CN112028993A - Nano antibody for resisting SARS-COV-2 virus N protein and its preparation method and use - Google Patents
Nano antibody for resisting SARS-COV-2 virus N protein and its preparation method and use Download PDFInfo
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Abstract
The invention discloses a nano antibody for resisting SARS-COV-2 virus N protein and a preparation method and application thereof, belonging to the field of biotechnology. A nano antibody for resisting SARS-COV-2 virus N protein comprises CDR1 shown by SEQ ID NO. 1, CDR2 shown by SEQ ID NO. 2 and SEQ ID NO: 3, CDR 3; the invention provides a nano antibody for resisting SARS-COV-2 virus N protein, a new type coronavirus N protein screening phage display nano antibody library which is recombined and expressed, a nano antibody which is specifically combined with N protein is screened out, the antibody still keeps the recognition capability to the antigen after being fused and expressed with humanized Fc protein, and the antibody can be used as a quality control antibody of a new crown antibody detection kit after being fused and expressed with Fc protein.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to a nano antibody for resisting SARS-COV-2 virus N protein, a preparation method and application thereof.
Background
The coronavirus belongs to the family of coronavirus and the genus coronavirus, and is a single-stranded positive-sense RNA virus; since the first virus was discovered in 1937, several coronaviruses were identified, belonging to the alpha, beta, gamma and genus, among which 7 coronaviruses can infect humans, HCoV-229E, HCoV-NL63 of the alpha genus and HCoV-OC43, HCoV-HKU1, SARS-CoV, MERS-CoV and the novel coronaviruses SARS-CoV-2 identified in 2019.
The genome of SARS-CoV-2 is similar to that of the coronavirus that caused the prevalence of SARS in 2003. The understanding of the SARS-CoV-2 protein is based, in large part, on the results of previous studies on SARS; the protein of SARS-CoV consists of two polymeric proteins: ORF1a and ORF1ab proteolytically form 16 nonstructural proteins, four structural proteins: spike protein (S), envelope (E), membrane (M) and nucleocapsid (N), and eight accessory proteins: ORF3a, ORF3b, ORF6, ORF7a, ORF7b, ORF8a, ORF8b, and ORF9 b. Although it is believed that the helper proteins are not essential for viral replication, they play an important role in the interaction of the virus with the host.
The nucleocapsid protein of coronavirus is a structural protein which is expressed in a large quantity in the infection process, and after the virus infects cells, the N protein and RNA enter the cells together to participate in the replication, assembly and release of the virus; the SARS-CoV N protein contains two RNA binding structural domains, which are N end NTD and C end CTD, and the middle is connected by a structural domain rich in serine and arginine; the novel coronavirus antigen N protein has stronger immunogenicity, and is an important detection target of a new crown antibody detection kit and an antigen detection kit, so that the development of a high-sensitivity new crown antigen detection method and a used quality control antibody are very important.
Disclosure of Invention
The invention aims to solve the problems that the high sensitivity detection of a new corona antigen needs to be further improved and a quality control antibody used by the new corona antigen needs to be developed in the prior art, and provides a nano antibody for resisting SARS-COV-2 virus N protein, a preparation method and application thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
a nano antibody for resisting SARS-COV-2 virus N protein comprises CDR1 shown by SEQ ID NO. 1, CDR2 shown by SEQ ID NO. 2 and SEQ ID NO: 3, CDR3 shown.
A nano antibody for resisting N protein of SARS-COV-2 virus has the amino acid sequence shown in SEQ ID NO. 5.
A nano antibody for resisting N protein of SARS-COV-2 virus has the nucleotide sequence shown in SEQ ID NO. 4.
A gene encoding the nanobody of claim 1.
A method of preparing the nanobody of claim 1.
Preferably, the method mainly comprises the following steps:
s1, constructing a nano antibody library;
s2, saving the phage surface to display a nano antibody library;
s3, elutriating the nano antibody;
s4, panning and recognizing the nano antibody of the target protein;
s5, selecting a positive clone recognizing the N protein.
A recombinant construct which is expressed by fusing the nanobody of claim 1 with an Fc protein.
A method of making the recombinant construct of claim 7, comprising the steps of,
a first step of amplifying the nanobody gene of claim 1 with a primer;
secondly, inserting the amplified nano antibody gene into an antibody expression vector pSecTag2A-fc through double enzyme digestion of sfII and NotI;
thirdly, extracting plasmids by using a plasmid extraction kit;
and fourthly, expressing the fusion protein.
