CN116375852B - Recombinant fluorescent nano antibody and application thereof in preparation of rabies virus detection reagent - Google Patents
Recombinant fluorescent nano antibody and application thereof in preparation of rabies virus detection reagent Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/08—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses
- C07K16/10—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from viruses from RNA viruses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- G01N33/56983—Viruses
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- 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
- C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/20—Fusion polypeptide containing a tag with affinity for a non-protein ligand
- C07K2319/21—Fusion polypeptide containing a tag with affinity for a non-protein ligand containing a His-tag
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/60—Fusion polypeptide containing spectroscopic/fluorescent detection, e.g. green fluorescent protein [GFP]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/185—Escherichia
- C12R2001/19—Escherichia coli
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/005—Assays involving biological materials from specific organisms or of a specific nature from viruses
- G01N2333/08—RNA viruses
- G01N2333/145—Rhabdoviridae, e.g. rabies virus, Duvenhage virus, Mokola virus or vesicular stomatitis virus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention relates to a recombinant fluorescent nano antibody and application thereof in preparation of rabies virus detection reagent, belonging to the technical field of biological molecular diagnosis. The nano antibody with fluorescent protein mark obtained by prokaryotic expression and purification is used for replacing the fluorescein mark antibody to be applied to the direct immunofluorescence Detection (DFA) of rabies virus.
Description
Technical Field
The invention relates to the technical field of biological molecular diagnosis, in particular to a recombinant nano antibody containing a green fluorescent protein label, which can be used for direct immunofluorescence detection of rabies virus antigen.
Background
Rabies (rabies) is a virulent zoonotic infectious disease caused by highly neurotropic rabies virus (RABV), and the death rate is up to 100%. Currently, the gold standard for rabies etiology detection is the World Health Organization (WHO) and world animal health organization (WOAH) approved gold standard for rabies virus detection is the direct immunofluorescence method (direct fluorescent antibody test, DFA), which allows rapid, sensitive, specific detection of viral antigens in human and animal brain tissues. However, this method still has some drawbacks in that it requires the use of Fluorescein Isothiocyanate (FITC) coupled labeled RABV-specific fluorescent antibodies, which can vary in specificity and background from lot to lot or from manufacturer to manufacturer during the production and preparation of fluorescent antibodies, and in that it is difficult to formulate standardized DFA protocols using different conjugates and different conjugate dilutions in different laboratories. In addition, commercially available FITC-labeled anti-RABV nucleoprotein monoclonal antibodies are very expensive, difficult to purchase, long in shelf life, and high in storage condition requirements, which further limit the popularization of DFA. At present, the fluorescent antibody used for rabies detection has high cost, high requirement on storage conditions and other problems, and innovation of a rabies detection method is urgent to be promoted, so that an economic and efficient diagnosis product is developed.
Disclosure of Invention
In order to solve the problems of high cost, difficult purchase, high requirement on storage conditions and the like of the fluorescent antibody used for detecting rabies at present, the invention aims to prepare the recombinant nano fluorescent antibody which can be directly used for detecting rabies virus antigen through a prokaryotic expression mode. G protein nano antibody with stronger rabies virus neutralization capacity is selected as a modification object through screening, an Enhanced Green Fluorescent Protein (EGFP) gene and 6 histidine (6 XHis) tags are connected at the C end of the G protein nano antibody, the antibody fusion protein gene is cloned to a prokaryotic expression vector pMal-p5x, and the expression is carried out through prokaryotic expression engineering bacteria Shuffle T7-B. The nano antibody with fluorescent protein mark obtained by prokaryotic expression and purification is used for replacing the fluorescein mark antibody to be applied to the direct immunofluorescence Detection (DFA) of rabies virus.
In order to achieve the above purpose, the invention adopts the following specific scheme:
in a first aspect, a nanobody that binds rabies virus G protein, the nanobody having an amino acid sequence as set forth in SEQ ID NO:02, the nucleotide sequence is shown as SEQ ID NO: 01.
In a second aspect, a recombinant fluorescent nanobody comprises the following elements:
(a) The amino acid sequence combined with rabies virus G protein is shown as SEQ ID NO:02, a nanobody shown in the specification;
(b) A green fluorescent protein EGFP gene connected to the C end of the nano antibody through a flexible peptide GGGGS multiplied by 3;
(c) A 6 XHis tag attached at the end.
