Nano antibody capable of combining SARS-CoV-2 and application thereof
阅读说明：本技术 可结合SARS-CoV-2的纳米抗体及其应用 (Nano antibody capable of combining SARS-CoV-2 and application thereof ) 是由 吴喜林 吴稚伟 于 2021-01-28 设计创作，主要内容包括：本发明涉及一种可结合SARS-CoV-2的多肽,包括3个互补决定区CDR1-3,CDR1序列为或包括SEQ I D NO:1-9所示序列之一,CDR2序列为或包括SEQ I D NO:10-20所示序列之一,CDR3序列为或包括SEQ I D NO:21-38所示序列之一。本发明针对SARS-CoV-2进行纳米抗体药物开发,通过免疫羊驼、利用噬菌体库展示纳米单抗的平台技术等,筛选到特异性结合病毒的S蛋白上受体结构域的纳米抗体,鉴定了其CDR序列,构建了人源化的抗体C9NB；利用病毒中和实验评估C9NB在治疗该病毒感染的疗效；同时,利用AAV载体装载了编码C9NB的表达序列。本发明为SARS-CoV-2的临床预防、治疗和检测提供了潜在的纳米抗体新药。(The invention relates to a polypeptide capable of binding SARS-CoV-2, comprising 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NO. 1-9, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NO. 10-20, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NO. 21-38. The invention carries out nano antibody drug development aiming at SARS-CoV-2, and through immune alpaca, platform technology which utilizes phage library to display nano monoclonal antibody, etc., nano antibody which is specifically combined with receptor structural domain on S protein of virus is screened, CDR sequence is identified, and humanized antibody C9NB is constructed; evaluating the efficacy of C9NB in treating the viral infection using a virus neutralization assay; simultaneously, an AAV vector was used to load the expression sequence encoding C9 NB. The present invention provides a new potential nano antibody medicine for the clinical prevention, treatment and detection of SARS-CoV-2.)
1. A polypeptide capable of binding SARS-CoV-2, comprising 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NOS: 1-9, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NOS: 10-20, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NOS: 21-38.
2. The polypeptide of claim 1, wherein said polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being sequentially staggered from said CDR 1-3.
3. The polypeptide of claim 2, wherein the polypeptide is a monoclonal antibody.
4. The polypeptide of claim 2, wherein the polypeptide is a VHH.
5. The polypeptide of claim 4, wherein the polypeptide is a VHH of camelid or humanized VHH.
6. Use of the polypeptide of any one of claims 1-5 in the manufacture of a therapeutic agent for SARS-CoV-2 infection.
7. Use of the polypeptide of any one of claims 1-5 in the preparation of a SARS-CoV-2 detection agent.
8. A nucleic acid encoding the polypeptide of any one of claims 1-5.
9. Use of the nucleic acid of claim 8 for the preparation of a medicament for the treatment of SARS-CoV-2.
10. An expression vector comprising the nucleic acid of claim 8.
Technical Field
The invention relates to the field of biomedicine. More particularly, it relates to a polypeptide capable of binding SARS-CoV-2 and the application of the above-mentioned polypeptide in the treatment, prevention and detection of SARS-CoV-2.
Background
In 12 months 2019, a novel coronavirus (SARS-CoV-2) appeared, resulting in severe and even fatal pneumonia in humans. Entry of the virus into the cell is dependent on binding between the receptor domain (RBD) of the viral spike protein (S protein) and the target cell receptor angiotensin converting enzyme 2(ACE2), indicating that disruption of the RBD-ACE2 interaction will prevent entry of SARS-CoV-2 into the cell. Therefore, screening for antibodies that bind RBD and inhibit the binding of RBD to ACE2, targeting RBD, would be an effective method for combating SARS-CoV-2.
