CN109438574B - Porcine epidemic diarrhea virus M protein specific heavy chain antibody - Google Patents
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Abstract
The invention discloses a porcine epidemic diarrhea virus M protein specific heavy chain antibody, and the nucleotide sequence of the antibody is as follows: SEQ ID NO. 4. The invention has the following effects: a phage display technology is utilized to construct a PEDV M protein bactrian camel source immune library, 8 strains from the hypervariable region anti-M protein VHH antibody gene coding sequences of bactrian camel IgG heavy chain antibody are obtained by screening, soluble expression and ELISA experiments are carried out on the sequences to prove that the sequences can be combined with M protein, and the difference of absorbance OD405 values shows that the sequences have different combining capacities with the M protein. Coli, and preparing the in vitro recombinant genetically engineered antibody.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a porcine epidemic diarrhea virus M protein single domain antibody.
Background
Porcine Epidemic Diarrheia (PED) is a highly contagious enteric infectious disease in pigs caused by infection with Porcine Epidemic Diarrhea Virus (PEDV). The sick pigs infected with PEDV transmit the virus mainly through feces, and the sick pigs mainly show clinical characteristics of diarrhea, vomiting, dehydration and the like and are one of the main infectious diseases causing death of suckling piglets. In 1971 PED first occurred in the uk and subsequently diffused around the world. PEDV is first isolated in 1980 in China, PED epidemic situation occurs in most pig raising areas in 2010, diseases occur from suckling piglets to breeding sows, and serious economic loss is caused to pig raising households. PEDV belongs to Coronav irus (Coronav) of Coronavi ridae (Coronav Ridae) of Nidovirales (Nidovirales), the genome is RNA with single-strand positive strand infectivity, and similar to other coronaviruses, the 5 'end of the genome has a cap structure (cap), the 3' end has 1 Poly (A) tail, the total length of the genome is 2,8033 nt, and the genome respectively encodes 4 virus structural proteins of S, E, M, N and the like. The S ensures that the preserved neutralizing epitope can induce an organism to generate a neutralizing antibody, and has good protection effect on piglets. In the early stages of PEDV infection, the body can generate high-level antibodies against N and N proteins, and the PEDV antigen is a candidate antigen for developing a virus serum antibody diagnostic method. The E protein has application prospect in antiviral research.
The heavy chain antibody (VHH) naturally lacking the light chain in the camel peripheral blood has biological characteristics which are not possessed by other terrestrial organisms, and the antigen binding site of the VHH is only composed of a single structural domain of a heavy chain variable region, but keeps good and specific antigen binding capability like the common IgG antibody. VHH is the smallest fully functional IgG antibody molecule fragment currently available with a molecular weight of 15kDa, 1/10 for conventional IgG, 1/3 for monoclonal antibody, 4.8nm in height and 2.2 nm in diameter, also known as nanobody (nanobody). The VHH antibody antigen binding site has an extended antigen complementary binding region and tissue penetrability, and can bind to an epitope which cannot be contacted by a common IgG antibody. In addition, the in vitro recombinant VHH has the unique properties of easy expression, good water solubility and the like, and has wide application prospect in the research and development field of miniature genetic engineering antibodies, small molecule antibody medicines and high-sensitivity diagnostic reagents.
Disclosure of Invention
The invention aims to provide a porcine epidemic diarrhea virus M protein specific heavy chain antibody;
it is another object of the present invention to provide a nucleotide sequence encoding the heavy chain antibody specific for the M protein of porcine epidemic diarrhea virus;
the invention also aims to provide the expression of the porcine epidemic diarrhea virus M protein specific heavy chain antibody or the coding sequence thereof in E.
The invention adopts the following technical scheme that a porcine epidemic diarrhea virus M protein specific heavy chain antibody, the nucleotide sequence of the coded antibody is as follows: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7 or SEQ ID NO. 8.
A recombinant expression vector contains the nucleotide sequence.
A host cell, the host cell is a prokaryotic cell containing the nucleotide sequence.
