CN116462752A - Recombinant nano antibody targeting BVDV nonstructural proteins and application thereof - Google Patents
Recombinant nano antibody targeting BVDV nonstructural proteins and application thereof 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
- C07K16/1081—Togaviridae, e.g. flavivirus, rubella virus, hog cholera virus
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/14—Antivirals for 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K39/00—Medicinal preparations containing antigens or antibodies
- A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
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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®
-
- 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 belongs to the technical field of biology, and particularly relates to a recombinant nano antibody targeting BVDV nonstructural proteins and application thereof. The amino acid sequence of the recombinant nano antibody is shown as SQE ID NO. 1. The recombinant nano antibody is obtained by expression of an escherichia coli expression system, and the preparation method is simple; compared with the existing BVDV nonstructural protein targeting nano-antibody, the recombinant nano-antibody can autonomously pass through cell membranes, can inhibit the replication of BVDV in cells, and provides a new technical means for BVDV prevention and control.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to a recombinant nano antibody targeting BVDV nonstructural proteins and application thereof.
Background
Bovine viral diarrhea (Bovine Viral Diarrhea, BVD) is an animal epidemic caused by infection with bovine viral diarrhea virus (Bovine Viral Di arrhea Virus, BVDV). BVDV has a genomic size of about 12.3kb and natural mutants of about 16.5kb have been found. The 5 'and 3' ends of which have an untranslated region (Untranslated region, UT R), respectively, are referred to as 5'-UTR and 3' -UTR. The open reading frame (Open Reading Frame, ORF) of BVDV encodes a precursor polyprotein of about 4000 amino acids which is processed into four structural proteins (C, erns, E1 and E2) and eight non-structural proteins (Npro, p7, NS2, NS3, NS4A, NS4B, NS5A and NS 5B) by a number of proteases, the genome of which is NH2-Npro/C/Erns/E1/E2/p7/NS2/NS3/NS4A/NS4B/NS5A/NS5B-COOH in sequence from the N-terminus to the C-terminus.
The virus can cause damage to the digestive system, respiratory system and reproductive system of cattle, and causes great economic loss for cattle industry. BVDV has a broad host and tissue tropism as other pestiviruses. BVDV has been reported to be infecting species other than the species from which they were originally isolated. BVDV has been successfully isolated to date in more than 40 species, including camels, goats, deer, sheep, pigs, and various ungulate wild animals, among others. The first description of BVDV-induced disease is mainly about the gastrointestinal system, but it was found that the virus could invade respiratory and reproductive systems and cause multiple organ damage, and then the cattle infected with BVDV would have low conception rate, abortion, malformed embryo and embryonic death, which seriously jeopardizes reproductive performance and productivity of the herd. The major cause of the major hazard to flocks is due primarily to their immunosuppressive and persistent nature. BVDV causes tremendous damage to the host's immune system, which makes it more difficult for the herd to resist infection by other viruses. In addition, vertical transmission from the mother to the fetus makes calves a host of persistent infection (Persistent Infection, PI) that continually detoxifies, which in turn results in greater transmission.
Nanobodies (Nbs), also known as Heavy chain antibodies (HcAbs) or Heavy chain variable domains of Heavy chain antibodies (Variable domain of Heavy chain of Heavy chain Antibody, VHH). In contrast to conventional antibodies, nbs lacks the light chain and first heavy chain constant region (Constant Region of Heavy chain, CH) domains, which is the smallest antibody with an intact antigen binding fragment. Due to the lack of light chains and the first CH, nbs has special properties that many traditional antibodies do not possess: including low relative molecular mass, high affinity, low immunogenicity, thermal stability, high yields, and the like. In addition, nbs are able to bind to recessed epitopes that traditional antibodies cannot recognize, such as catalytic sites for enzymes. Based on the advantages, nbs have great application potential in the fields of disease diagnosis, treatment and the like. However, most of the nano antibodies prepared at present cannot penetrate through cell membranes, cannot play a role in inhibiting viruses in vivo, and severely limit the application of the nano antibodies.