In 1993, a novel natural antibody derived from camelidae was found. The antibody naturally lacks a light chain and consists only of a heavy chain comprising two constant regions (CH2 and CH3), a hinge region and a heavy chain Variable region (VHH, i.e., antigen binding site) with a relative molecular mass of about 13KDa, which is only 1/10 of conventional antibodies, and with a molecular height and diameter at the nanometer level, is the smallest functional antibody fragment currently available, and thus is also referred to as Nanobody (Nb). Because the nano monoclonal antibody has the characteristics of high stability (not degraded at 90 ℃), high affinity, homology of more than 80 percent with a human antibody, low toxicity and immunogenicity and the like, the nano monoclonal antibody is widely applied to the research and development of immunodiagnosis kits, the research and development of imaging, and the research and development of antibody drugs aiming at the fields of tumors, inflammations, infectious diseases, nervous system diseases and the like.
It has been reported that the binding mechanism of RBD-ACE2 of SARS-CoV-2 is almost the same as that of SARS-CoV, and that immunization of animals with RBD protein can produce strong and effective polyclonal antibody against SARS-CoV, thereby inhibiting virus invasion. It was concluded from these studies that anti-RBD antibodies were also effective in preventing the entry of SARS-CoV-2. Therefore, it is expected that a specific detection antibody capable of detecting SARS-CoV-2 and a highly efficient and stable SARS-CoV-2 specific neutralizing antibody with low IC50 can be obtained by a new technical means.
Disclosure of Invention
The invention obtains alpaca source nanometer monoclonal antibody and VHH thereof by immunizing alpaca with antigen, and is used for treating SARS-CoV-2 infected patients. Based on these studies, the present invention provides a polypeptide capable of binding SARS-CoV-2, comprising 3 complementarity determining regions CDR1-3, wherein the sequence of CDR1 is or comprises one of the sequences shown in SEQ ID NOS: 1-9, the sequence of CDR2 is or comprises one of the sequences shown in SEQ ID NOS: 10-20, and the sequence of CDR3 is or comprises one of the sequences shown in SEQ ID NOS: 21-38.
In a specific embodiment, the polypeptide further comprises 4 framework regions FR1-4, said FR1-4 being staggered with respect to said CDR 1-3. For example, the FR1-4 sequence can be designed as shown in SEQ ID NOS: 39-42, but the scope of the present invention is not limited thereto. The specific recognition and binding ability of an antibody is mainly determined by the CDR region sequences, and the FR sequences have little influence and can be designed according to species, which is well known in the art. For example, FR region sequences of human, murine or camelid origin may be designed to link the above CDRs, such that a polypeptide or domain that binds SARS-CoV-2. For example, the sequence of FR1-4 of human origin can be designed to be 43-46.
In a preferred embodiment, the polypeptide is a monoclonal antibody.
In a preferred embodiment, the polypeptide is VHH.
In a preferred embodiment, the polypeptide is a VHH of camelid origin or a humanized VHH.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) the sequence of CDR1 is FSISSXDT, where X at position 6 represents isoleucine or valine; and is
II) the sequence of CDR2 is SEQ ID NO 10 and the sequence of CDR2 is SEQ ID NO 21; or
The sequence of CDR1 is SEQ ID NO. 11 and the sequence of CDR2 is SEQ ID NO. 22.
Preferably, the sequence of CDR1 is SEQ ID NO 1, the sequence of CDR2 is SEQ ID NO 10, and the sequence of CDR3 is SEQ ID NO 21; or
The sequence of CDR1 is SEQ ID NO 2, the sequence of CDR2 is SEQ ID NO 11, and the sequence of CDR3 is SEQ ID NO 22.
In one embodiment, the CDR sequences of the polypeptides are as follows:
I) CD1 having the sequence of SEQ ID NO 6
II) the sequence of CD2 is selected from SEQ ID NO: 15-17; and is
III) the sequence of CD3 is selected from SEQ ID NO 26-35.