Preferably, the host cell is a prokaryotic cell containing the above expression vector.
Expression of the porcine epidemic diarrhea virus M protein specific heavy chain antibody or the coding sequence thereof in E.
The invention has the following effects: a phage display technology is utilized to construct a PEDV M protein bactrian camel source immune library, 8 strains from the hypervariable region anti-M protein VHH antibody gene coding sequences of bactrian camel IgG heavy chain antibody are obtained by screening, soluble expression and ELISA experiments are carried out on the sequences to prove that the sequences can be combined with M protein, and the difference of absorbance OD405 values shows that the sequences have different combining capacities with the M protein. Coli, and preparing the in vitro recombinant genetically engineered antibody.
Drawings
FIG. 1 is a diagram showing the alignment of the amino acid sequences of clones resistant to the VHH protein of PEDV M;
coli anti-PEDV M protein VHH antibody SDS-PAGE analysis.
Detailed Description
The following examples further illustrate the invention but are not intended to limit the invention in any way.
Example 1 preparation of heavy chain antibody specific for porcine epidemic diarrhea Virus M protein
1. Materials and methods
1.1 laboratory animals and reagents
Taking 2-peak 10-month-old female bactrian camel as an experimental animal, bovine lymphocyte separation fluid and a 96-hole enzyme label plate; restriction enzyme, DNA chipThe segment recovery kit and the plasmid extraction kit are OMEGA products and nickel affinity chromatography resin (Qiagen);E.colirecombinant expression of PEDV M antigen (His tag and GST tag); the strain, the helper phage and the reagents Escherichia coli TG1 and BL21 Star (DE3), the helper phage M13K07 and the vector pHEN2 were purchased from NEB company, and other reagents were all domestic analytical purifiers.
1.2 methods
1.2.1 Camel immunization and antibody monitoring
For the first immunization, the camels were inoculated with a subcutaneous multiple spot (dorsal, hind leg and cervical bilateral muscles) of 500 μ g recombinant M protein emulsified with 206 adjuvant (purchased from seepic, france). The booster immunization is carried out 21 days after the first immunization, the dosage is 206 emulsified 1000 mu g M protein, the booster immunization is carried out 35 days, 42 days and 63 days after the first immunization, and the antigen inoculation dosage is 1000 mu g M protein. Venous blood was collected and sera were isolated before the first immunization and before each booster immunization, and the PEDV antibodies were detected by indirect ELISA using M protein (GST tag) as antigen. In the immune experiment process, camel feeding management, immunity and blood sample collection are all completed under the supervision and guidance of professional veterinarian workers.
1.2.2 isolation of peripheral blood
And 7 days (namely 63 days) after the last immunization, 10mL of anticoagulation blood (the anticoagulant is heparin sodium and is 10 IU/mL) is collected from the jugular vein, equal amount of Han's solution is added for 1-time dilution, then equal volume of lymphocyte separation solution is added, and the total lymphocytes in the peripheral blood are separated by density gradient centrifugation. The isolated lymphocytes were resuspended in MEM and added to a six-well plate (37 ℃, 5% CO)2) Standing for 30 min, adsorbing to remove cell debris and macrophage, centrifuging to collect lymphocyte, adding MEM, and storing in liquid nitrogen for cell RNA extraction.
1.2.3 VHH antibody immune library construction
Design 3 pairs of primers (see Table 1-primers for amplification of VHH genes) genes encoding VHH proteins of interest were obtained by three rounds of amplification. Extracting lymphocyte total RNA by Trizol reagent, synthesizing first strand cDNA by using G1 as reverse transcription primer, amplifying by using cDNA as template and H1 and G1 as primers to obtain 2 DNA bands with the size of 600bp and 900bp, recovering the 600bp band as template, and carrying out 2 nd round PCR amplification.
