CN108359009B - Brucella single-chain antibody and application thereof - Google Patents
Brucella single-chain antibody and application thereof Download PDFInfo
- Publication number
- CN108359009B CN108359009B CN201711457652.8A CN201711457652A CN108359009B CN 108359009 B CN108359009 B CN 108359009B CN 201711457652 A CN201711457652 A CN 201711457652A CN 108359009 B CN108359009 B CN 108359009B
- Authority
- CN
- China
- Prior art keywords
- antibody
- brucella
- seq
- variable region
- sequence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
- C07K16/1221—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria from Brucella (G)
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
- C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2317/00—Immunoglobulins specific features
- C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
- C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
- C07K2317/622—Single chain antibody (scFv)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Urology & Nephrology (AREA)
- Medicinal Chemistry (AREA)
- Organic Chemistry (AREA)
- Hematology (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Virology (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Pathology (AREA)
- Biophysics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Cell Biology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention relates to the technical field of biological product detection, and discloses a brucella single-chain antibody and application thereof, wherein the single-chain antibody comprises a light chain variable region, a heavy chain variable region and a joint for connecting the light chain variable region and the heavy chain variable region, wherein the sequence of the light chain variable region is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 1 at amino acids 130-252. The invention prepares the single-chain antibody which can be specifically combined with the brucella antigen.
Description
Technical Field
The invention relates to the technical field of biological product detection, in particular to a brucella single-chain antibody and application thereof.
Background
Brucellosis (hereinafter referred to as brucellosis) is a zoonosis caused by brucellosis infection, and is basically characterized in that fever and abortion of sick individuals cause serious threats to the life health of people and various animals such as pigs, cattle, sheep and the like. Therefore, the rapid and accurate diagnosis of brucella and the treatment and elimination of brucella are always one of the most important public health targets in countries and regions where brucella is epidemic.
Research related to diagnosis of the disease has been long, important progress is made in the technical methods of diagnosis, particularly serological diagnosis, and the method has better application from early agglutination test, complement fixation test to later enzyme-linked immunosorbent assay (ELISA) and Fluorescence Polarization Assay (FPA) reported in recent years. Agglutination tests are a serological diagnostic method commonly used in the early days, among which standard tube agglutination tests (STAT or SAT) and plate agglutination tests (plate agglutination test) are more typical. These two tests were later gradually replaced by the rose-bengal agglutination test (RPBT), which has high sensitivity, low cost, rapid detection, easy operation and is suitable for high throughput screening. Because of the relatively low specificity of RPBT, it can only be used as a preliminary screening test. The Complement Fixation Test (CFT) has high specificity, but the operation steps are complicated, time-consuming and less applicable. The development of mature ELISA methods in recent years has become recognized by those skilled in the art. Specifically, the ELISA used for the detection of the brucellosis antibody is divided into indirect ELISA (indirect enzyme-linked immunological assay, iELISA) and competitive enzyme-linked immunological assay (cELISA). Both of these methods are the test methods prescribed by World Organization for Animal Health (OIE) standards, which are also the prescribed test methods for Animal disease testing in international trade. The iELISA method completes detection through the steps of brucella LPS coating-antibody specific binding in a serum sample-HRP labeled secondary antibody binding-color development, is simple to operate, has high sensitivity, is convenient for high-throughput detection, and is a screening method. The cELISA also has the characteristic of high flux, judges the result according to the chromogenic reaction by the combination of the standardized brucella antigen specific antibody and the antibody possibly existing in the serum sample for competition and the coating antigen, can effectively avoid the false positive result caused by the cross reaction, has higher detection specificity, and is generally used as a diagnostic method. The cELISA reagents and development for the detection of the disease-spreading antibodies are receiving increasing attention and importance from the skilled person.
In the cELISA reagent for detecting brucellosis, the most central component is brucella antigen-specific antibody (competitive antibody). The antibody needs to bind specifically with high affinity to the brucella antigen in order to compete with the corresponding antibody in the serum sample in the detection system. At present, commercial kits and literature reports that the monoclonal antibody is directed against brucella antigen. Compared with a polyclonal antibody, the monoclonal antibody has obvious improvement on performance parameters such as specificity, stability and the like, and the detection performance of the corresponding cELISA kit is guaranteed to a certain extent. However, in the process of preparing monoclonal antibodies, hybridoma screening is complicated, hybridoma cell preservation cost is high, and experimental mice need to be fed. The monoclonal antibody prepared by the existing mouse ascites induction method has different batch differences, thereby bringing greater workload for quality control of products. In this regard, the single-chain antibody prepared based on the genetic engineering technology is more advantageous than the monoclonal antibody, once the corresponding DNA sequence and expression plasmid are obtained, the large-scale production can be carried out through prokaryote-Escherichia coli, so that the mass preparation of the antibody with low cost, high efficiency and high quality is realized, and therefore, the preparation of the single-chain antibody has more important practical significance.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provide a brucella single-chain antibody and application thereof.
In order to achieve the above object, the present invention provides, in a first aspect, a single chain antibody comprising a light chain variable region, a heavy chain variable region and a linker connecting the light chain variable region and the heavy chain variable region, wherein the sequence of the light chain variable region is as shown in SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 1 at amino acids 130-252.