Preferably, the sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 15, and the sequence of CD3 is selected from SEQ ID NO 26-33 and 35; or
The sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 16, and the sequence of CD3 is SEQ ID NO 27; or
The sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 17, and the sequence of CD3 is SEQ ID NO 34.
In one embodiment, the CDR sequences of the polypeptides are as follows:
the sequence of CD1 is SEQ ID NO 3, the sequence of CDR2 is SEQ ID NO 12, and the sequence of CD3 is SEQ ID NO 23; or
The sequence of CD1 is SEQ ID NO 4, the sequence of CDR2 is SEQ ID NO 13, and the sequence of CD3 is SEQ ID NO 24; or
The sequence of CD1 is SEQ ID NO 5, the sequence of CDR2 is SEQ ID NO 14, and the sequence of CD3 is SEQ ID NO 25; or
The sequence of CD1 is SEQ ID NO 7, the sequence of CDR2 is SEQ ID NO 18, and the sequence of CD3 is SEQ ID NO 36; or
The sequence of CD1 is SEQ ID NO 8, the sequence of CDR2 is SEQ ID NO 19, and the sequence of CD3 is SEQ ID NO 37; or
The sequence of CD1 is SEQ ID NO 9, the sequence of CDR2 is SEQ ID NO 20, and the sequence of CD3 is SEQ ID NO 38.
The present invention also provides the application of the polypeptide in preparing SARS-CoV-2 infection treating agent.
In a preferred embodiment, the CDR sequences of the above polypeptides are as follows:
the sequence of CD1 is SEQ ID NO 3, the sequence of CDR2 is SEQ ID NO 12, and the sequence of CD3 is SEQ ID NO 23; or
The sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 16, and the sequence of CD3 is SEQ ID NO 27; or
The sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 17, and the sequence of CD3 is SEQ ID NO 34.
The present invention also provides the application of the polypeptide in preparing SARS-CoV-2 detecting agent.
In a preferred embodiment, the CDR sequences of the above polypeptides are as follows:
the sequence of CD1 is SEQ ID NO 3, the sequence of CDR2 is SEQ ID NO 12, and the sequence of CD3 is SEQ ID NO 23; or
The sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 16, and the sequence of CD3 is SEQ ID NO 27; or
The sequence of CD1 is SEQ ID NO 6, the sequence of CDR2 is SEQ ID NO 17, and the sequence of CD3 is SEQ ID NO 34.
The invention also provides nucleic acids encoding the above polypeptides.
The invention also provides the application of the nucleic acid in preparing SARS-CoV-2 treating and preventing medicine.
In a specific embodiment, the nucleic acid is DNA or RNA.
In a specific embodiment, the nucleic acid is present in a gene expression cassette.
The invention also provides an expression vector containing the expression cassette of the nucleic acid.
In a preferred embodiment, the expression vector is a viroid expression vector.
In a preferred embodiment, the expression vector is an adeno-associated viral expression vector (AAV vector).
The invention carries out nano antibody drug development aiming at SARS-CoV-2, and through immune alpaca, platform technology which utilizes phage library to display nano monoclonal antibody, etc., nano antibody VHH which is specifically combined with receptor structure domain (RBD) on SARS-CoV-2S protein is screened, CDR sequence is identified, and humanized VHH-huFc1(C9NB) is constructed; the virus neutralization experiment is utilized to evaluate the curative effect of C9NB in treating SARS-CoV-2 infection; simultaneously, an AAV vector was used to load the expression sequence encoding C9 NB. The present invention provides a new potential nano antibody medicine for the clinical prevention, treatment and detection of SARS-CoV-2.