Use of the nanobody of claim 1 in a detection kit for detecting neocorona antibody, colloidal gold kit.
Compared with the prior art, the invention provides a nano antibody for resisting SARS-COV-2 virus N protein, a preparation method and application thereof, and has the following beneficial effects:
1. the invention provides a nano antibody against N protein of SARS-COV-2 virus, a new type coronavirus N protein screening phage display nano antibody library which is recombined and expressed, nano antibody which is specifically combined with N protein is screened out, the antibody still keeps the recognition capability to antigen after being fused and expressed with humanized Fc protein, the antibody can be used as quality control antibody of a new crown antibody detection kit after being fused and expressed with Fc protein, and the EC50 of the antibody combined with N protein is measured to be 2.15ng/ml by ELISA method; the antibody can be continuously transformed, the affinity of the antibody is provided, the antibody can be directly used for developing a high-sensitivity new crown antigen detection kit, the antibody can be used for debugging the new crown antibody detection kit, the specificity of the antibody detected by a colloidal gold kit is good, and the concentration of the detected antibody is as low as 62.5 ng/ml.
Drawings
FIG. 1 is a diagram showing the results of the detection of the binding of nanobodies to N protein according to the present invention;
FIG. 2 is a diagram showing the results of colloidal gold assay according to the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments.
Example 1:
the preparation method of the nano antibody for resisting SARS-COV-2 virus N protein comprises the following steps:
1. construction of Nanobody libraries
1.1, taking a 15ml centrifuge tube, firstly adding a separation solution with the same amount as the blood sample, carefully sucking the blood sample and adding the blood sample on the liquid surface of the separation solution, and centrifuging for 20-30min at 450-650 g; after centrifugation, the centrifuge tube is divided into four layers from top to bottom, namely a plasma layer, an annular milky white lymphocyte layer, a transparent separation liquid layer and a red blood cell layer; carefully sucking the second annular milky white lymphocyte layer into another 15ml centrifuge tube by using a pipette; adding 10ml of cleaning solution into the obtained centrifugal tube, uniformly mixing the cells, 250g, and centrifuging for 10 min; after centrifugation, the supernatant is clarified, and the centrifugation time is prolonged if the supernatant is not clarified; cells were washed 2 times with 1xPBS and lysed by addition of trizol and stored at-80 ℃.
1.2, taking the peripheral lymphocyte lysate stored at minus 80 ℃ to melt at room temperature; adding 0.2ml chloroform, covering, shaking vigorously for 15s, standing at room temperature for 2min, and centrifuging at 12000g and 4 deg.C for 15 min; carefully transferring the supernatant into another centrifuge tube, adding isopropanol with the same volume, uniformly mixing, and standing at room temperature for 10 min; centrifuging at 12000g at 4 deg.C for 15 min; centrifuging, removing supernatant, rinsing precipitate with 75% ethanol, and centrifuging at 12000g at 4 deg.C for 5 min; centrifuging, removing supernatant, placing the centrifuge tube at room temperature, and dissolving RNA in RNase-free water after drying; a small amount of dissolved RNA is taken out and is run on agarose gel, and the concentration is measured to judge whether the RNA is degraded or not.
1.3, taking out the total RNA from-80 ℃ and thawing the total RNA on ice; opening the secondary structure of RNA at 65 ℃ for 5min on a PCR instrument; sequentially adding buffer, reverse transcriptase and primer at 37 deg.C for 30min and at 98 deg.C for 5 min; verifying the obtained cDNA by using an internal reference primer; the temperature is-20 ℃, and the long-term storage needs-80 ℃; preparing a PCR reaction system, and amplifying antibody genes from cDNA by using alpaca antibody library primers; carrying out 2% agarose gel electrophoresis analysis on the PCR product, cutting a target band about 500bp, and recovering a target fragment amplification product by using a gel recovery column; carrying out double enzyme digestion on the pCANTAB5E vector and the antibody gene by using Sfi I and Not I; cutting 1.5% agarose gel, recovering target fragment with Tiangen DNA recovery kit, subpackaging, and freezing at-20 deg.C; preparing a reaction system, and connecting antibody genes into a pCANTAB5E vector after enzyme digestion; the ligation system was formulated as follows: vector 1.5ug, antibody 0.5ug, T4 ligase 2. mu.L, 10xbuffer 10. mu.L, water to 100. mu.L, and ligation overnight at 16 ℃ on a PCR instrument.