In a third aspect, the method for constructing the recombinant fluorescent nanobody includes the following steps:
step one, screening VHH fragments of rabies virus G protein nanobody: cloning a G protein gene of rabies virus CVS-11 onto a pGBKT7 vector to construct a plasmid pGBKT7-RABV-JM-G, taking the plasmid pGBKT7-RABV-JM-G as a bait, screening a nanobody VHH fragment combined with rabies virus G protein in a yeast two-hybrid nanobody library, and screening to obtain a nanobody VHH fragment RABV-Nb04 with an amino acid sequence shown in SEQ ID NO:02, the nucleotide sequence is shown as SEQ ID NO:01 is shown in the figure;
step two, constructing a recombinant plasmid pMal-RABVNb-EGFP: c end of RABV-Nb04 obtained in the first step is connected with a flexible peptide GGGGS multiplied by 3 and green fluorescent protein gene EGFP, 6 XHis and stop codon are added at the tail end to synthesize a gene fragment RABVNb-EGFP, enzyme cutting sites of Nco I and Hind III are introduced at the two ends, and the gene fragment RABVNb-EGFP is inserted into pMal-p5x plasmid to construct recombinant plasmid pMal-RABVNb-EGFP;
step three, expression and purification of fusion protein: and (3) converting the pMal-RABVNb-EGFP obtained in the step (II) into SHuffleT7 competent bacteria for expression and purification to obtain the recombinant fluorescent nanobody.
In a fourth aspect, a gene encoding the recombinant fluorescent nanobody has a nucleotide sequence as set forth in SEQ ID NO: shown at 03.
In a fifth aspect, an expression vector comprising the above gene is a pMal-p5x plasmid.
In a sixth aspect, a host cell comprising the expression vector described above is E.coli SHuffleT7.
In a seventh aspect, the use of the nanobody or recombinant fluorescent nanobody described above for the preparation of a rabies detection reagent.
In an eighth aspect, a rabies detection kit comprises the recombinant fluorescent nanobody described above.
Compared with the prior art, the invention has the following advantages:
1. the recombinant fluorescent nano antibody has small molecular weight and simple structure, can be prepared by using prokaryotic expression systems such as escherichia coli and the like, and greatly reduces the cost.
2. The green fluorescent protein is directly fused with the nano antibody for expression, and compared with the traditional fluorescent antibody, the fluorescent protein does not need to use fluorescein for post-labeling, thereby further saving the cost and the preparation process.
3. The recombinant fluorescent nano antibody obtained by the invention detects the G protein of the RABV virus, and the protein can be expressed on the surface of an infected cell, so that a cell sample is not required to be fixed during a cell immunofluorescence test, the detection is directly carried out, and the test operation process is simplified.
Drawings
FIG. 1 is a graph of the results of direct immunofluorescence assays of recombinant fluorescent nanobodies on immobilized samples; in the figure: a: commercialized fluorescent antibodies; b: RABVNb-EGFP;
FIG. 2 is a graph of the results of direct immunofluorescence assays of recombinant fluorescent nanobodies on non-immobilized samples; in the figure, a: commercialized fluorescent antibodies; b: RABVNb-EGFP.
Detailed Description
The invention screens out nanometer antibody with strong binding force with rabies virus G protein by utilizing a yeast two-hybrid system, connects and Enhances Green Fluorescent Protein (EGFP) gene and 6 histidine (His) labels at the C end of the nanometer antibody, clones the antibody fusion protein gene onto a prokaryotic expression vector pMal-p5x, and expresses the antibody fusion protein gene by prokaryotic expression engineering bacteria Sheffle T7-B. The nano antibody with fluorescent protein mark obtained by prokaryotic expression and purification is used for replacing the fluorescein mark antibody to be applied to the direct immunofluorescence Detection (DFA) of rabies virus.
The technical scheme of the invention will be clearly and completely described in the following in connection with the embodiments of the invention.
1. And (3) screening VHH fragments of rabies virus G protein nanobodies.
Cloning a G protein gene of rabies virus CVS-11 onto a pGBKT7 vector to construct a plasmid pGBKT7-RABV-JM-G, taking the plasmid pGBKT7-RABV-JM-G as a bait, and screening a nanobody VHH fragment combined with rabies virus G protein from a yeast two-hybrid nanobody library (artificially synthesized library).