Drawings
FIG. 1 shows the antiserum titers against the S protein (A) and RBD protein (B), respectively, after one week of SARS-CoV-S protein immunization and 2 nd and 3 rd alpaca immunization, with the preimmune serum as a control;
FIG. 2 is an electrophoretogram of PCR products amplified using SARS-CoV-S-VHH phage antibody library as a template;
FIG. 3 is the panning identification of SARS-CoV-S-VHH phage antibody library, wherein A is the ELISA detection statistical chart after phage library panning against S protein; b is the second wheel (2)nd) And a third wheel (3)rd) 96 clones are selected from the panned phage antibody library respectively, and phage ELISA detection statistical chart is carried out aiming at RBD protein;
FIG. 4 is a statistical chart of ELISA detection of eukaryotic expressed VHH antibodies, each dot representing a clone, with OD450 for RBD/OD 450 for blank on the ordinate, a positive being defined by a ratio greater than 5.0;
FIG. 5 is a statistical SPR detection of eukaryotic expressed VHH antibodies;
FIG. 6 is a statistical plot of OD450 of the binding of positive VHH antibodies to RBD proteins at different purified concentrations detected by ELISA;
FIG. 7 is a statistical chart of SARS-CoV-2 virus infection experiment for neutralization of positive VHH antibody, wherein A is a statistical chart of a virus infection experiment for neutralization of positive VHH antibody; b is a statistical chart of positive antibody neutralization euvirus infection experiments.
Detailed Description
1. Preparation of alpaca immune and antiserum
Priming alpaca with 250 μ g of emulsified mixture of S protein of SARS-CoV-2 and 250 μ l Freund 'S complete adjuvant, boosting 2 times with 125 μ g S protein and 250 μ l Freund' S incomplete adjuvant on 14 days and 28 days, collecting blood and detecting antiserum titer 1 week after 2 times of immunization; after 1 week of the 3 rd immunization, 200ml of blood was collected for phage antibody library construction.
Antiserum titers were determined by ELISA using the S protein at a concentration of 0.5. mu.g/ml and theRBD protein coating detection plate, adding gradient diluted antiserum (control is pre-immune alpaca serum) into each well, incubating for 1.5h at 37 deg.C, washing for 2 times, adding 1: 10000 diluted second antibody of horse radish peroxidase labeled Goat anti-Llama IgG (H + L) is incubated for 1H at 37 ℃, after washing for 4-6 times, 100 mu L of TMB substrate is added, incubation is carried out for 10min at 37 ℃, and 50 mu L of 0.2M H is added2SO4The reaction was stopped and the OD450nm was measured. ELISA assay serum titers were specified at the highest dilution of OD450 above 2.1-fold of blank and greater than 0.2.
The results are shown in FIG. 1, where the antisera from 2 and 3 immunizations gave a titer of 2.19X 10, respectively, against S protein6And 6.56X 106The titer of the RBD protein is 2.43 multiplied by 10 respectively4And 2.19X 105. It can be seen that this antigen can induce camels to produce high titers of antiserum specific for the RBD region on the S protein.
Construction and panning of VHH phage library
Collecting 200ml of camel peripheral blood after immunization, separating by using lymphocyte separation liquid (GE Ficoll-Paque Plus) to obtain camel PBMC, extracting RNA according to a TRIzol operation manual, inverting by using oligo (dT) into cDNA, cloning the camel VHH gene to phagemid plasmid by using techniques such as primer amplification, molecular cloning and the like, and transforming TG1 bacteria to obtain the VHH phage library. To further identify whether the construction of the CoV-VHH phage library was successful, the VHH target gene of the immune S protein alpaca was amplified by PCR, and it was found that the target band was 500bp and the size was as expected (FIG. 2), indicating that the CoV-VHH phage antibody library contains VHH gene. Selecting 50 clones for sequencing, wherein the sequencing result shows that the sequenced sequences do not have completely consistent repeated sequences; the alignment results show that the most of the different sequences are in the CDR binding region. Through detection, the experiment constructs a CoV-VHH phage antibody library with the library capacity of 2.0 multiplied by 109The positive rate is 100%, the sequence Diversity (Diversity) is 100%, and the effective insertion rate (In frame rate) is greater than 95%.