In a second aspect, the present invention provides a gene encoding the single-chain antibody of the first aspect.
In a third aspect, the present invention provides an expression vector comprising the gene of the second aspect.
In a fourth aspect, the present invention provides a host cell transformed with the expression vector of the third aspect.
The fifth aspect of the present invention provides a kit for detecting a brucella antibody, comprising: the single chain antibody of the first aspect.
In a sixth aspect, the invention provides (for non-diagnostic purposes) a method for detecting brucella antibodies in a sample, the method comprising performing a competitive ELISA assay on the sample using a single chain antibody as described in the first aspect.
In the invention, a brucella antigen is used for immunizing a mouse, splenocytes of the mouse are collected, RNA is extracted, an immune antibody library is constructed, and a single-chain antibody capable of being specifically combined with the brucella antigen is prepared by phage display and panning technologies. The prepared prokaryotic expression plasmid is used for transforming escherichia coli, so that the large-scale preparation of the antibody with low cost, high efficiency and high quality can be realized, and a core raw material foundation is laid for preparing a better and cheaper brucellosis cELISA kit.
Drawings
FIG. 1 shows the results of detection of Brucella antibody by competitive ELISA (relative antibody content vs. absorbance at 405 nm);
FIG. 2 shows the results of detection of Brucella antibody by competitive ELISA (inhibition of antibody relative content and absorbance at 405 nm).
Detailed Description
The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.
The single-chain antibody provided by the invention comprises a light chain variable region, a heavy chain variable region and a joint for connecting the light chain variable region and the heavy chain variable region, and is characterized in that the sequence of the light chain variable region is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 1 at amino acids 130-252.
In a preferred embodiment of the invention, the linker sequence is as set forth in SEQ ID NO: 1 at amino acids 115-129.
The sequence of the single-chain antibody provided by the invention is shown as SEQ ID NO: 1, and the following components:
EIVLTQSPVTLSVSPGERATLSCRASQSVIGNLAWYQQKPGQAPRLLIYGASARAAGIPDRFSGSGSGTDFTLTISSLKSEDFAVYYCQQYNNWPPYTFGQGTKVEIKRSGGSTGGGGSGGGGSGGGGSVQLLESGGGLVQPGGSLRLSCAVSGLNLRRYAMSWVRQAPGKGLEWVSVIGASDGNVYYADSVKGRFIISRDNSKDTLDLQMNSLRVEDTAVYYCTKDHWLASWTDEMDVWGQGTLVTVSSAS(SEQ ID NO：1)。
according to a preferred embodiment of the invention, the sequence of said single chain antibody consists of SEQ ID NO: 1 and a sequence linked to SEQ ID NO: 1, a V5tag and a His tag (shown in SEQ ID NO: 3).
The invention also provides a gene for coding the single-chain antibody.
According to a preferred embodiment of the invention, the sequence of said gene may be as set forth in SEQ ID NO: 2, and can also be represented by SEQ ID NO: 2 (containing the V5tag coding sequence and the His tag coding sequence). In this preferred embodiment, SEQ ID NO: 2, shown at positions 1-342 of SEQ ID NO: 2 position 343-387 (or SEQ ID NO: 5) is a linker coding sequence, SEQ ID NO: positions 388-756 show the heavy chain variable region coding sequence.
gaaattgtgttgacacagtctccagtcaccctgtctgtgtctccaggggagagagccaccctctcctgcagggccagtcagagtgttatcggcaacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccgccagggccgccggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagcctgaagtctgaagattttgcagtttattactgtcagcagtataataactggcctccgtacacttttggccaggggaccaaggtggaaatcaaacgttccggagggtcgaccggtggaggcggttcaggcggaggtggctct ggcggtggcggatcggtgcagctgttggagtctgggggaggcttggtacagcctggggggtccctgagactctcctgtgcagtctctggactcaaccttagaagatatgccatgagttgggtccgccaggctccagggaaggggctggagtgggtctcggtaataggtgccagtgatggaaatgtctactacgctgactccgtgaagggccgattcatcatctcaagagacaattccaaggacacgttggatctgcaaatgaacagcctgagagtcgaggacacggccgtatattactgtacgaaagatcattggctcgccagttggaccgacgagatggacgtctggggccagggcaccctggtcactgtctcctcagctagcggcaaaccaatcccaaacccactgctgggcctggatagtactcaccatcaccatcaccat(SEQ ID NO：2)
The expression vector provided by the invention comprises the gene. The expression vector may be pET32a into which the above gene is inserted.
The host cell provided by the invention is transformed with the expression vector. The host cell may be a recombinant bacterium. The recombinant strain may be a strain containing the expression vector of the present invention, and for example, may be obtained by transferring the expression vector of the present invention into a competent strain, such as escherichia coli competent strain BL21(DE 3).
The kit for detecting the brucella antibody provided by the invention comprises: single chain antibodies as described above. The kit is a kit for detecting brucella antibody based on the competitive ELISA principle, and thus may contain various reagents required for performing competitive ELISA, for example, the kit may further include a washing solution, a diluent, a horseradish peroxidase (HRP) -labeled mouse anti-V5 monoclonal antibody, a chromogenic substrate for HRP, a reaction stop solution, and the like.