The 2 nd round PCR product was used as a template, and H2 and H3 were used as primers to obtain a 450bp DNA band. Taking 2 rounds of PCR products as templates and P1, TN as upstream and downstream primers to carry out 3 rd round amplification to obtain a 450bp band. Cloning the PCR product of round 3 by SfiI/NotI double enzyme digestion to pHEN2 vector, converting the ligation product into TG1 by electrotransformation method, culturing at 37 deg.C and 180r/min for 1h, centrifuging, concentrating, and spreading on ampicillin-resistant 2 XYTG plate (2% glucose, 100. mu.g/mL Amp)r). Simultaneously, 10. mu.L of the extract was diluted to 1X 104The plates were plated, incubated overnight at 37 ℃ and the stock capacity was calculated. After transformation, the plate was incubated at 37 ℃ for 16 hours in an incubator, the lawn grown on the plate was scraped clean with 10mL of 2 XYT medium, glycerol was added to the final concentration of 25%, and the resultant was stored at-70 ℃ in portions as a bank of the obtained anti-PEDV M protein VHH immunoantibody. The electrically transformed clones were randomly selected for sequencing and the library size was calculated from the diversity and number of clones.
1.2.4 obtaining specific anti-PEDV M protein VHH antibody by secondary screening process
The antibody screening procedure was as follows:
(1) antigen coating: m antigen (GST tag) was diluted to 20ug/ml, 50 ul/well, overnight at 4 ℃. PBS washing for 3 times;
(2) and (3) sealing: adding 300 ul/hole 2% M-PBS, sealing at 37 deg.C for 1h, washing with PBS 3 times;
(3) combining: taking 20ul phase +180ul 0.1MPBS, mixing uniformly, standing at 50 ul/hole for 1h at 37 ℃; throwing out redundant phage solution, inverting the plate, beating and throwing on a paper towel to remove residual liquid, and washing for 3 times by using 0.1% PBST;
(4) a first antibody: 1 part by weight with 0.1M PBS: standing the rabbit antibody with the concentration of 1000 and M1350 ul/hole at 37 ℃ for 1 h; throwing out redundant liquid, inverting the flat plate, beating and throwing on a paper towel to remove residual liquid, and washing for 3 times by using 0.1 percent PBST;
(5) secondary antibody: using 0.1M PBSS as 1: 10000-diluted HRP-goat anti-rabbit is kept standing for 1h at 37 ℃; throwing out redundant liquid, inverting the flat plate, beating on a paper towel to remove residual liquid, and washing for 4 times by using 0.1 percent PBST;
(6) color development: 0.5ml of 0.2M/L Na2HPO4 + 0.1M/L citric acid is taken and added with a small amount of OPD, 10ulH2O2 and mixed evenly for 50 ul/hole; 2M sulfuric acid stopped 25 ul/well, detection was done at 490nm wavelength.
1.2.5 soluble expression of VHH and ELISA detection
40 clones were randomly selected, transformed into E.coli BL21 Star (DE3) expression competence, and then selected in 1ml culture medium for rapid induction of expression, TG1 empty cells were used as blank control. Collecting induced 1ml bacterial liquid, centrifuging at 12000rpm for 2min, discarding supernatant, and resuspending the precipitate with 0.5ml TBS; 400W (6 s ultrasound, 4 is interval) ultrasound for 10 min; adding 50ul of ultrasonic supernatant into the mixture per hole, standing the mixture for 1h at 37 ℃, and washing the mixture for 3 times by TBS; adding 2% M-TBS, 200 ul/hole, sealing at 37 ℃ for 1h, and washing with 0.1% TBST for 4 times; 4mg PNPP was added to 1M diethanolamine + 0.5mM MgCl2 at 25 ul/well, stopped with 3M NaOH at 25 ul/well. And detecting the result under the condition of 405nm wavelength. Positive reaction clones were selected for sequence determination and alignment.
2. Results
2.1 detection of M antibody levels in Camel serum
Female bactrian camels of 2-peak 10-month age were used as experimental control animals.