The phage antibody library was recovered from VHH-phagemid transformed bacteria with the help of M13KO7 helper phage and precipitated with PEG/NaCl. The S-His protein coated with 50. mu.g/ml was subjected to phage antibody library enrichment three times. And (3) carrying out combined identification on the enriched phage, eluting, transforming, coating a plate, selecting a single clone to carry out ELISA (enzyme-linked immuno sorbent assay) on the phage and S protein and RBD protein respectively, sequencing the clone with the combined reading value of more than 1.0, cloning to an expression vector pVAX1, and transfecting 293F cells to express to produce the nano monoclonal antibody.
The panning library was tested for binding to protein S. The phage ELISA results showed that the binding reading values of the CoV-VHH phage library and S protein before enrichment were 0.79, and the reading values of the phage library after one round, two rounds and three rounds of enrichment were 1.58, 2.43 and 2.78 respectively (FIG. 3A). To further verify the positive phage rate of binding of CoV-VHH proteins in the enriched library, 94 clones were selected from each of the 2 nd and 3 rd round enriched libraries for single phage ELISA detection. The results showed that 36.1% of the individual phage clones bound to S protein in round 2, 52.9% of them bound to RBD simultaneously, 31.9% of the phage clones bound to RBD in round 3, and the binding/blank reading was greater than 5 (FIG. 3B), successfully enriching the high binding CoV-VHH phage library by S protein panning.
Eukaryotic expression of VHH-huFc (C9NB)
Through molecular cloning technology, the 21 nanometer monoclonal antibody VHH genes with different sequences obtained through sequence analysis are fused with human Fc genes and inserted into pCDNA3.4 to construct Nb-huFc-pCDNA3.4 expression plasmids. The constructed Nb-huFc-pCDNA3.4 was transfected into 293tt cells, expressed to produce Nb-huFc (C9NB), and purified using Protein G. Purified VHH-huFc1(C9NB) was collected and tested in ELISA, with some antibodies having good binding capacity (fig. 4). The respective sequence numbers of the constructed humanized antibodies are shown in Table 1.
TABLE 1 original antibody numbering and CDR sequences corresponding to humanized antibodies SNB
All clones tested by the above ELISA were tested for affinity of the antibody to RBD protein.Affinity was detected using the Fortebio biomolecular interaction platform. And (2) solidifying the antibody VHH-hfc1 to be detected onto an Anti-human IgG Fc Capture Biosensors (AHC) probe, solidifying for 400s, then combining with an antigen RBD-his protein, combining for 180s, dissociating for 180s, observing the combination dissociation condition of the antibody and the antigen, and fitting a curve by an instrument to derive data. The results of the affinity assay are shown in Table 2, and the affinity (KD) of most antibodies can reach 10-8To 10-9M (nM scale), the binding dissociation curve of the partial antibodies is shown in FIG. 5. Therefore, the obtained C9NB nano antibodies have higher affinity and can specifically recognize and combine the RBD structural domain on the S protein of SARS-CoV-2 virus. Among them, the antibodies C9NB15, C9NB22 and C9NB31 obtained higher response values at steady state, indicating that the binding capacities of the three antibodies are stronger, and further studies were performed on the three antibodies.
Table 2 summary of C9NB affinities.