The method for detecting brucella antibodies in a sample (not the destination of diagnosis) provided by the invention comprises performing competitive ELISA detection on the sample by using the single-chain antibody.
According to a more specific embodiment, the detection method comprises: separating brucella antigen Lipopolysaccharide (LPS) from the brucella culture, and coating the holes of an enzyme-labeled plate; properly diluting a sample to be detected, mixing the diluted sample with the single-chain antibody, standing the mixture, and adding the mixture into the hole; washing after incubation, and adding a mouse anti-V5 monoclonal antibody marked by horseradish peroxidase (HRP); washing after incubation, and adding a chromogenic substrate (ABTS) of HRP; and (3) terminating the reaction after incubation, carrying out color comparison by an enzyme-labeling instrument at 405nm, wherein the absorbance is in negative correlation with the concentration of the brucella antibody in the sample.
Generally, the present invention provides a method of making a single chain antibody comprising:
(1) immunizing a mouse by using lipopolysaccharide derived from brucella, separating splenocytes of the mouse, extracting total RNA, and preparing cDNA by using a reverse transcription technology;
(2) using cDNA as template, respectively synthesizing mouse IgG heavy chain coding sequence and light chain coding sequence, and connecting the heavy chain coding sequence and the light chain coding sequence by using overlap extension PCR technology;
(3) inserting the coding sequence obtained after the connection in the step (2) into a phagemid vector to construct an immune antibody library; screening to obtain a single-chain antibody specifically binding to lipopolysaccharide derived from brucella through phage display and antigen specificity panning;
(4) optionally, the DNA fragment encoding the single-chain antibody is subcloned into a prokaryotic expression vector to construct a recombinant plasmid, the recombinant plasmid is transformed into escherichia coli, and the single-chain antibody can be obtained through induction expression and affinity purification. Step (4) is a step selectively performed, and in order to obtain a large amount of single-chain antibodies, step (4) is preferably performed.
According to a more specific embodiment, the above preparation method comprises: separating brucella antigen Lipopolysaccharide (LPS) from the culture of the brucella, and immunizing Balb/c mice; separating mouse spleen cells, extracting total RNA, connecting an immunoglobulin heavy chain coding fragment and a light chain coding fragment by using a PCR (polymerase chain reaction) and molecular cloning technology, and inserting the immunoglobulin heavy chain coding fragment and the light chain coding fragment into a phagemid vector to construct an immune antibody library; obtaining a single-chain antibody capable of specifically binding with brucella antigen LPS through phage display and antigen specificity panning; subcloning the DNA fragment coding the antibody into a prokaryotic expression vector pET32a to construct a plasmid pET32 a-Bru-ScFv; the plasmid is transformed into escherichia coli BL21(DE3), isopropyl-beta-d-thiogalactoside (IPTG) is induced to express and affinity purified, and the single-chain antibody is obtained.
The present invention will be described in detail below by way of examples. In the following examples, room temperature means "25 ℃.
Example 1
This example illustrates the preparation of a Brucella antigen-specific ScFv
(1) Preparation of Brucella antigen LPS
Weighing Brucella melitensis S99 sterilized bacteria (purchased from IDEXX company, product number P00120) with wet weight of about 2.5g, adding 17mL double distilled water for resuspension, transferring into 100mL blue-covered wire-mouth bottle, and heating to 66 ℃; the suspension was slowly added to 19mL of a 90% (V/V) phenol (analytically pure) solution preheated to 66 ℃ and shaken at 66 ℃ for 20 minutes (5 seconds per 30 seconds).
Secondly, taking out the reaction bottle from the water bath kettle, and placing the reaction bottle in a refrigerator at 4 ℃ for cooling for 30 minutes; transferring the cooled solution into a round-bottom centrifuge tube, centrifuging for 2.5 hours at 4 ℃ and 10000 g; the third layer of phenol phase was pipetted into a round bottom centrifuge tube with a glass syringe, centrifuged at 10000 g for 3 minutes at 4 ℃.
③ transferring the phenol phase into a 100mL blue-cap silk-mouth bottle, adding 50mL cold methanol (analytically pure) solution containing 0.5mL saturated sodium acetate (analytically pure), and standing for 2.5 hours at 4 ℃; centrifugation was carried out at 4 ℃ and 10000 g for 15 minutes, and the supernatant was discarded.
Resuspending the precipitate in 8mL of sterilized double distilled water, stirring at room temperature for 2.5 hours in the dark, at 4 ℃, 10000 g, centrifuging for 15 minutes, and taking the supernatant; the precipitate was resuspended in 8mL of sterile double distilled water, stirred at room temperature in the dark for 1 hour, at 4 ℃ and 10000 g, centrifuged for 15 minutes, and the supernatant was collected.
Fifthly, mixing the two supernatants evenly, adding 0.8g of trichloroacetic acid (analytically pure), and stirring for 10 minutes at room temperature; centrifuging at 4 deg.C for 15 min at 10000 g, collecting supernatant, transferring into 3.5kDa dialysis bag (Beijing Solebao Co.), dialyzing with 1L double distilled water at 4 deg.C in dark for 4 days, and changing solution every 12 hr to obtain Brucella antigen-LPS.