1.4, streaking TG1 to a minimum culture plate for overnight culture at 37 ℃; inoculating single TG1 colony to 5mL2YT culture solution, and performing shaking culture at 37 ℃ overnight; adding 5mL of the overnight inoculated culture solution into 300mL of 2YT culture solution the next day, and performing shaking culture until OD600 reaches 0.4-0.5; after the bacterial liquid is subjected to ice bath for 30min, centrifuging the bacterial liquid in a precooled centrifuge at 4000g for 15min at 4 ℃; gently resuspending the precipitate in ice water with 300mL of pre-cooled sterile deionized water until the precipitated cells are completely and uniformly dispersed in the water; centrifuging at 4000g for 15min at 4 ℃ in a precooled centrifuge; resuspending the cells twice as described above sequentially with 150mL of pre-chilled sterile deionized water and 30mL of pre-chilled 10% glycerol; finally, resuspending the cells in 1mL of pre-cooled 10% glycerol, and placing on ice for immediate use or subpackaging; freezing at-80 deg.C; adding 5uL of the ligation product into 100uL of competence, placing on ice for precooling, and transferring into a precooled electric rotor cup; adjusting the voltage of an electrotransformation machine to 2.5KV, shocking for 5ms, quickly adding 0.9ml of 2YT culture medium after shocking, and carrying out shake culture at 37 ℃ for 2 hours; and (3) taking 10uL of the diluted gradient, coating the diluted gradient on an SOBAG plate, calculating the storage capacity, coating the rest bacterial liquid on 10 SOBAG plates, and culturing at 37 ℃ overnight.
1.5, counting the colonies subjected to gradient dilution, and calculating the storage capacity of the antibody library established this time; randomly picking 20 clones from the SOBAG plate, and detecting the efficiency of the antibody gene insertion into the vector by colony PCR; 20 randomly selected clones were subjected to sequencing analysis to detect the antibody library capacity, and the integrity and diversity of antibody genes.
2. Display nano antibody library for rescuing phage surface
2.1, the constructed nano antibody library is stored in a host bacterium in the form of phagemid, and the library should be rescued to become a phage display antibody library before the panning process is started. The specific method comprises the following steps:
1.5mL of E-tag-labeled antibody library was inoculated into 300mL of 2YT-AG medium to OD600nmAbout 0.3 to about 0.4; shaking culture at 37 deg.C for about 1.5h to OD600nm＝0.5-0.6; according to the bacteria: adding helper phase helper phage (M13K07) to the phase 1:5, and culturing at 37 ℃ for about 1h with shaking; centrifuging at 4000rpm and 15 deg.C for 15min, and removing culture medium; adding 200mL of 2YT-AK (100. mu.g/mL Amp, 50. mu.g/mL Kan) culture medium to resuspend the bacteria, and culturing at 37 ℃ for 2 h; centrifuging at 10000rpm for 20min to remove precipitate; adding 40mL of PEG/NaCl into the supernatant to precipitate phage, and carrying out ice bath overnight; centrifuging at 10000rpm for 20min, and removing supernatant; suspending the phase with 0.6mL of 2YT medium, and keeping the phase at 4 ℃; if large amounts of phase are required, the culture time is extended from two hours to overnight culture after changing the kan-resistant medium(ii) a The obtained phage were subjected to gradient dilution, infected with TG1 bacteria, coated with SOBAG plates, and phage pool titers were calculated by colony counting.
3. Panning of Nanobodies
In the experiment, His Bind to Resin and antigen protein are utilized to pan the antibody from the phage displayed antibody library, and the specific process is as follows: activation of His Bind Resin: putting 200 mu L of His Bind Resin into a 1.5mL centrifuge tube, centrifuging for 1min at 1000g, and removing the preservation solution; add 200. mu.L of ddH2Cleaning the resin once, centrifuging for 1min at 1000g, removing the supernatant, and repeating the step once; adding 200 μ L of ionized buffer solution, resuspending the resin, standing for 10min, centrifuging to remove supernatant; adding 200 μ L binding buffer solution, resuspending the resin, and standing for 10 min; add 40. mu.L of the resin to a 1.5mL centrifuge tube and centrifuge to remove the supernatant.