Clone ID
Ka(1/MS)
Kd(1/S)
KD(M)
Response(nM)
C9NB01
6.48E+04
2.97E-03
4.59E-08
0.0156
C9NB02
6.85E+05
1.69E-02
2.47E-08
0.0302
C9NB04
4.40E+05
6.30E-03
1.43E-08
0.1824
C9NB05
7.62E+05
1.11E-03
1.46E-09
0.0195
C9NB07
1.84E+05
2.40E-02
1.30E-07
0.0166
C9NB09
5.16E+05
4.67E-03
9.04E-09
0.1254
C9NB12
3.79E+05
4.70E-03
1.24E-08
0.2218
C9NB13
3.04E+05
2.49E-04
8.19E-10
0.0203
C9NB14
5.76E+04
<1.0E-07
<1.0E-12
0.0159
C9NB15
6.94E+05
4.22E-03
6.09E-09
0.2314
C9NB19
3.48E+05
1.31E-02
3.76E-08
0.0366
C9NB22
4.92E+05
2.94E-03
5.99E-09
0.2322
C9NB24
8.30E+05
3.53E-03
4.25E-09
0.1684
C9NB25
3.12E+05
1.92E-03
6.14E-09
0.0168
C9NB27
2.46E+05
5.61E-03
2.28E-08
0.016
C9NB28
2.04E+03
5.82E-02
2.86E-05
0.0068
C9NB29
1.34E+05
5.95E-04
4.45E-09
0.0169
C9NB30
5.46E+05
1.16E-02
2.12E-08
0.0469
C9NB31
6.37E+05
6.64E-03
1.04E-08
0.246
C9NB33
4.34E+05
1.95E-03
4.51E-09
0.1328
Note: ka is the binding constant, KD is the dissociation constant, KD is the affinity, and Response is the Response value at which steady state is obtained.
4. Antibody gradient dilution ELISA
Coating the detection plate with 0.5. mu.g/ml RBD protein, 100. mu.l per well, incubating at 37 ℃ for 2h, washing for 2-4 times, blocking with 4% bovine serum, 250. mu.l per well, incubating at 37 ℃ for 1h, washing for 2-4 times, adding 100. mu.l of purified antibody diluted in gradient to each well, incubating at 37 ℃ for 1.5h, washing for 2 times, adding 1: 100. mu.l of horseradish peroxidase-labeled anti-human antibody diluted 10000 was incubated at 37 ℃ for 1 hour, washed 4 to 6 times, 100. mu.l of TMB substrate was added, incubated at 37 ℃ for 10min, 50. mu.l of 0.2M H2SO4 was used to stop the reaction, and OD450nm was determined. The results show (fig. 6) that when the concentrations of antibodies C9NB15, C9NB22, and C9NB31 were as low as 0.001526, 0.000763, and 0.000763ug/ml, respectively, the ratio of OD450 bound by RBD protein/OD 450 of blank was still greater than 2.
Neutralization of SARS-CoV-2 pseudovirus by C9NB
SARS-CoV-2, SARS-CoV and MERS-CoV pseudoviruses were generated by co-transfection of expression vectors expressing firefly luciferase (pNL43R-E-luciferase) and pcDNA3.1(Invitrogen) into 293T cells (ATCC), respectively. Viral supernatants were collected after 48 h. Viral titers were determined by luciferase activity in relative light units (Bright-Glo luciferase assay vector system, Promega Biosciences). The control monoclonal antibody was anti-SFTS antibody SNB02(1 mg/ml). C9NB15, C9NB22 and C9NB31 in VHH-huFc1 were selected for in vitro neutralization experiments. The antibodies were serially diluted to different concentrations, along with SARS-CoV-2, SARS-CoV and MERS-CoV pseudoviruses, in 5% CO2Incubated at 37 ℃ for 1 hour, and 1.5X10 added4Huh7 cells, 5% CO2Evaluation of monoclonal antibodies by assay for luciferase Activity after incubation in 37 ℃ incubator for 48 hoursHalf maximal inhibitory concentration of body (IC50)
As shown in FIG. 7A, the neutralizing activity of C9NB15, C9NB22 and C9NB31 was very good, and the inhibition rate reached 90% at antibody concentrations of 0.00395, 0.00794 and 0.02029. mu.g/ml, respectively.