Sixthly, coating the prepared LPS on an enzyme-labeled plate hole, identifying by adopting a commercial Brucella antibody (Abcam company) through an Indirect ELISA (iELISA) method, setting a negative control and a positive control, and judging whether the prepared LPS is identified and combined by the corresponding antibody, namely whether the prepared LPS has immunogenicity.
Seventhly, subpackaging the qualified products into brown penicillium bottles with the volume of 1 mL/bottle, freeze-drying, and storing at room temperature in a dark place for later use.
(2) Preparation of immune antibody library
Emulsifying LPS purified from Brucella melitensis S99 with equivalent Freund' S complete adjuvant (Sigma company) by double-injector mutual pushing method, and performing basic immunization (100 μ g/mL) on 2 Balb/c mice of about 8 weeks old by four-limb subcutaneous multipoint injection and intraperitoneal injection; after 2 weeks, LPS was emulsified with an equal amount of Freund's incomplete adjuvant (Sigma Co.) and mice were further immunized by the routes of subcutaneous multi-point injection and intraperitoneal injection into the limbs (100. mu.g/ml); after 1 week, the mice were sacrificed and their spleens were removed, and splenocytes were separated by a milling method, and total RNA of mouse splenocytes was prepared by a Trizol (Bilun biosystem) method.
Preparing cDNA by Reverse Transcription (RT) technology; mouse IgG heavy chain coding sequence and light chain coding sequence were synthesized by PCR using cDNA as template and mouse IgG light chain specific primer and heavy chain specific primer, respectively (see sequence: Antibody Engineering, R.Kontermann and S.Dubel, Springer Verlag, Heidelberg, Germany (2010) pp.21-44) (RT and PCR kits from NEB Corp.); connecting the two by using overlapping extension PCR (SOE PCR) technology, wherein a linker (linker) is arranged in the middle, and the two ends are respectively provided with an sfII enzyme cutting site and an NotI enzyme cutting site, and finally forming a PCR product sequence mode as follows: sfiI cleavage site (ggcccagccggcc, SEQ ID NO: 4) -VL encoding fragment (shown as bases 1-342 of SEQ ID NO: 2) -linker encoding fragment (ggtggaggcggttcaggcggaggtggctctggcggtggcggatcg, SEQ ID NO: 5) -VH encoding fragment (shown as bases 388-756 of SEQ ID NO: 2) -NotI cleavage site (gcggccgc, SEQ ID NO: 6).
③ using sfiI and NotI (NEB) to double-cut the PCR product, recovering, then performing ligation reaction with phagemid vector pCANTAB5E (eurobacillus), and electrotransfering the ligation reaction product into competent escherichia coli TG1 (NEB), and culturing overnight, which is the original antibody library. The bacterial culture system is added with auxiliary phage M13K07(NEB company) for co-culture, culture supernatant is collected, and the phage antibody library is obtained after filtration sterilization.
Coating an enzyme-labeled plate hole with brucella antigen LPS, adding phage, washing to remove unbound components after incubation, adding an HRP-labeled phage specific antibody (Abcam company), washing to remove unbound components after incubation, and adding an HRP substrate (Sigma company) for color development (phage ELISA); collecting the phage in the positive hole, and entering the next round of panning; collecting the positive phages after 4 rounds of panning, transferring the positive phages into escherichia coli TG1, spreading the escherichia coli TG1 on an agar plate, and culturing overnight; selecting bacterial clone liquid for culture, extracting plasmids, carrying out DNA sequencing (Shanghai Yingjun biotechnology limited) identification, and selecting correct phagemids for storage.
(3) Preparation of Brucella antigen-specific ScFv expression vector
The ScFv coding fragment in the phagemid is subcloned into a prokaryotic expression vector pET32a (vast Lingzi) by a PCR technology and a DNA ligation reaction, and is provided with a V5 label to prepare a prokaryotic expression plasmid pET32a-Bru-ScFv, and correct clone is selected through DNA double digestion and DNA sequencing identification. The pattern of the protein coding fragment in the plasmid is as follows: ScFv coding fragment-V5 tag coding fragment-His tag coding fragment, DNA sequence (5 '-3') as shown in SEQ ID NO: 2, respectively. The translated protein amino acid sequence mode is ScFv-V5Tag-His Tag, and the amino acid sequence (N end-C end) is shown as SEQ ID NO: 3, showing:
EIVLTQSPVTLSVSPGERATLSCRASQSVIGNLAWYQQKPGQAPRLLIYGASARAAGIPDRFSGSGSGTDFTLTISSLKSEDFAVYYCQQYNNWPPYTFGQGTKVEIKRSGGSTGGGGSGGGGSGGGGSVQLLESGGGLVQPGGSLRLSCAVSGLNLRRYAMSWVRQAPGKGLEWVSVIGASDGNVYYADSVKGRFIISRDNSKDTLDLQMNSLRVEDTAVYYCTKDHWLASWTDEMDVWGQGTLVTVSSASGKPIPNPLLGLDSTHHHHHH(SEQ ID NO：3)
(4) expression, purification and identification of brucella antigen-specific ScFv
Prokaryotic expression plasmid pET32a-Bru-ScFv transformation competent Escherichia coli BL21(DE3) is cultured, 800mg/mL IPTG is cultured by shaking at 28 ℃, and protein expression is induced; collecting bacterial culture supernatant, and purifying by nickel column affinity chromatography to obtain single-chain antibody Bru-ScFv, wherein the purification process is as follows:
BL21(DE3) (NEB) transformed with the recombinant plasmid pET32a-Bru-ScFv was expressed by induction with 800mg/mL IPTG (Sigma), and then centrifuged at 6000rpm to collect cell pellets.