4. Nano antibody for elutriating and identifying target protein
Adding 35 μ L (about 10 μ g) of purified RBD protein into 165 μ L PBS, mixing, adding into EP tube filled with activated resin, mixing for 1h, centrifuging for 1min at 1000g, and removing supernatant; adding 200 μ L rinsing buffer solution, resuspending the resin, centrifuging at 1000g for 1min, removing supernatant, and repeating the steps once; taking 300 mu L of the rescued phage display antibody library solution, adding 0.3 mu L of Triton X-100, and gently mixing by using a micropipette; adding 40 mu L of unactivated resin, and slightly rotating for reaction for 1 h; centrifuging at 1000g for 1min, collecting supernatant, adding 40 μ L resin coated with antigen protein, and slightly rotating for 2 hr; centrifuging at 1000g for 1min, and removing supernatant; adding 500 μ L of rinsing buffer (containing 0.1% Triton X-100), resuspending the resin, rinsing with gentle shaking for 5min, centrifuging at 1000g for 1min, removing supernatant, and repeating this step for 5 times;
adding 500 μ L of rinsing buffer (0.1% Tween-20), resuspending the resin, rinsing with gentle shaking for 5min, centrifuging at 1000g for 1min, removing supernatant, and repeating the steps for 5 times; after the last rinsing, the resin was transferred to a new EP tube, centrifuged at 1000g for 1min and the supernatant removed; adding 200 μ L elution buffer, and slightly rotating for 20 min; centrifuging for 1min at 1000g, taking supernatant, adding into 5mL TG1 bacterial liquid, and infecting for 1h at 37 ℃; coating the infected bacterial liquid on an SOBAG plate, and performing inverted culture at 30 ℃ overnight; the next day, colonies on the plates were scraped with 2YT-AG medium and rescued as phages for the next round of panning.
5. Selection of positive clones for the N-recognition protein
Randomly picking single colony from the SOBAG plate and inoculating the single colony into a 96-well bacterial culture plate, adding 200 mu L of 2YT-AG into each well, and culturing at 37 ℃ overnight; sucking 25 μ L bacterial liquid into a new bacterial culture plate, adding 175 μ L2 YT-AG culture medium, and culturing at 37 deg.C for 3 hr; centrifuging at 3500rpm for 10min, removing supernatant, resuspending the bacterial pellet in 200 μ L2 YT-AI (100 μ g/mL Amp, 1mM IPTG) culture medium, and inducing at 30 deg.C overnight; centrifuging at 3500rpm for 10min, and storing the supernatant at 4 deg.C; adding the purified target protein into an ELISA plate, and coating overnight at 4 ℃; after the coating solution was decanted, the cells were washed 3 times with PBS and blocked with 4% PBSM (PBS containing 4% skim milk) for 1 h; washing with PBS for 1 time, adding 50 μ L above prepared nanometer antibody supernatant and 50 μ L4% PBSM per well, and reacting at 37 deg.C for 1 h; after washing 3 times with PBS and PBST, 100. mu.L anti-E/HRP conjugation (diluted 1: 5000 with 4% PBSM) was added to each well and incubated at 37 ℃ for 1 h; washing with PBST and PBS three times, adding 100 μ L TMB substrate solution, reacting for 15min in dark, adding 25 μ L2 mol/L H2SO4The reaction was terminated and OD was measured with a microplate reader450nmThe value determines the concentration of the protein of interest.
6. Positive clone sequencing and sequence analysis
And (3) identifying the target protein with a larger OD450nm value obtained by the panning through ELISA, sequencing the positive monoclonal Cherey-fed forward, and using a sequencing universal primer S1: 5'-GACCATGATTACGCCAAGC-3', the variable regions of the heavy and light chains of the antibody were sequenced using DNAstar and Clustalw 1.8.
Clone number E9 antibody sequence is as follows:
nucleotide sequence of E9 antibody:
amino acid sequence of the E9 antibody:
example 2:
fusion expression of Nanobody and Fc protein
A recombinant construct, which is expressed by fusing the nano antibody and Fc protein;
1. construction of recombinant expression vectors
Amplifying the antibody gene with a primer (F: CGGCCCAGCCGGCCATGGCC, R: GGACTAGTGCGGCC GCTGAGGAGACGGTGACCTG) according to the sequencing result; the antibody gene is subjected to double enzyme digestion by sfiI and NotI, inserted into an antibody expression vector pSecTag2A-fc, and extracted into a plasmid by using a plasmid extraction kit.
pSecTag2A-Fc was modified with pSecTag2A (Thermo Fisher, V90020) and the human IgG1 Fc gene was inserted into the hind III and BamH I cleavage sites of the vector.