1.1 self-activation detection
RABV-JM-G, pGBKT7 was transformed into AH109, smeared onto SD/-Trp plates and incubated at 30℃for 3-4d. 6 spots were randomly picked and PCR verified with pGBKT7 vector primers, all were correctly cloned, three cloning spots were randomly picked onto SD/-Trp, SD/-Trp/-His/-Ade, SD/-Trp/-His/-Ade+X-alpha-gal plates and incubated at 30℃for 3-5d. Wherein pGBKT7 is the negative control.
1.2 library DNA transformation
(1) Single strain was picked from SD-T plate and inoculated into 50mL of liquid SD-T medium at 30℃and 225rpm, followed by shaking culture for 24 hours.
(2) The cells were transferred to 500mL of YPDA liquid, and the cells were cultured with shaking at 30℃and 225rpm for 4-5 hours at an initial OD 600=0.2 to an OD 600=0.6.
(3) The bacteria were harvested by centrifugation at 4000rpm for 5min at room temperature.
(4) The cells were resuspended in 30mL of sterile water, mixed well, harvested by centrifugation, room temperature, 4000rpm,5min, and the supernatant discarded.
(5) The cells were resuspended in 20ml of 0.1M LiAc, mixed well, harvested by centrifugation, at room temperature, 4000rpm,5min and the supernatant discarded.
(6) The cells were resuspended in 10mL of 0.1M LiAc, mixed, harvested by centrifugation, at room temperature, 4000rpm,5min, and the supernatant discarded.
(7) Sequentially adding the following reagents into the centrifuge tube, and blowing and mixing by using a gun head, or shaking vigorously for about 1min until the reagents are completely mixed.
(8) Incubated in a water bath at 30 ℃ for 30min.
(9) And (5) carrying out heat shock in a water bath at 42 ℃ for 25min.
(10) Resuscitates in a water bath at 30 ℃ for 1h.
(11) The cells were collected by centrifugation at room temperature at 4000rpm for 5min, the supernatant was discarded, the cells were resuspended in 6mL of sterile water and mixed as gently as possible, and 20. Mu.L of the culture was diluted therefrom and applied to SD-TL plates for detection of library transformation efficiency. The rest is coated with SD-TLH, and the total number of the blocks is 20.
(12) Culturing at 30 deg.c for 3-7d to observe colony growth.
(13) The single colony of the clone was picked up and transferred to SD-TLHA+X-alpha-gal screening plate for further culture for 3-5d.
1.3 screening library results
Positive yeast clones were obtained by screening in SD-TLHA+X-alpha-gal screening plates, and 100 clones were picked up from the plates for PCR verification. To identify what genes are the positive clones screened in SD-TLHA+X-alpha-gal plates, respectively, these positive clones need to be amplified from yeast cells for DNA sequencing and BLAST comparison analysis. 100 positive yeast clones are amplified by all PCR, sequenced, and finally 5 gene sequences are obtained through Seqman and BLAST comparison.
1.4 Yeast Positive cloning rotation verification
Positive clones grown on SD-TLHA+X-alpha-gal defective plates were streaked with sterile water and then spotted onto SD-TL, SD-TLH, SD-TLHA, SD-TLHA+X-alpha-gal defective plates, respectively, and incubated at 30℃for 3-4 days, respectively. The 5 positive yeast clones obtained by screening can grow normally on SD-TL, SD-TLH, SD-TLHA and SD-TLHA+X-alpha-gal defect plates, and can develop blue on SD-TLHA+X-alpha-gal plates.