Neutralization of SARS-CoV-2 true virus by C9NB
The SARS-CoV-2 focused reduction neutralization assay (FRNT) was performed in a biosafety tertiary certification laboratory. Neutralization assays were performed on live SARS-CoV-2 using clinical isolates previously obtained from nasopharyngeal swabs from one infected patient (Beta/Shenzhen/SZTH-003/2020, EPI _ ISL _406594at GISAID). The detection antibody was serially diluted and mixed with 75. mu.l of SARS-CoV-2 (8X 103 focused forming units/ml, FFU/ml) in a 96-well microwave plate and incubated at 37 ℃ for 1 hour. The mixture was then transferred to a 96-well plate, seeded with Vero E6 cells and allowed to absorb for 1 hour at 37 ℃. The inoculum was then removed and overlay medium (100. mu.l containing 1.6% hydroxymethyl cellulose, CMC) was added. Then incubated at 37 ℃ for 24 hours. Cells were fixed for 30 min in 4% paraformaldehyde solution and the cover removed. Cells were permeabilized with 0.2% Triton X-100 and incubated with cross-reactive rabbit anti-sars-cov-n IgG (Sino Biological, Inc) for 1 hour at room temperature, followed by addition of HRP-labeled goat anti-rabbit IgG (H + L) antibody (Jackson ImmunoResearch). The cells were further incubated at room temperature. The reaction was performed with KPL TrueBlue peroxidase substrate (Seracare life sciences). The number of SARS-CoV-2 lesions was counted using an EliSpot analyzer (Cellular Technology Ltd.).
As shown in FIG. 7B, C9NB15, C9NB22 and C9NB31 exhibited excellent neutralizing activity, and the inhibition rate reached 90% at antibody concentrations of 0.0308, 0.1716 and 0.0411. mu.g/ml, respectively.
10. AAV viral vector loaded VVH
Adeno-associated virus (AAV) is derived from non-pathogenic wild adeno-associated virus, and is considered one of the most promising gene transfer vectors due to its high safety, wide host cell range (dividing and non-dividing cells), low immunogenicity, and long time for expressing foreign genes in vivo, and is widely used in gene therapy and vaccine research worldwide.
AAV Helper-Free viral packaging system was purchased from Cell Biolabs, San Diego USA. Inserting the DNA coding sequence of the VHH into the pAAV-MCS plasmid by a molecular cloning technology; after the successful construction is proved by sequencing, the constructed plasmid pAAV-Ab and pHelper and pAAV-DJ plasmids are used for co-transfecting AAV-293T cells by using a PEI transfection reagent according to the mass ratio of 1:1: 1. Supernatants were collected at 48, 72, 96 and 120 hours post transfection and concentrated with 5xPEG8000(sigma) and finally purified with 1.37g/ml cesium chloride. Purified AAV was dissolved in PBS, identified and stored at-80 ℃ after packaging.
The antibody can specifically recognize and combine S protein of SARS-CoV-2, and C9NB15, C9NB22 and C9NB31 have good neutralization activity, so after the AAV vector loaded with VHH is introduced into human body, the antibody with neutralization activity can be expressed by human body, so that the human body can prevent SARS-CoV-2 infection, and can treat SARS-CoV-2 infected patient.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Sequence listing
<110> Nanjing Tuofeng Biotech Co., Ltd
<120> nano antibody capable of binding SARS-CoV-2 and its application
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<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 15
Ile Asn Ile Ile Asn Arg Thr
1 5
<210> 16
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 16
Ile Asp Val Ile Asn Arg Ala
1 5
<210> 17
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 17
Ile Asn Ile Ile Asn Arg Pro
1 5
<210> 18
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 18
Val Thr Ser Gly Gly Ser Thr
1 5
<210> 19
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 19
Ile Thr Asn Ser Gly Ser Thr
1 5
<210> 20
<211> 7
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 20
Ile Thr Ser Gly Gly Ser Thr
1 5
<210> 21
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 21
Tyr Gly Gln Asp Val Leu Gly Gln Ile Tyr