Disclosure of Invention
Aiming at the technical problems, the invention provides a recombinant nanobody targeting BVDV nonstructural proteins, which can autonomously cross cell membranes and inhibit BVDV replication, and can be used for preparing medicines for preventing or treating bovine viral diarrhea virus infection, and specifically comprises the following contents:
in a first aspect, the invention provides a recombinant nanobody targeting BVDV nonstructural proteins, wherein the amino acid sequence of the recombinant nanobody is shown as SEQ ID NO. 1.
Preferably, the recombinant nanobody contains HA and a histidine tag, and the amino acid sequence of the recombinant nanobody is shown as SEQ ID NO. 2.
In a second aspect, the present invention provides a nucleotide encoding the recombinant nanobody of the first aspect above.
Preferably, the nucleotide sequence is as shown in SQE ID No. 3.
In a third aspect, the present invention provides a recombinant vector or recombinant cell comprising a nucleotide encoding the recombinant nanobody of the first aspect above.
Preferably, the vector is selected from pET21b.
Preferably, the recombinant cell is recombinant E.coli BL21 (DE 3).
In a fourth aspect, the present invention provides a primer for amplifying the nucleotide sequence of the recombinant nanobody of the second aspect.
Preferably, the primer comprises:
F：CCGCATATGTACGGTCGTAAGAAACGTCGCCAGCGTCGCCGTGGAGGCGGTGGC TCGGGCGGTGGCGGCTCGGGTGGCGGTGGTTCTCAGGTCCAACTGCAGGAG (SEQ ID NO. 4);
R：TGCTCGAGAGCGTAATCTGGAACATCGTATGGGTATGAGGAGACGGTGACCTGG GTCCCCT (SEQ ID NO. 5).
In a fifth aspect, the present invention provides a method for preparing the recombinant nanobody according to the first aspect, the method comprising:
(1) Synthesizing a nucleotide sequence encoding the recombinant nanobody of the first aspect;
(2) Ligating the nucleotide sequence of step (1) with a vector/plasmid, transforming the bacteria, and constructing recombinant cells;
(3) And (3) inducing and expressing the recombinant cells constructed in the step (2), and purifying to obtain the recombinant nanobody.
Preferably, the method comprises:
(1) Synthesizing a nucleotide sequence encoding the recombinant nanobody of the first aspect;
(2) Connecting the nucleotide sequence in the step (1) with pET21b to obtain a recombinant vector, and constructing a recombinant cell by using competent BL21 (DE 3) of the recombinant vector;
(3) And (3) inducing and expressing the recombinant cells constructed in the step (2), and purifying to obtain the recombinant nanobody.
Preferably, the method is as follows: synthesizing a fragment of interest encoding the nucleotide sequence of the second aspect; cutting a target fragment and a pET21b empty vector by using Nde I and Xho I, recovering the cut target fragment and vector, connecting and converting the target fragment and vector to competent Trans5a, and selecting positive recombinant plasmid; transferring the recombinant plasmid into expression competent BL21 (DE 3) to induce expression; purifying by using Ni-NTA affinity chromatographic column, and finally obtaining the recombinant nano antibody after renaturation and concentration.
In a sixth aspect, the present invention provides an application of the recombinant nanobody of the first aspect in preparing a medicament for preventing or treating bovine viral diarrhea virus infection.
Preferably, the recombinant nano antibody is added with any pharmaceutically acceptable auxiliary material to prepare any pharmaceutically acceptable dosage form.
In a seventh aspect, the present invention provides a composition comprising the recombinant nanobody of the first aspect above.
The beneficial effects of the invention are as follows: the invention firstly provides a recombinant nano antibody targeting BVDV nonstructural proteins, wherein the amino acid sequence of the recombinant nano antibody is shown as SQE ID NO. 1; the recombinant nano antibody can independently pass through a cell membrane and inhibit the replication of BVDV, so that the problem that most of the nano antibodies prepared at present cannot pass through the cell membrane and cannot inhibit viruses in vivo is solved, and a novel technical means is provided for the prevention and control of BVDV; the recombinant nano antibody is expressed by an escherichia coli expression system, and the preparation method is simple.