The four times of M antigen (His label) immune camel serum antibody 1: 50 dilution detection shows (see table 2-M protein antibody detection (OD 450 nm)), after 2 times of boosting immunization, 2 experimental control animals have positive anti-M antibodies, and when the antibody titer is measured 7 days after the last immunization, the 2 peak camel serum antibody OD450nm reaches 1.58 and 1.92, so that the organism in-vivo humoral and cellular immune levels can be judged to be in a continuous rising stage at the moment, and the experiment requirements for collecting peripheral blood lymphocytes are met.
2.2 anti-M protein VHH immune library construction and screening
After four rounds of selection, a more pronounced enrichment effect could be detected (see table 3 — enrichment index for each round of phage selection). The result of the monoclonal phage ELISA experiment shows 40 positive clones, and the OD450nm value of each clone is shown in Table 4, and M13KO7 and 1% M-PBS in Table 4 are negative controls. DNA sequencing and nucleotide sequence comparison are carried out on the clones to obtain 8 VHH gene base sequences, and 6 idiotype anti-M protein VHH antibody genes are obtained after amino acid comparison.
2.3 soluble expression of VHH and ELISA detection
The M protein is used as an antigen coated ELISA plate, and the soluble expression and ELISA detection are carried out on 6 idiotypic anti-M protein VHH antibody genes obtained under the conditions of 30 ℃ and 37 ℃, so that the idiotypic anti-M protein VHH antibody genes can be combined with the M protein, and the combination ability of the idiotypic anti-M protein VHH antibody genes with the antigen is different according to the height of an absorbance value (OD 405 nm) (see table 5-M protein ELISA identification).
2.4 alignment of anti-M protein VHH Gene sequences and expression in E.coli
The nucleotide sequencing and sequence analysis of the obtained VHH gene are shown in FIG. 1, and the amino acid sequence alignment results are shown in FIG. 2. The analysis was performed by The Kabat et al. (Kabat, 1991) alignment method, The amino acids in The boxes of fig. 1 represent The characteristic marker amino acids of The bactrian camel-derived VHH antibody, The underlined amino acids represent The different amino acids between The different VHH clones. In order to stably obtain VHH antibodies, VHH gene sequences were cloned into pET-28a vectors, transferred into E.coli BL21 (DE3) competent cells, induced by 1.0 mM IPTG for expression, and SDS-PAGE protein electrophoretic analysis revealed a band of interest with a molecular weight of about 25kDa (VHH of about 13.6Kda, histidine tag of 8-10 kDa), as indicated by the arrows in the figure, confirming successful expression of all VHH antibodies.
Illustrated by figure 1 and table 4: the screening of phage display technology is utilized to obtain 8 strains of antibody sequences of PEDV M protein VHH coded by the antibodies, and the amino acid sequence alignment results show that the FR2 region sequences of the antibodies have the characteristic of typical heavy chain antibody amino sequence, namely the amino acid residues in the sequences are respectively F37/E44/R45/G47 (figure 1), which proves that the antibody sequences are from the hypervariable region of the heavy chain antibody. The CDR1 region of the antibody has the amino acid residue 31S andor N, 32S andor N, and the CDR2 amino acid residue 54D or N has variation, which indicates that the diversity of the amino acid sequences of the 8 strains of antibodies has significant difference.