1.5 determination of the neutralizing Capacity of VHH Virus
After 5 selected VHHs were expressed by yeast purification, RABV virus neutralization assay was performed and the measured neutralizing antibody titers are as follows:
TABLE 1
We selected RABV-Nb04 as the object of modification of recombinant fluorescent nanobody, and the nucleic acid sequence of RABV-Nb04 after optimization according to the codon preference of Escherichia coli is as follows:
>RABV-Nb04 DNA sequence(SEQ ID NO：01)
CAAGTACAGCTACAAGAATCAGGTGGAGGGTTGGTTCAGGCGGGTGGCTCTT TACGTCTGAGCTGTGCCGCAAGCGGCACCATCTTTCGTTATGGTGTTGGTGGTTGGTATCGTCAAGCGCCGGGCAAAGAGAGAAGCGTCGATTGGTCCGGCAGCCGCACTTATTACGCGGACTCTGTGAAGGGCGCGGACTCGGTGAAGGGTCGCTTCACCATCAGCCGTGATAATGCAAAAAACACCGTGTACCTGCAAATGAACAGCCTGAAACCGGAAGATACCGCTGTGTACTACTGCGATAGTTCCGTAGTTCCGGGTATTGAGAAGTACGACGACTGGGGTCAGGGCACGCAGGTTACCGTCAGCTCC
the translated amino acid sequence is as follows:
>RABV-Nb04 amino acid sequence(SEQ ID NO：02)
QVQLQESGGGLVQAGGSLRLSCAASGTIFRYGVGGWYRQAPGKERSVDWSGSR TYYADSVKGADSVKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCDSSVVPGIEKYDD WGQGTQVTVSS
2. construction of recombinant plasmid pMal-RABVNb-EGFP
The RABV-Nb04 gene fragment is optimized according to the preference of the escherichia coli codon by the Souzhou Jin Weizhi biotechnology limited company, a flexible peptide GGGGS multiplied by 3 and a green fluorescent protein gene EGFP are connected at the C end of the RABV-Nb04 gene fragment, and a 6 XHis and a stop codon are added at the tail end of the RABV-Nb04 gene fragment. And (3) synthesizing a gene fragment RABVNb-EGFP, introducing enzyme cutting sites of Nco I and HindIII at two ends, and inserting the gene fragment into a pMal-p5x plasmid to construct a recombinant plasmid pMal-RABVNb-EGFP. pMal-RABVNb-EGFP was transformed into SHuffleT7 competent bacteria.
>RABVNb-EGFP DNA sequence(SEQ ID NO：03)
CAAGTACAGCTACAAGAATCAGGTGGAGGGTTGGTTCAGGCGGGTGGCTCTT TACGTCTGAGCTGTGCCGCAAGCGGCACCATCTTTCGTTATGGTGTTGGTGGTTGGTATCGTCAAGCGCCGGGCAAAGAGAGAAGCGTCGATTGGTCCGGCAGCCGCACTTATTACGCGGACTCTGTGAAGGGCGCGGACTCGGTGAAGGGTCGCTTCACCATCAGCCGTGATAATGCAAAAAACACCGTGTACCTGCAAATGAACAGCCTGAAACCGGAAGATACCGCTGTGTACTACTGCGATAGTTCCGTAGTTCCGGGTATTGAGAAGTACGACGACTGGGGTCAGGGCACGCAGGTTACCGTCAGCTCCGGATCCGCGGCCGCGGGCGGTGGCGGCAGTGGCGGTGGCGGCAGTGGCGGTGGCGGCAGCGTGAGCAAAGGCGAAGAACTGTTTACCGGCGTGGTGCCGATTCTGGTGGAACTGGATGGCGATGTGAACGGCCATAAATTTAGCGTGAGCGGCGAAGGCGAAGGCGATGCGACCTATGGCAAACTGACCCTGAAATTTATTTGCACCACCGGCAAACTGCCGGTGCCGTGGCCGACCCTGGTGACCACCCTGACCTATGGCGTGCAGTGCTTTAGCCGCTATCCGGATCATATGAAACAGCATGATTTTTTTAAAAGCGCGATGCCGGAAGGCTATGTGCAAGAACGCACCATTTTTTTTAAAGATGATGGCAACTATAAAACCCGCGCGGAAGTGAAATTTGAAGGCGATACCCTGGTGAACCGCATTGAACTGAAAGGCATTGATTTTAAAGAAGATGGCAACATTCTGGGCCATAAACTGGAATATAACTATAACAGCCATAACGTGTATATTATGGCGGATAAACAGAAAAACGGCATTAAAGTGAACTTTAAAATTCGCCATAACATTGAAGATGGCAGCGTGCAGCTGGCGGATCATTATCAGCAGAACACCCCGATTGGCGATGGCCCGGTGCTGCTGCCGGATAACCATTATCTGAGCACGCAGAGCGCGCTGAGCAAAGATCCGAACGAAAAACGCGATCATATGGTGCTGCTGGAATTTGTGACCGCGGCGGGCATTACCCTGGGCATGGATGAACTGTATAAACATCACCATCACCATCATTAA。
3 expression and purification of fusion proteins
3.1 expression of fusion proteins
(1) The preserved positive SHuffleT 7-expressing bacteria were removed from the refrigerator, inoculated into 10mL EP tube, and 5mL Amp was added + LB culture medium is placed on a constant temperature shaking table at 30 ℃ for culturing for 12-16 h at 200 r/min. As a first seed liquid.