1 5 10
<210> 22
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 22
Tyr Gly Gln Asp Ile Leu Gly Gln Ile Tyr
1 5 10
<210> 23
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 23
Ala Gly Val Val His Asp Val Gln Ala Met Cys Val Met Asn Pro
1 5 10 15
<210> 24
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 24
Thr Ala Arg Pro Ser Leu Trp Ala Val Val Ala Gly Cys Pro Leu Asp
1 5 10 15
Gln Asn Thr Tyr Phe Ser
20
<210> 25
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 25
Ala Ala Pro His Ser Gly Ser Val Cys Pro Arg Trp Ala Glu Tyr Tyr
1 5 10 15
Gly Val Asp His
20
<210> 26
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 26
Ala Ala His Phe Val Pro Pro Gly Ser Arg Leu Arg Asp Cys Leu Val
1 5 10 15
Asn Glu Leu Tyr Asn Tyr
20
<210> 27
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 27
Ala Ala His Phe Val Pro Pro Gly Ser Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Glu Leu Tyr Asn Tyr
20
<210> 28
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 28
Ala Ala His Phe Val Pro Pro Gly Ser Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Glu Ala Tyr Asn Tyr
20
<210> 29
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 29
Ala Ala His Phe Val Pro Pro Gly Gly Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Asp Leu Tyr Asn Tyr
20
<210> 30
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 30
Ala Ala His Phe Val Pro Pro Gly Ser Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Asp Leu Tyr Asn Tyr
20
<210> 31
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 31
Ala Ala His Phe Val Pro Pro Gly Ser Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Asp Val Tyr Asn Tyr
20
<210> 32
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 32
Ala Ala His Phe Val Pro Pro Glu Ser Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Glu Leu Tyr Asn Tyr
20
<210> 33
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 33
Ala Ala His Phe Val Pro Pro Glu Ser Arg Leu Arg Gly Cys Leu Val
1 5 10 15
Asn Glu Ala Tyr Asn Tyr
20
<210> 34
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 34
Ala Ala His Phe Val Pro Pro Gly Ser Arg Leu Gly Gly Cys Leu Val
1 5 10 15
Asn Glu Leu Tyr Asn Tyr
20
<210> 35
<211> 22
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 35
Ala Ala His Phe Val Pro Pro Gly Ser Arg Phe Arg Gly Cys Ser Val
1 5 10 15
Asn Glu Leu Tyr Asn Tyr
20
<210> 36
<211> 9
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 36
Asn Ala Arg Leu Phe Asp Pro Gly Tyr
1 5
<210> 37
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 37
Asn Thr Phe His Tyr
1 5
<210> 38
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 38
Thr Thr Ala Gly Ser Trp Gln Gly Asp Tyr
1 5 10
<210> 39
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 39
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Pro
20 25
<210> 40
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 40
Met Gly Trp Tyr His Gln Ala Pro Gly Lys Gln Arg Glu Leu Val Ala
1 5 10 15
Ala
<210> 41
<211> 38
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 41
Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
1 5 10 15
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp
20 25 30
Thr Ala Val Tyr Tyr Cys
35
<210> 42
<211> 20
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 42
Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Glu Pro Lys Thr Pro
1 5 10 15
Lys Pro Gln Pro
20
<210> 43
<211> 25
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 43
Gln Val Arg Leu Val Glu Ser Gly Gly Gly Ser Val Gln Ala Gly Glu
1 5 10 15
Thr Leu Arg Leu Ser Cys Thr Ala Ser
20 25
<210> 44
<211> 17
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 44
Met Gly Trp Tyr Arg Gln Gly Pro Gly Asn Glu Cys Glu Met Val Ala
1 5 10 15
Tyr
<210> 45
<211> 36
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 45
Ala Asp Ser Thr Lys Gly Arg Phe Thr Ile Ser Gln Asp Asn Ala Lys
1 5 10 15
His Thr Leu Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly
20 25 30
Val Tyr Tyr Cys
35
<210> 46
<211> 10
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 46
Gly Gln Gly Thr Arg Val Thr Val Ser Ser
1 5 10