Sequence listing
<110> Lanzhou veterinary research institute of Chinese academy of agricultural sciences
<120> a porcine epidemic diarrhea virus M protein specific heavy chain antibody
<130>2015
<160>8
<170>PatentIn version 3.3
<210>1
<211>372
<212>DNA
<213>Camelus bactrianus
<400>1
gaggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctgggtt cccctttact agctccgtca tgggatggtt ccgccaggct 120
ccagggaagg aacgcgaggg ggtcgcagct atttcggttg atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc ggctagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210>2
<211>372
<212>DNA
<213>Camelus bactrianus
<400>2
gatgtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctggatt cccctttagt aacaacgtca tgggctggtt ccgccaggct 120
ccagggaagg aacgcgaggg ggtcgcagct atttcggtta atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gactagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggggccaggg gaccctggtc 360
accgtctcct ca 372
<210>3
<211>372
<212>DNA
<213>Camelus bactrianus
<400>3
gaggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctggatt cccctttagt agcaacgtca tgggctggtt ccgccaggct 120
ccagggaagg aacgcgaggg gatcgcagct atttcggttg gtagtggtag cacatggtat 180
ggcgactccg tgaagggccg attcaccatc tccctggaca acgccaacaa cacgctgtat 240
ctccaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gactagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210>4
<211>372
<212>DNA
<213>Camelus bactrianus
<400>4
gaggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctgggtt ctcctttagt agcaacgtca tgggctggtt ccgccaggct 120
ccagggaagg aacgcgaggg ggtcgcagct atttcggttg atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gactagacgt 300
ggagttattc ttacactgag cccagagacc tatgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210>5
<211>372
<212>DNA
<213>Camelus bactrianus
<400>5
caggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctggatt cccctttagt aacaacgtca tgggctggtt ccgccaggct 120
ccagggaagg aacgcgaggg ggtcgcagct atttcggtta atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc gactagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggggccaggg gaccctggtc 360
accgtctcct ca 372
<210>6
<211>372
<212>DNA
<213>Camelus bactrianus
<400>6
caggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctgggtt cccctttact agctccgtca tgggatggtt ccgccaggct 120
ccagggaagg aacgcgaggg ggtcgcagct atttcggttg atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccatgt actactgtgc ggctagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
<210>7
<211>372
<212>DNA
<213>Camelus bactrianus
<400>7
gaggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctggatt cccctttagt aacaacgtca tgggctggtt ccgccaggct 120
ccagggaagg aacgcgaggg ggtcgcagct atttcggtta atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccaacaa cacgctgtat 240
ctgcaaatga acagcctgaa acctgaggac actgccattt actactgtgc gactagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggagccaggg gaccctggtc 360
accgtctcct ca 372
<210>8
<211>372
<212>DNA
<213>Camelus bactrianus
<400>8
gaggtgcagc tggtggagtc tgggggaggc tcggtgcaga ctggagggtc tctgagactc 60
tcctgcgcag cctctggatt cccctttagt agcagcgtca tgggctggtt ccgccaggct 120
ccaggaaagg aacgcgaggg ggtcgcagct atttcggtag atagtggtag cacatggtat 180
gccgactccg tgaagggccg attcaccatc tccctggaca gcgccagcaa cacactgtat 240
ctgcaaatga acggcctgaa acctgaggac actgccatgc actactgtgc gactagacgt 300
ggagttattc ttacactaag cccagagacc tatgactact ggggccaggg gacccaggtc 360
accgtctcct ca 372
Claims (5)
1. A porcine epidemic diarrhea virus M protein specific VHH antibody, characterized in that the nucleotide sequence coding the antibody is as follows: SEQ ID NO. 4.
2. A recombinant expression vector comprising the nucleotide sequence of claim 1.
3. A host cell characterized in that said host cell is a prokaryotic cell comprising a nucleotide sequence according to claim 1.
4. The host cell of claim 3, wherein the host cell is a prokaryotic cell comprising the expression vector of claim 2.
5. The use of the gene encoding the porcine epidemic diarrhea virus M protein-specific VHH antibody of claim 1 in Escherichia coli (E.coli) (II-H-E)Escherichia coli) The expression of (1).
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| CN108892723B (en) * | 2018-07-12 | 2021-09-21 | 内蒙古农业大学 | Single-domain heavy chain antibody for detecting porcine epidemic diarrhea virus, preparation method and application |
| CN109678953A (en) * | 2018-12-24 | 2019-04-26 | 西北农林科技大学 | A kind of construction method and purposes of the VHH phage display library of anti-PEDV N protein |
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| CN109438573A (en) | 2019-03-08 |
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| CN105504053A (en) | 2016-04-20 |
| CN109438574A (en) | 2019-03-08 |
| CN109517060A (en) | 2019-03-26 |
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| CN109438573B (en) | 2020-11-10 |
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