(2) Inoculating 100 μL of the primary seed solution into 5mL of Amp + LB culture medium, continuing to culture at the constant temperature of 30 ℃ and 200r/min for 12-16 h.
(3) Adding 1-5% Amp into 1L TB culture medium, adding 10mL of cultured secondary seed solution, and culturing at 30deg.C with a constant temperature shaking table at 200r/min for 3-4 hr until bacterial liquid OD 600 After reaching about 0.5, 10mL of 0.1mol/L IPTG was added to induce expression for 24 hours.
(4) And taking out the bacterial liquid after 1d, pouring the bacterial liquid into 6 50mL centrifuge tubes, centrifuging for 25min at 4 ℃ at 5000r/min, discarding the supernatant, and continuously pouring the bacterial liquid for repeated operation until all bacterial liquids are collected.
(5) The enriched cells were resuspended in 20mL bs or BufferA to prepare a total of 3 tubes of 20mL per tube suspension.
(6) An ice box or a 1L beaker is added with an ice water mixture, a bacterial suspension is inserted into ice, and an ultrasonic probe is placed in an ultrasonic crusher at a distance of 1-3 cm and 300w from the bottom of a tube, and is subjected to ultrasonic treatment for 3s and 5s for 30min. The distance between the probe and the bottom of the tube is noted in the ultrasonic process, so that the probe is prevented from contacting the tube wall. Optionally adding lysate.
(7) After ultrasonication, it was observed whether the suspension became transparent and clear, and the suspension was centrifuged at 5000r/min at 4℃for 30min, and the supernatant was filtered through a 0.22 μm filter to prepare a stock solution.
3.2 purification of fusion proteins
(1) Placing the filtered stock solution in ice, and adding HisTrap TM HP (5 mL) affinity column was connected to GE AKTA pure chromatograph or NGC Quest 10Plus chromatograph (with advanced flushing system) to check if the loading well was connected correctly and if gas was in the feed tube.
(2) Pa is less than or equal to 0.3,3mL/min, 30mL of 20% ethanol is introduced to wash the chromatographic column, and 30mL of ultrapure water is introduced to wash the ethanol.
(3) Pa is less than or equal to 0.3,3mL/min, a 30mL BufferA equilibrium chromatographic column is introduced, the ultraviolet absorption peak value is observed, and the ultraviolet absorption peak value is zeroed after being leveled.
(4) Pa is less than or equal to 0.3,2mL/min, and the sample is loaded by the pump A until all samples are sucked.
(5) Pa is less than or equal to 0.3,3mL/min, 30mL Buffer A is introduced to balance the chromatographic column again, and the mixed protein and the protein with weak binding force are washed away.
(6) Pa is less than or equal to 0.3,2mL/min, the liquid inlet pipe of the pump A is arranged in the Buffer A solution, the liquid inlet pipe of the pump B is arranged in the Buffer B solution, and a linear elution program of 0-100% of Buffer B and 50mL is arranged. The collection tubes are placed in sequence into collection wells (NGC Fraction Collector).
(7) And identifying the collected protein liquid according to the elution peak of the linear elution profile.
(8) After the linear elution procedure is finished, 30mL of 100% Buffer solution is continuously introduced, and Pa is less than or equal to 0.3 and 3mL/min.
(9) Pa is less than or equal to 0.3,3mL/min, and 30mL of ultra-pure water is introduced.
(10) Pa is less than or equal to 0.3,3mL/min, 20 percent ethanol is introduced into a sealed chromatographic column, and the chromatographic column is taken down and stored at 4 ℃.
(11) The dialysis bag (10 kDa) was boiled with ultra pure water for 10min, optionally with Na2CO3 or NaHCO3.