② resuspend the thalli precipitation by using a binding buffer solution (20mmol/L sodium dihydrogen phosphate, pH5.8, analytically pure) of a cation chromatographic column HiTrap SP.F.and then ultrasonically breaking the thalli in ice bath (power 300W, work 10s, gap 10s, repeat 15 times, and complete cycle 3 rounds).
③ collecting the ultrasonic crushing product, centrifuging for 15 minutes at 4 ℃, 10000 rpm, collecting the supernatant, filtering by a 0.45 mu m microporous filter membrane, and collecting the filtrate.
Fourthly, the filtrate is loaded on a column AKTAprime (Amersham company), balanced by combining with a buffer solution, and then linearly eluted by using an eluent (20mmol/L sodium dihydrogen phosphate, 1M NaCl, pH5.8, analytically pure), and a main elution peak is collected.
Diluting the elution peak containing the target protein by using a His-binding buffer solution (20mmol/L sodium dihydrogen phosphate, 0.5M NaCl, pH 7.4 and analytical purity) according to the volume ratio of 1:9, purifying the elution peak again by using a HisTrap HP column, balancing the His-binding buffer solution, eluting by using a His-eluent (20mmol/L sodium dihydrogen phosphate, 0.5M NaCl, 0.5M imidazole, pH 7.4 and analytical purity) by using a 10% buffer solution B to remove non-specifically bound hybrid protein, and washing the target protein by using a 100% buffer solution B after balancing.
Sixthly, protein quantitative determination is carried out by using a BCA (Bluey biology) method, and the purity of the product is identified by using an SDS-PAGE method.
Seventhly, identifying the LPS specific single-chain antibody by an indirect ELISA method: the purified brucella LPS coating diluent is diluted, 100 mu l of the diluted solution is added to each hole of the ELISA plate, and the solution is coated overnight at 4 ℃. Taking out the enzyme label plate the next day, discarding the antigen, and washing the plate. Diluting the single-chain antibody to be identified by 1:1000, 1:2000, 1:4000, 1:8000, 1:16000 and the like, adding 100 mu l/hole into the corresponding hole, and making blank, negative and positive control holes. Incubate at 37 ℃ for 1 hour, wash the plate, add mouse anti-V5 monoclonal antibody (1:2000, from Invitrogen, cat # MA5-15253), incubate at 37 ℃ for 1 hour, wash the plate, add HRP-labeled goat anti-mouse IgG (1:3000, from Invitrogen, cat # 31430), add to the corresponding wells at 100. mu.l/well, incubate at 37 ℃ for 40 minutes. The liquid is discarded, the plate is washed,patting to dry, adding 100 μ l/well ABTS substrate color development solution (Sigma Co.), developing in dark for 10 min, adding stop solution (2M H)2SO4Analytically pure) 50. mu.l/well. The absorbance value was measured at 405nm and the titer of the single chain antibody (maximum dilution with positive results) was judged.
Example 2
This example illustrates the detection of Brucella antibodies in a sample by competitive ELISA
(1) Brucella LPS coated enzyme label plate
24mL of coating solution (NaHCO) is prepared for each ELISA plate3 2.93g，Na2CO31.59g, double distilled water dissolution, total volume 1L, pH 9.6, assay pure), LPS antigen (LPS coating concentration determined by ELISA quantitation and matrix titration data of LPS) was added to 24mL of antigen coating solution, and 200. mu.L of diluted antigen was added to each well. Sealing strips are attached and coated for about 20 hours at 4 ℃.
And secondly, throwing off the antigen, cleaning the ELISA plate for 5 times by clean tap water, tapping on absorbent paper after the washing is finished until no obvious liquid residue exists, and then drying for 18 hours in a dark place.
And thirdly, the high-value positive samples, the medium-value positive samples and the negative samples are used for identifying the coated enzyme label plates in different batches.
And fourthly, the coated enzyme label plate can be used immediately or stored in a sealed manner at room temperature in a dark place for 12 months.
(2) HRP-labeled goat anti-mouse IgG
With NaIO4Firstly oxidizing sugar molecules on the surface of HRP into aldehyde groups, and then combining the aldehyde groups with amino groups of antibody protein, wherein the obtained enzyme-labeled antibody has high yield and comprises the following specific steps:
preparing HRP enzyme: 10mg of HRP enzyme (Sigma Co.) was weighed, and mixed with 2mL of pure water to prepare a 5mg/mL HRP liquid, and then 340. mu.L of HRP enzyme was added thereto in an amount of 34. mu.L/mg of HRP enzyme4After 1 hour, 250. mu.L of ethylene glycol (analytical grade) was added to the HRP enzyme in an amount of 250. mu.L per mg of the HRP enzyme, and after 4 ℃ in the absence of light, the mixture was transferred to a dialysis bag and dialyzed overnight against 1mM acetic acid buffer (pH 4.0-4.4) 2 times, during which the solution was changed, and the light was required.
