmid
According to conventional molecular cloning method (with reference to chief editors' such as J. Sa nurse Brooker " molecular cloning experiment guide "), single-chain antibody encoding gene and pOPE101-XP carrier are respectively behind NotI and NcoI double digestion, the single-chain antibody encoding gene is inserted pOPE101-XP carrier (Changzhou peace provides than gloomy bio tech ltd), make up recombinant expression plasmid (referring to Fig. 1), and with its Transformed E .coli JM109 competent cell (Beijing Quanshijin Biotechnology Co., Ltd), obtain recombinant expressed host.The clone who transforms carries out bacterium liquid PCR evaluation and the plasmid double digestion is identified.
6: the abduction delivering of single-chain antibody
With the positive colony after identifying in that to contain the Amp+(final concentration be 100 μ M) the LB liquid nutrient medium in be cultured to bacterium liquid OD600 to 0.6.Bacterium liquid is divided into three parts, is respectively and induces group, TES treatment group and the non-group of inducing.Induce group and TES treatment group in bacterium liquid, to add IPTG (final concentration 100 μ M), induce 4h, collection bacterium liquid in 30 ℃.Bacterium liquid is through centrifugal collecting precipitation, and is a for inducing group, and the ice-cold TES (50mmol/LTrils-HCl, 1mmol/L EDTA, 250g/L Sucrose) of another part usefulness is resuspended, places 15min on ice, and centrifugal collection supernatant is the TES treatment group." pET System Manual " according to Merck ﹠ Co., Inc. carries out separation and purification.
The indirect ELISA screening of embodiment 2 antigen-specific single-chain antibodies
Get the NDV P albumen (Changzhou peace provides than gloomy bio tech ltd) of the prokaryotic expression of purifying, spent the night in 4 ℃ of bags with 50mmol/L sodium bicarbonate salts solution (pH9.6), wash 3 times with PBST (containing 0.1%Tween20, as follows) behind the 5% skim-milk solution sealing 1h; Get TES and handle bacterium liquid supernatant 50 μ L, and add above-mentioned bag behind 4% skim-milk solution, the 50 μ L mixings by good P albumen, 37 ℃ of reaction 2h, PBST washing; Add Myc-Tag Mouse mAb(Myc-label mouse monoclonal antibody available from Ray Biotech company) 37 ℃ of reactions of 100 μ L (1:2000) 2h, the PBST washing; Add Peroxidase-conjugated Affinipure Goagt Anti-Mouse IgG(available from Abmart company) 100 μ L (1:4000), 37 ℃ of reaction 1h, PBST washing; The TMB colour developing, 2mol/L sulfuric acid termination reaction, microplate reader reads the OD450 value, establishes simultaneously and does not induce bacterium liquid supernatant for expressing negative control, and BSA is the antigen negative contrast.Indirect ELISA result's judgement is with P/N (the OD450 value in the positive hole of P, the OD450 value in the negative hole of N) expression, and P/N 〉=2.1 are positive; 1.5≤P/N＜2.1 are suspicious; P/N＜1.5 are negative.Positive colony is verified (with reference to Yao Huochun chief editor " veterinary microbiology experiment instruction ") through 3 replica tests, establishes infectious bronchitis virus, infections chicken cloacal bursa virus etc. simultaneously, is the specificity of antigen control checking single-chain antibody.The indirect ELISA result shows, under the same terms, single-chain antibody ZL1 is 0.465 with the reaction OD value mean value of the Avian pneumo-encephalitis virus P albumen of bag quilt, and with the reaction OD value mean value of control group antigen of bag quilt be 0.106, proof single-chain antibody Zl1 can identify newcastle disease virus P albumen specifically, and with infections chicken cloacal bursa virus, avian infectious bronchitis virus etc. cross-immune reaction does not take place.
In addition, the single-chain antibody ZL2 that another invention similar with the present invention obtains, its reaction OD value mean value with the Avian pneumo-encephalitis virus P albumen of bag quilt is 0.663, has significant difference (P＜0.05) with ZL1OD value mean value 0.465 of the present invention.
The single-chain antibody encoding gene of 3 pairs of acquisitions of embodiment checks order, and proves that it reaches 243 amino acid inferring accordingly by 729 Nucleotide and forms, and described nucleotide sequence is shown in SEQ ID No.4, and described aminoacid sequence is shown in SEQ ID No.3.
The instruction book chain antibody has certain anti-new castle disease virus activity, can be used for diagnosis and the treatment of newcastle disease virus.
Claims (8)
1. the single-chain antibody ZL1 of a chicken source property anti-newcastle disease virus P albumen, be and the protein bound single-chain antibody of the Avian pneumo-encephalitis virus P of prokaryotic expression, it has the variable region of light chain of aminoacid sequence shown in SEQ ID No.1 at least, shown in SEQ ID No.2 the variable region of heavy chain of aminoacid sequence, and the middle connection peptides between variable region of light chain and variable region of heavy chain.
2. single-chain antibody ZL1 as claimed in claim 1 is characterized in that it has the aminoacid sequence shown in SEQ ID No.3.
3. the gene of a coding single-chain antibody as claimed in claim 1 is characterized in that it has the nucleotide sequence shown in the SEQ ID No.4.
4. gene as claimed in claim 3 is characterized in that further comprising in the described nucleotide sequence restriction enzyme site NotI, NcoI, and wherein NcoI is CCATGG, and NotI is GCGGCCGC.
5. contain each described expression carrier of claim 3-4.
6. expression vector as claimed in claim 5 is characterized in that described carrier is prokaryotic expression carrier.
7. expression vector as claimed in claim 6 is characterized in that described carrier is the pOPE101-XP carrier.
8. the single-chain antibody ZL1 of chicken as claimed in claim 1 or 2 source property anti-newcastle disease virus P albumen is in the application in the medicine of preventing and/or treating for the preparation of newcastle disease.
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