(12) Adding the protein solution of the collecting pipe corresponding to the elution peak into a dialysis bag, clamping the two ends, adding 900mL of dialysis Buffer into a 1L beaker, dialyzing for 6h by using a magnetic stirrer at the temperature of 4 ℃ and 300r/min in a biological refrigerated cabinet, and continuing to dialyze for 6h by replacing a new dialysis Buffer.
(13) The dialysis Buffer in the beaker was poured out, 900mL of PBS Buffer solution was added for further dialysis for 6 hours, and the operation was repeated once.
(14) The dialyzed crude protein was filtered through a 0.22 μm filter, and Factor Xa protease (Factor Xa) was added at 0.5. Mu.L/mL, followed by cleavage at 23℃for 12 to 16 hours.
(15) And (3) placing the protein liquid after enzyme digestion into a centrifuge, and centrifuging for 10min at 4 ℃ at 5000 r/min.
(16) Amylose resin was packed into a gravity column having a column volume of 3mL, and 6 times the column volume of ultrapure water was introduced.
(17) The Column was flushed with 5 Column volumes of Column Buffer A.
(18) Adding the centrifuged supernatant into a gravity column, and collecting the flow-through liquid until the supernatant flows through.
(19) Adding 5 times of Column volume Column buffer B, and collecting eluent.
(20) 3 volumes of ultrapure water, 3 volumes of 0.1% SDS,1 volume of ultrapure water, and 5 volumes of 20% ethanol were added in this order. Pouring out the filler and preserving at 4 ℃.
(21) Purified recombinant proteins were quantified using BCA protein quantification kit.
4. Application of recombinant fluorescent nano antibody
4.1 recombinant fluorescent nanobody RABVNb-EGFP for direct fluorescent antibody Detection (DFA)
(1) Two sets of direct immunofluorescence experiments were designed with and without 80% acetone fixation.
(2) Preparing BHK cells, culturing in a culture dish until the BHK cells are single-layered, removing supernatant, washing with PBS for 1 time, adding 1mL of pancreatin for digestion for 45-50 s until the cells are loose, removing pancreatin, adding 3mL of complete culture medium for blowing to disperse, calculating the required cell dilution according to the number of plates, adding the culture medium for dilution, taking a 96-well plate, adding the prepared cell liquid into the plate by a discharge gun, and adding 100 mu L of cell liquid into each hole. After plating, cells were observed under a microscope for uniformity and proper density. Then placed in an incubator at 37℃overnight. The diluted virus solution was added to the wells with a gun, 100. Mu.L of diluted virus solution per well, and then placed in an incubator at 37℃for 48 hours.
(3) After 48h of CVS-11 virus infection of BHK cells, direct immunofluorescence experiments were performed with commercial RABV antibody (FITC Anti-Rabies Monoclonal Globulin, available from Fujirebio Diagnostics, USA) and RABVNb-EGFP after fixation with 80% acetone.
The results show that the fluorescent spots of the recombinant fluorescent nanobody RABVNb-EGFP are larger and dispersed compared with the commercial fluorescent antibody after the CVS-11 infected cells are fixed by detecting the sample, and the bright specific fluorescent spots can be seen (figure 1).
(4) After 48h infection of BHK cells with CVS-1 virus, direct immunofluorescence experiments were performed using commercial RABV antibodies, RABVNb-EGFP, on 96-well plates without 80% acetone fixation. The results showed that commercial fluorescent antibodies failed to see green foci and that RABVNb-EGFP observed bright, specific green spots of fluorescence (fig. 2).
4.2RABVNb-EGFP application to RABV Virus titre determination
(1) Taking cells with good growth state, digesting into single cells by pancreatin, and obtaining the cell density of 2×10 6 Each of the cells was spread on a 96-well cell plate with 100. Mu.L of each well, and placed at 37℃with 5% CO 2 Culturing in a cell culture incubator. After the cells adhere, the virus solution to be detected is diluted by a multiple ratio (10 -1 ～10 -8 ) 100. Mu.L of diluted virus solution was added to 96-well cell plates along the walls, 4 wells were repeated for each dilution, and the wells were placed in a 5% CO2 cell incubator at 37℃for 48 hours.