Preparing an antibody: commercial goat anti-mouse IgG (Zhongshan organism), 0.01M PBS buffer (Biyunnan organism) was purchased and dialyzed overnight at 4 ℃ and the concentration was determined.
Marking: mixing the antibody and HRP enzyme at a mass ratio of 1:1, adding 1M Na2CO3Buffer (1:80, volume ratio, assay pure), adjusting the pH of the reaction system to 9.5, and reacting at 25 ℃ for 2.5 hours.
Fourthly, terminating: fresh preparation of 0.1M NaH4B (4mg/mL, analytical grade), 47. mu.L per mg of HRP enzyme was added. After standing at 4 ℃ for 2 hours, the cells were transferred to a dialysis bag. Dialyzed overnight against 0.01M PBS buffer (pH 7.0-7.2) during which the solution was changed twice and protected from light.
And fifthly, subpackaging and storing: the labeled antibody was aliquoted in brown EP tubes and the antibody titer was determined and stored at-20 ℃ in the dark.
(3) Detection of brucella antibody based on competitive ELISA principle
Taking an enzyme label plate coated with brucella LPS, and balancing to room temperature for later use.
And mixing 100 mu L of appropriately diluted animal serum sample to be detected, positive control sample and negative control sample with 100 mu L of the brucella antigen-specific single-chain antibody prepared in the invention, and incubating for 15 minutes at room temperature.
③ adding the sample-ScFv mixture into an enzyme-labeled plate hole coated with LPS, and incubating for 1 hour at room temperature.
(iv) washing solution (Na)2HPO40.14g, Tween-201 mL, double distilled water to 1L, assay pure) plate wash 5 times, pat dry, add 200. mu.L of mouse anti-V5 monoclonal antibody (1:2000, Invitrogen) per well, incubate for 1 hour at room temperature.
Fifthly, washing the plate for 5 times by using washing liquor, beating to dry, adding goat anti-mouse IgG (1:3000) marked by HRP into each hole, and incubating for 40 minutes at room temperature.
Sixthly, washing the plate for 5 times by using washing liquid, patting the plate dry, adding 100 mu L of ABTS color developing agent mixture into each hole, and incubating the mixture for 10 minutes at room temperature in a dark place; reaction stop solution (2M H) was added to each well2SO4Analytically pure) 50. mu.L.
At 405nm, reading and recording the absorbance value of each hole by using an enzyme-labeling instrument.
And calculating results: the inhibition rate (PI) is (uninhibited control empty absorbance Ac-sample detection hole absorbance As)/uninhibited control empty absorbance multiplied by 100%, the PI is more than or equal to 20% positive, and the maximum dilution multiple showing the positive result is the titer of the Brucella antibody in the sample.
Wherein the Brucella antibody positive mixed bovine serum (positive by tiger red plate agglutination test) is diluted at a ratio of 1:100 to obtain basic sample, and 0 μ L, 50 μ L, 100 μ L, 150 μ L, 200 μ L, 250 μ L and 300 μ L of basic sample are respectively diluted with sample diluent (Na)2HPO4 1.4g,NaCl 7g,KCl 0.2g,KH2PO40.2g, Tween-200.5 mL, 1mL of 1% phenol red, double distilled water to a constant volume of 1L, analytically pure) to a total volume of 600 μ L, the relative contents of brucella antibody in each diluted sample were 0, 50, 100, 150, 200, 250 and 300, the absorbance of each sample at 405nm was measured according to the above steps (i) - (i), the result is shown in fig. 1, and the inhibition ratio was calculated according to the calculation method described in step (i), and the result is shown in fig. 2. As can be seen from FIGS. 1 and 2, there is a quantitative relationship between the absorbance and the inhibition rate and the concentration of the test object, indicating that the single-chain antibody obtained by the present invention can be used for the detection of a brucellosis antibody.
The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention, including combinations of various technical features in any other suitable way, and these simple modifications and combinations should also be regarded as the disclosure of the invention, and all fall within the scope of the invention.