2. Expression of fusion proteins
All reagents were left at room temperature for 10 minutes before transfection, and 6-well petri dishes were used for the following operations; diluting 3 mu g of plasmid DNA to 250 mu L of serum-free DMEM medium, and blowing and sucking for 3-4 times by using a pipette gun; diluting 5 mu L PEI reagent to 250 mu L serum-free DMEM medium, and blowing and sucking for 3-4 times by using a pipette gun; note that: the serum-free DMEM medium is a diluent, DNA can not be carried out by using the serum-containing medium, the diluted PEI transfection reagent is added into the diluted plasmid DNA at one time, and the diluted PEI transfection reagent is blown and sucked by a pipette 3-4 times; standing at room temperature for 10-15 minutes to form PEI-DNA complex; counting and plating of cells 18-24 hours before transfection to achieve around 80% confluence of adherent cells at the time of transfection;
discarding the original culture medium in the wells, and adding 1ml of fresh DMEM complete culture medium; preparing a PEI-DNA complex; uniformly dripping the PEI-DNA mixed solution into a cell culture medium, and slightly performing cross motion to uniformly disperse the PEI-DNA compound; petri dish with 5% CO2In a constant temperature incubator at 37 ℃, collecting fine particles after 72 hoursAnd (4) detecting the protein expression quantity.
Transforming 100ml of cells according to the same proportion, and purifying the recombinant expressed antibody; taking the supernatant to carry out affinity chromatography purification and SDS-PAGE electrophoresis analysis, and preliminarily determining the activity of the purified nano antibody by indirect competition ELISA.
The target protein expression quantity can be further improved by optimizing induction expression conditions such as host bacteria, expression vectors, induction culture time, temperature, IPTG concentration and the like, and a way is provided for preparing a large amount of nano antibodies.
And (3) experimental verification results:
1. nanobody binding to N protein
The E9 nanobody obtained by screening in example 1 was combined with N protein, and the EC50 value of the antibody combined with N protein was detected, and the plate was coated with N protein at 2. mu.g/ml, and the detection results are shown in FIG. 1.
2. Application of recombinant antibody fused with nano antibody and Fc protein in colloidal gold detection
The purified recombinant antibody was diluted at 1000ng/ml, 500ng/ml, 250ng/ml, 125ng/ml, 62.5ng/ml, 32.25ng/ml and loaded at 100. mu.L to a colloidal gold detection card. The detection results are shown in figure 2 below: the antibody can recognize N protein, the detection signal is reduced along with the increase of the dilution ratio, the specificity of the antibody is good, and the concentration of the detected antibody is as low as 62.5 ng/ml.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (9)
1. A nano antibody for resisting SARS-COV-2 virus N protein is characterized in that the nano antibody comprises CDR1 shown by SEQ ID NO. 1, CDR2 shown by SEQ ID NO. 2 and SEQ ID NO: 3, CDR3 shown.
2. A nano antibody for resisting SARS-COV-2 virus N protein is characterized in that the amino acid sequence is shown in SEQ ID NO. 5.
3. A nano antibody for resisting SARS-COV-2 virus N protein is characterized in that the nucleotide sequence is shown in SEQ ID NO. 4.
4. A gene encoding the nanobody of claim 1.
5. A method of preparing the nanobody of claim 1.
6. The method for preparing nanobody according to claim 5, characterized by mainly comprising the steps of:
s1, constructing a nano antibody library;
s2, saving the phage surface to display a nano antibody library;
s3, elutriating the nano antibody;
s4, panning and recognizing the nano antibody of the target protein;
s5, selecting a positive clone recognizing the N protein.
7. A recombinant construct, which is expressed by fusing the nanobody of claim 1 with an Fc protein.
8. A method of making the recombinant construct of claim 7, comprising the steps of,
a first step of amplifying the nanobody gene of claim 1 with a primer;
secondly, inserting the amplified nano antibody gene into an antibody expression vector pSecTag2A-fc through double enzyme digestion of sfII and NotI;
thirdly, extracting plasmids by using a plasmid extraction kit;
and fourthly, expressing the fusion protein.
9. Use of the nanobody of claim 1 in a detection kit for detecting neocorona antibody, colloidal gold kit.
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