(2) After 48h, the 96-well plate was removed, the solution in the wells was discarded, 100 μl of pre-chilled 80% acetone was added to each well, and immediately discarded. 100. Mu.L of pre-chilled 80% acetone was then added and the mixture was placed in a-20℃refrigerator for 30min or overnight at 4 ℃. After fixation, the plates were removed, acetone was discarded, 200. Mu.L of pre-chilled PBS buffer was added to each well, gently shaken, discarded, and the wash repeated 3 times. Taking diluted fluorescent antibody, adding 100 mu L of the diluted fluorescent antibody into each hole, incubating for 1h, discarding liquid in the holes, and washing for 3 times by 200 mu L of PBS buffer solution.
(3) Observation results: and observing the rabies virus fluorescence oven under a fluorescence microscope, recording the maximum dilution of the fluorescent hole and the number of the fluorescent oven at the secondary dilution, and calculating the virus titer according to a Reed-Muench method. The results show (Table 2) that the prokaryotic expression recombinant fluorescent nanobody RABVNb-EGFP can be used for detecting the titer of RABV virus, and the effect is more sensitive than that of commercial fluorescent antibody.
TABLE 2RABVNb-EGFP detection of viral titres
It should be noted that the above-mentioned embodiments are to be understood as illustrative, and not limiting, the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made to the present invention without departing from its spirit or scope.
Claims (9)
1. A nanobody that binds rabies virus G protein, characterized in that: the amino acid sequence of the nano antibody is shown as SEQ ID NO:02, the nucleotide sequence is shown as SEQ ID NO: 01.
2. A recombinant fluorescent nanobody, characterized in that: comprising the following elements:
(a) The amino acid sequence combined with rabies virus G protein is shown as SEQ ID NO:02, a nanobody shown in the specification;
(b) A green fluorescent protein EGFP gene connected to the C end of the nano antibody through a flexible peptide GGGGS multiplied by 3;
(c) A 6 XHis tag attached at the end.
3. A gene encoding the recombinant fluorescent nanobody of claim 2, which has a nucleotide sequence as set forth in SEQ ID NO: shown at 03.
4. A recombinant expression vector comprising the gene according to claim 3.
5. The expression vector of claim 4, wherein: the recombinant expression vector is constructed by introducing the gene of claim 3 into a pMal-p5x plasmid.
6. A host cell comprising the recombinant expression vector of claim 4.
7. The host cell of claim 6, wherein: the host cell is escherichia coli SHuffleT7.
8. Use of the nanobody of claim 1 or the recombinant fluorescent nanobody of claim 2 for the preparation of a rabies detection reagent.
9. A rabies detection kit, characterized in that: comprising the recombinant fluorescent nanobody of claim 2.
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| CN104911195A (en) * | 2015-05-22 | 2015-09-16 | 华南农业大学 | Modified rabies virus resisting HEP-Flury strain M protein and preparing method and application of monoclonal antibody thereof |
| CN104928302A (en) * | 2015-05-22 | 2015-09-23 | 华南农业大学 | Expressed rabies virus glycoprotein optimized through gene modification and monoclonal antibody and application thereof |
| CN109369803A (en) * | 2018-09-07 | 2019-02-22 | 深圳市国创纳米抗体技术有限公司 | A kind of nano antibody of rabies poison G-protein and its application |
| CN112390880A (en) * | 2020-11-20 | 2021-02-23 | 南开大学 | Lupin allergen Lupan1 specific nano antibody and application thereof |
| WO2021226728A1 (en) * | 2020-05-15 | 2021-11-18 | Universidad Austral De Chile | Rapid single-step gradient method for the generation of nanoantibodies |
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| CN104911195A (en) * | 2015-05-22 | 2015-09-16 | 华南农业大学 | Modified rabies virus resisting HEP-Flury strain M protein and preparing method and application of monoclonal antibody thereof |
| CN104928302A (en) * | 2015-05-22 | 2015-09-23 | 华南农业大学 | Expressed rabies virus glycoprotein optimized through gene modification and monoclonal antibody and application thereof |
| CN109369803A (en) * | 2018-09-07 | 2019-02-22 | 深圳市国创纳米抗体技术有限公司 | A kind of nano antibody of rabies poison G-protein and its application |
| WO2021226728A1 (en) * | 2020-05-15 | 2021-11-18 | Universidad Austral De Chile | Rapid single-step gradient method for the generation of nanoantibodies |
| CN112390880A (en) * | 2020-11-20 | 2021-02-23 | 南开大学 | Lupin allergen Lupan1 specific nano antibody and application thereof |
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