Sequence listing
<110> Chongqing Prospection science and technology Limited of Jilin medical college
<120> Brucella single-chain antibody and use thereof
<141> 2017-12-27
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 252
<212> PRT
<213> Artificial Sequence
<400> 1
Glu Ile Val Leu Thr Gln Ser Pro Val Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ile Gly Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Ala Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Lys Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly
100 105 110
Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Asn Leu Arg Arg Tyr
145 150 155 160
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ser Val Ile Gly Ala Ser Asp Gly Asn Val Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Asp
195 200 205
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Lys Asp His Trp Leu Ala Ser Trp Thr Asp Glu Met Asp Val Trp
225 230 235 240
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser
245 250
<210> 2
<211> 816
<212> DNA
<213> Artificial Sequence
<400> 2
gaaattgtgt tgacacagtc tccagtcacc ctgtctgtgt ctccagggga gagagccacc 60
ctctcctgca gggccagtca gagtgttatc ggcaacttag cctggtacca gcagaaacct 120
ggccaggctc ccaggctcct catctatggt gcatccgcca gggccgccgg catcccagac 180
aggttcagtg gcagtgggtc tgggacagac ttcactctca ccatcagcag cctgaagtct 240
gaagattttg cagtttatta ctgtcagcag tataataact ggcctccgta cacttttggc 300
caggggacca aggtggaaat caaacgttcc ggagggtcga ccggtggagg cggttcaggc 360
ggaggtggct ctggcggtgg cggatcggtg cagctgttgg agtctggggg aggcttggta 420
cagcctgggg ggtccctgag actctcctgt gcagtctctg gactcaacct tagaagatat 480
gccatgagtt gggtccgcca ggctccaggg aaggggctgg agtgggtctc ggtaataggt 540
gccagtgatg gaaatgtcta ctacgctgac tccgtgaagg gccgattcat catctcaaga 600
gacaattcca aggacacgtt ggatctgcaa atgaacagcc tgagagtcga ggacacggcc 660
gtatattact gtacgaaaga tcattggctc gccagttgga ccgacgagat ggacgtctgg 720
ggccagggca ccctggtcac tgtctcctca gctagcggca aaccaatccc aaacccactg 780
ctgggcctgg atagtactca ccatcaccat caccat 816
<210> 3
<211> 272
<212> PRT
<213> Artificial Sequence
<400> 3
Glu Ile Val Leu Thr Gln Ser Pro Val Thr Leu Ser Val Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ile Gly Asn
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Ala Ser Ala Arg Ala Ala Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Lys Ser
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asn Trp Pro Pro
85 90 95
Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Ser Gly Gly
100 105 110
Ser Thr Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
115 120 125
Ser Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
130 135 140
Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Leu Asn Leu Arg Arg Tyr
145 150 155 160
Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
165 170 175
Ser Val Ile Gly Ala Ser Asp Gly Asn Val Tyr Tyr Ala Asp Ser Val
180 185 190
Lys Gly Arg Phe Ile Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Asp
195 200 205
Leu Gln Met Asn Ser Leu Arg Val Glu Asp Thr Ala Val Tyr Tyr Cys
210 215 220
Thr Lys Asp His Trp Leu Ala Ser Trp Thr Asp Glu Met Asp Val Trp
225 230 235 240
Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Gly Lys Pro Ile
245 250 255
Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr His His His His His His
260 265 270
<210> 4
<211> 13
<212> DNA
<213> Artificial Sequence
<400> 4
ggcccagccg gcc 13
<210> 5
<211> 45
<212> DNA
<213> Artificial Sequence
<400> 5
ggtggaggcg gttcaggcgg aggtggctct ggcggtggcg gatcg 45
<210> 6
<211> 8
<212> DNA
<213> Artificial Sequence
<400> 6
gcggccgc 8
Claims (9)
1. A Brucella single-chain antibody comprises a light chain variable region, a heavy chain variable region and a joint for connecting the light chain variable region and the heavy chain variable region, and is characterized in that the sequence of the light chain variable region is shown as SEQ ID NO: 1, and the sequence of the heavy chain variable region is shown as SEQ ID NO: 1 at amino acids 130-252.
2. The single chain antibody of claim 1, wherein the sequence of the single chain antibody is as set forth in SEQ ID NO: 1 is shown.
3. A single chain antibody according to claim 1 or 2, wherein the sequence of the single chain antibody consists of SEQ ID NO: 1 and a sequence linked to SEQ ID NO: 1 and a V5tag and a His tag at the C terminal, and the sequence is shown as SEQ ID NO: 3, respectively.
4. A gene encoding the single chain antibody of any one of claims 1 to 3.
5. The gene of claim 4, wherein the sequence of the gene is shown in SEQ ID NO: 2, respectively.
6. An expression vector comprising the gene of claim 4 or 5.
7. A host cell transformed with the expression vector of claim 6.
8. A kit for detecting a Brucella antibody, characterized by comprising: the single chain antibody of any one of claims 1 to 3.