Drawings
Identification of the expression results of the recombinant nanobody rNb1 of fig. 1;
FIG. 2 shows the detection result of the membrane penetration effect of the recombinant nanobody rNb 1;
FIG. 3 cytotoxicity assay results of recombinant nanobody rNb 1;
FIG. 4 results of recombinant nanobody rNb1 inhibiting BVDV 5' UTR mRNA;
FIG. 5 results of recombinant nanobody rNb1 inhibiting BVDV titer in supernatant.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without undue burden are within the scope of the invention
EXAMPLE 1 preparation of recombinant nanobodies
The amino acid sequence of the recombinant nanobody is shown as SQE ID No.1, the HA and His tags are conveniently introduced into the sequence for purification in the embodiment, the corresponding amino acid sequence is shown as SQE ID No.2, the gene sequence is shown as SQE ID No.3, and the primers, plasmids and the like are provided by Sean Optimazethapyr Biotech Co., ltd, and are specifically as follows:
the primer design is shown in table 1, and the upstream and downstream primers respectively have Nde I and Xho I restriction sites; amplifying by using pTrip-CMV-Nb1 plasmid as a template and using primers shown in table 1 to obtain a target fragment of the recombinant nanobody;
and (3) carrying out double digestion on the target fragment and the pET21b empty vector by using Nde I and Xho I, recovering the digested target fragment and vector, connecting and converting to competent Trans5a, identifying positive clones by bacterial liquid PCR, and randomly picking 3 positive clones for sequencing analysis by Seiran and Castosia biotechnology Co.
The recombinant plasmid sequenced for the correct sequence was transferred into expression competent BL21 (DE 3). Table 4h was induced at IPTG concentration of 0.4mM at 37 ℃. And purifying by utilizing a Ni-NTA affinity chromatographic column, and finally, obtaining the recombinant nano antibody with good purity and higher concentration after renaturation and concentration. Identification was performed by SDS-PAGE and Western Blot. As shown in FIG. 1, SDS-PAGE (left panel) and Western Blot (right panel) showed that the concentrated recombinant nanobody was single and bright, indicating that the recombinant nanobody rNb1 with high content and good purity was obtained.
TABLE 1 amplification primers
Example 2 detection of Membrane penetration Effect of recombinant nanobody
Recombinant nanobody (rNb) with a final concentration of 10 mu M was added to MDBK cells, after 24 hours of incubation, the 24-well plate was removed from the incubator, the medium was discarded, and PBS was washed 3 times; adding 4% paraformaldehyde, 500 μl/well, fixing at 37deg.C for 10min, and washing with PBS for 3 times; adding 500 μL of 0.25% Triton-X-100, rupture of membranes at 37deg.C for 10min, and washing with PBS for 3 times; 500. Mu.L of 1% BSA was added and blocked at 37℃for 30min; will beThe Anti-His Mouse Monoclonal An tibody monoclonal antibody is diluted by 1% skimmed milk powder and added into cells, 200 mu L of the antibody is added into each well, and incubated for 1h at 37 ℃; discarding the primary antibody, washing 3 times with PBS, diluting Alexa Fluor@488Goat@Mouse with 1% skimmed milk powder at a ratio of 1:200, adding 200 μl of the diluted powder into cells per well, and incubating at 37deg.C in dark place for 1 hr; discarding the secondary antibody, washing 3 times with PBS, diluting DAPI with PBS according to a ratio of 1:1000, adding 200 mu L of DAPI into cells in each hole, and incubating for 10min at 37 ℃ in a dark place; the DAPI was discarded, washed 3 times with PBS, the slide was removed, fixed on a slide with a capper, observed with a fluorescence microscope, and photographed for storage. The IFA results are shown in fig. 2, where green fluorescence was detected in MDBK cells containing recombinant nanobody (rNb 1), while no green fluorescence was detected in MDB K cells of the control Mock, indicating that recombinant nanobody (rNb 1) was able to efficiently cross MDBK cell membranes.
EXAMPLE 3 recombinant nanobody cytotoxicity assay
Well conditioned MDBK cells were plated in 96 well plates and placed in 5% CO at 37 degrees Celsius 2 Fine of (2)Culturing in a cell incubator. When the cell density reaches about 70%, discarding the culture medium, washing 3 times with PBS, inoculating recombinant nanobody with different concentrations, setting 5 repetitions of each concentration, simultaneously setting a control group, continuing culturing, discarding the culture medium after 24 hours, changing to fresh culture medium, adding 10 μl of CCK8 reagent into the well, taking care of avoiding generating bubbles, placing into an incubator, continuing culturing for 2 hours, and measuring OD 450 Cell viability was calculated according to the instructions of the CCK8 kit.
As shown in FIG. 3, the recombinant nanobody (rNb 1) was non-toxic to MDBK cells and safe when the concentration was within the range of 0 to 30. Mu.M.
Example 4 recombinant nanobodies inhibit replication of BVDV 5' UTR mRNA
Well-conditioned MDBK cells were grown at 1X 10 5 The cells were spread in 24-well plates and placed in 5% CO at 37 ℃C 2 Is cultured in a cell culture incubator. When the cell density reached about 70%, after 0.01MOI BVDV was inoculated for 2 hours, the cells were washed 3 times with PBS, replaced with DMEM containing 3% FBS, and 0. Mu.M and 20. Mu.M of recombinant nanobodies were inoculated, and cell samples were collected at 36hpi by RT-qPCR and TCID 50 The effect of different concentrations of recombinant nanobodies on BVDV replication in MDBK cells was determined.
The results are shown in fig. 4, and the relative BVDV mRNA levels in the cells were significantly reduced after 36h treatment with 20 μΜ recombinant nanobody (rNb 1) compared to the untreated group, indicating that both recombinant nanobodies were effective in inhibiting BVDV mRNA synthesis.
EXAMPLE 5 recombinant nanobody inhibition of BVDV Virus titres in supernatant
Well-conditioned MDBK cells were grown at 1X 10 5 The cells were spread in 24-well plates and placed in 5% CO at 37 ℃C 2 Is cultured in a cell culture incubator. When the cell density reached about 70%, after 0.1MOI BVDV was inoculated for 2 hours, the cells were washed 3 times with PBS and replaced with DMEM containing 3% FBS, and 0. Mu.M and 20. Mu.M recombinant nanobodies rNb1 were inoculated, and the cell supernatant was collected at 36hpi and passed through TCID 50 The effect of recombinant nanobody rNb1 on BVDV in cell supernatant was determined.
The results are shown in fig. 5, and the virus titer in the cell supernatant was significantly reduced after 36h treatment with 20 μm recombinant nanobody (rNb 1) compared to the untreated group, indicating that both recombinant nanobodies were effective in inhibiting BVDV replication.
Claims (10)
1. A recombinant nano antibody targeting BVDV nonstructural proteins has an amino acid sequence shown in SEQ ID NO. 1.
2. The recombinant nanobody of claim 1, further comprising HA and a histidine tag, wherein the amino acid sequence of the recombinant nanobody is shown in SEQ ID No. 2.
3. A nucleotide encoding the recombinant nanobody of claim 1 or 2.
4. A nucleotide according to claim 3, wherein the nucleotide sequence is as set forth in SQE ID No. 3.
5. A recombinant vector/plasmid, or recombinant cell, comprising nucleotides encoding the nanobody of any of claims 1-2.
6. The method of producing a recombinant nanobody according to any one of claims 1 to 2, wherein the method comprises:
(1) Synthesizing a nucleotide sequence encoding the recombinant nanobody of any of claims 1-2;
(2) Ligating the nucleotide sequence of step (1) with a vector/plasmid to transform an expression competent cell BL21 (DE 3);
(3) And (3) performing induced expression on the expression competence in the induced expression step (2), and purifying to obtain the recombinant nano antibody.
7. The method of preparing a recombinant nanobody of claim 6, wherein the method is: synthesizing a fragment of interest encoding the nucleotide sequence of claim 3; cutting a target fragment and a pET21b empty vector by using Nde I and Xho I, recovering the cut target fragment and vector, connecting and converting the target fragment and vector to competent Trans5a, and selecting positive recombinant plasmid; transferring the recombinant plasmid into expression competent BL21 (DE 3) to induce expression; purifying by using Ni-NTA affinity chromatographic column, and finally obtaining the recombinant nano antibody after renaturation and concentration.
8. Use of the recombinant nanobody according to any one of claims 1-2 for the preparation of a medicament for the prevention or treatment of bovine viral diarrhea virus infection.
9. A composition comprising the recombinant nanobody of any of claims 1-2.
10. The recombinant nanobody of any one of claims 1-2, wherein any pharmaceutically acceptable excipient is added to make any pharmaceutically acceptable dosage form.
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