9. A method for detecting brucella antibodies in a sample for non-diagnostic purposes, comprising performing a competitive ELISA assay on the sample using a single chain antibody according to any one of claims 1 to 3.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711457652.8A CN108359009B (en) | 2017-12-28 | 2017-12-28 | Brucella single-chain antibody and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711457652.8A CN108359009B (en) | 2017-12-28 | 2017-12-28 | Brucella single-chain antibody and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN108359009A CN108359009A (en) | 2018-08-03 |
| CN108359009B true CN108359009B (en) | 2021-06-04 |
Family
ID=63010396
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711457652.8A Active CN108359009B (en) | 2017-12-28 | 2017-12-28 | Brucella single-chain antibody and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN108359009B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108433A2 (en) * | 1999-12-15 | 2001-06-20 | The Minister Of National Defence Of Her Majesty's Canadian Government | Combination vaccine for enhancing immunity against brucellosis |
| JP2008222590A (en) * | 2007-03-09 | 2008-09-25 | Obihiro Univ Of Agriculture & Veterinary Medicine | Method for preventing miscarriage and sterility by brucella infection |
| CN102532271A (en) * | 2011-12-14 | 2012-07-04 | 南方医科大学 | Brucella B cell epitope and monoclonal antibody and application thereof |
| CN103864928A (en) * | 2012-12-13 | 2014-06-18 | 中国农业科学院哈尔滨兽医研究所 | Preparation of common antigen monoclonal antibody of Brucella sLPS (lipopolysaccharides) and establishment of c-ELISA (competitive enzyme-linked immuno sorbent assay)method |
| CN104086628A (en) * | 2014-06-11 | 2014-10-08 | 南方医科大学 | Monoclonal antibody of brucella omp31 protein and application thereof |
-
2017
- 2017-12-28 CN CN201711457652.8A patent/CN108359009B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1108433A2 (en) * | 1999-12-15 | 2001-06-20 | The Minister Of National Defence Of Her Majesty's Canadian Government | Combination vaccine for enhancing immunity against brucellosis |
| JP2008222590A (en) * | 2007-03-09 | 2008-09-25 | Obihiro Univ Of Agriculture & Veterinary Medicine | Method for preventing miscarriage and sterility by brucella infection |
| CN102532271A (en) * | 2011-12-14 | 2012-07-04 | 南方医科大学 | Brucella B cell epitope and monoclonal antibody and application thereof |
| CN103864928A (en) * | 2012-12-13 | 2014-06-18 | 中国农业科学院哈尔滨兽医研究所 | Preparation of common antigen monoclonal antibody of Brucella sLPS (lipopolysaccharides) and establishment of c-ELISA (competitive enzyme-linked immuno sorbent assay)method |
| CN104086628A (en) * | 2014-06-11 | 2014-10-08 | 南方医科大学 | Monoclonal antibody of brucella omp31 protein and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| "Development of an improved competitive ELISA based on a monoclonal antibody against lipopolysaccharide for the detection of bovine brucellosis";Xiaolei Wang et al.;《BMC Veterinary Research》;20150521;第11卷;第1-9页 * |
| 光滑型布鲁氏菌LPS单克隆抗体的制备及鉴定;王加兰 等;《中国预防兽医学报》;20090831;第31卷(第8期);第642-645、649页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN108359009A (en) | 2018-08-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US6030792A (en) | Assays for measurement of protein fragments in biological media | |
| CN111153991A (en) | Human SARS-CoV-2 monoclonal antibody and its preparation method and use | |
| JP2016512044A (en) | Protocol for identification and isolation of antigen-specific B cells and production of antibodies against the desired antigen | |
| CN109678958B (en) | Human NT-proBNP specific recombinant goat monoclonal antibody, and preparation method and application thereof | |
| CN111793132A (en) | Monoclonal antibody of human procalcitonin and preparation method and application thereof | |
| WO2015089881A1 (en) | Human anti-cd26 antibody and application thereof | |
| CN114276445B (en) | Rotavirus recombinant protein specific antibody, plasmid vector and method | |
| CN109593130B (en) | Hepatitis B virus pre-S1 antigen specific single-chain antibody and application thereof | |
| CN108359009B (en) | Brucella single-chain antibody and application thereof | |
| KR101241667B1 (en) | Novel peptide tag and uses thereof | |
| CN116284352A (en) | Bovine leukemia virus antibody and detection kit | |
| CN110872354A (en) | Chicken-derived monoclonal antibody and single-chain antibody of mammal cell recombinant anti-human TK1, and preparation method and application thereof | |
| CN113004397B (en) | Antibodies that specifically bind to novel coronavirus NP proteins | |
| CN112979790B (en) | Antibodies and use in detecting novel coronaviruses | |
| CN113621079A (en) | Fusion protein of Fab antibody and calf intestinal alkaline phosphatase and preparation method thereof | |
| CN110903359A (en) | Preparation of campylobacter jejuni recombinant protein and monoclonal antibody thereof | |
| CN116987194B (en) | Anti-idiotype nano antibody of mimic epitope peptide of human ST2 antigen and application thereof | |
| CN110078820A (en) | Bovine leukemia virus antibody and detection kit | |
| CN110128512B (en) | Antigenic determinants and uses thereof | |
| CN117886891B (en) | Polypeptide targeting macrophage CD169 receptor and application thereof | |
| CN110054675B (en) | Immunogenic polypeptide, anti-TTC 36 antibody CP4-3 and application | |
| CN112979792B (en) | Antibodies against novel coronaviruses, encoding nucleic acids, vectors, host cells, derivatives and uses thereof | |
| KR102511816B1 (en) | c-Myc peptide specific antibody and uses thereof | |
| CN112898415B (en) | Antibody for detecting novel coronavirus and detection kit | |
| CN110054676B (en) | Immunogenic polypeptide, anti-TTC 36 antibody AP3-5 and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| CB03 | Change of inventor or designer information |
Inventor after: Cai Jianhui Inventor after: Wang Huiyan Inventor after: Liu Lei Inventor after: Fang Fang Inventor after: Hao Feng Inventor after: Li Yan Inventor after: Liu Jing Inventor after: Xu Song Inventor after: Qin Yili Inventor before: Cai Jianhui Inventor before: Li Yan Inventor before: Wang Huiyan Inventor before: Fang Fang Inventor before: Hao Feng Inventor before: Liu Jing Inventor before: Xu Song Inventor before: Qin Yili |
|
| CB03 | Change of inventor or designer information | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |