Cre-specific shark single-domain antibody and application thereof

Abstract

The invention discloses a Cre specific shark source single-domain antibody, the Cre specific shark source single-domain antibody comprises a Cre specific shark source single-domain antibody 1 to a Cre specific shark source single-domain antibody 5, and the amino acid sequences of variable regions VNAR of the Cre specific shark source single-domain antibody 1 to the Cre specific shark source single-domain antibody 5 are respectively as shown in SEQ ID NO. 1 to SEQ ID NO. 5. The nucleotide sequences of variable regions VNAR of the shark source single-domain antibody 1 to the shark source single-domain antibody 5 with the coded Cre specificity are respectively as shown in SEQ ID NO.6 to SEQ ID NO.10. The invention further discloses a preparation method of the shark source single-domain antibody 5 with the coded Cre specificity. The shark-source single-domain antibody disclosed by the invention is high in stability and low in immunogenicity, can be expressed in cells and exert functions, provides the shark-source single-domain antibody for marking Cre tool enzyme, and further promotes the development of a Cre tool enzyme system.

Description

Technical Field

[0001] The present invention belongs to the field of single domain antibody technology development, and specifically relates to a Cre-specific shark single domain antibody, and more specifically, to a shark single domain antibody that specifically binds to the Cre recombinase and its nucleotide sequence. The present invention also relates to a recombinant plasmid comprising the nucleotide coding sequence. Background Art

[0002] Antibodies (also called immunoglobulins) are proteins produced by B cells that can recognize and bind to specific antigens. Antibodies have a wide range of functions, including immune response, antibody-dependent cell-mediated cytotoxicity, antibody-dependent cellular phagocytosis, etc. Traditional antibodies are mainly composed of two heavy chains and two light chains, forming a Y-shaped structure, and specifically recognize antigens through their variable regions. Single-domain antibodies (sdAbs) are a special form of the antibody family. They contain only one antigen-binding domain and are usually composed of heavy chain single-domain antibodies (VHH) or shark single-domain antibodies (VNARs). Due to their advantages such as small size, stability and high affinity, single-domain antibodies have broad application prospects in biomedical research and clinical treatment.

[0003] Shark single-domain antibodies (VNAR, Variable New Antigen Receptor) are antibody molecules unique to the shark immune system and belong to the single-domain antibody family. VNAR is composed of a single variable region and has antigen-binding ability similar to traditional antibodies, but its structure is significantly different from mammalian antibodies. The advantages of shark single-domain antibodies are their small size, excellent stability, strong thermal stability and adaptability to extreme environments. These characteristics make VNAR more flexible in application, especially in experimental and clinical environments that require high stability and affinity. VNAR shows unique advantages as an antibody substitute.

[0004] Cre recombinase is a DNA recombinase protein derived from bacteriophage P1 that can specifically recognize and cut loxP sites in DNA, thereby achieving directional recombination of the genome. The Cre / loxP system is widely used in the field of gene editing, especially in model organisms such as mice. By activating Cre recombinase in specific tissues or cell types, conditional knockout or activation of genes can be achieved. Cre recombinase not only has important applications in basic research, but also has shown great potential in many fields such as gene therapy and cell research.

[0005] Antibodies against Cre recombinase are mainly used to detect the expression and localization of Cre protein in vitro, such as analysis by Western blot (WB), immunofluorescence (IF) and other techniques. In addition, anti-Cre recombinase antibodies can also be used to label Cre recombinase in vivo to assist in studying its distribution and function in vivo. At present, the antibodies against Cre recombinase on the market are mainly traditional IgG antibodies, while single-domain antibodies (such as VNAR) have not yet been widely used in this field. Since single-domain antibodies have the advantages of small size, high stability, low immunogenicity and good affinity, the development of specific antibodies against Cre recombinase based on shark single-domain antibodies has important research value and potential application prospects. Summary of the invention

[0006] The technical problem to be solved by the present invention is to provide a shark-derived single-domain antibody that specifically binds to the Cre recombinase, and at the same time provide a coding sequence of the single-domain antibody and a plasmid containing the coding sequence. The shark-derived single domain provided by the present invention is obtained by multiple methods including next-generation sequencing technology (NGS), cell screening, phage display screening and mass spectrometry, has high stability, small molecular weight, and is easy to transform.

[0007] Therefore, on one hand, the present invention discloses a Cre-specific shark-derived single domain antibody, wherein the Cre-specific shark-derived single domain antibody includes Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5, wherein the amino acid sequences of the variable regions VNAR of Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5 are shown as SEQ ID NO.1 to SEQ ID NO.5, respectively.

[0008] Preferably, the nucleotide sequences encoding the variable regions VNAR of Cre-specific shark-derived single domain antibodies 1 to Cre-specific shark-derived single domain antibodies 5 of the present invention are shown as SEQ ID NO.6 to SEQ ID NO.10, respectively.

[0009] In one aspect, the present invention further discloses a recombinant plasmid, wherein the recombinant plasmid contains any one of the nucleotide sequences described in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10.

[0010] In one aspect, the present invention further discloses a use of the Cre-specific shark-derived single-domain antibody 1 to the Cre-specific shark-derived single-domain antibody 5 in the detection of Cre protein.

[0011] Preferably, the Cre protein detection of the present invention is Western-Blot detection of Cre protein.

[0012] Preferably, the Cre protein detection of the present invention is flow cytometry or immunofluorescence detection of Cre protein.

[0013] Preferably, the Cre protein detection of the present invention is ELISA detection of Cre protein.

[0014] Beneficial effects of the present invention: The present invention provides shark-derived single-domain antibodies for in vitro and in vivo detection and labeling of Cre recombinase, which can be widely used in various scenarios such as Western-Blot, flow cytometry and immunofluorescence to achieve specific labeling of Cre protein. Compared with traditional antibodies, the changes in amino acids in the FR2 region of VH make VNAR more excellent in terms of anti-enzymatic hydrolysis, polymorphic stability, and protein folding and unfolding, and its physicochemical properties are more stable. In addition, shark single-domain antibodies are expressed and function in mammalian cells, which is convenient for rapid Cre recombinase labeling directly in vivo. At the same time, it can exert binding activity in cells, which also shows that single-domain antibodies have strong anti-interference ability under complex conditions. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the SDS-PAGE result of Cre antigen purification.

[0016] Figure 2 This is a statistical analysis of shark-derived single-domain antibodies that were screened for Cre specificity by NGS.

[0017] Figure 3 This is the result of phage ELISA screening of Cre-specific shark-derived single-domain antibodies through phage display.

[0018] Figure 4 This is the result of monoclonal ELISA screening of Cre-specific shark-derived single-domain antibodies by phage display.

[0019] Figure 5 These are partial peptide fragments of Cre-specific shark-derived single-domain antibodies detected by mass spectrometry in shark serum after immunization.

[0020] Figure 6 It is the Western-Blot result of detecting the expression of 5 Cre-specific shark single-domain antibody proteins (SEQ ID NO.1 to SEQ ID NO.5) in 293T cells.

[0021] Figure 7 This is a fluorescence image of five Cre-specific shark single-domain antibody proteins (SEQ ID NO.1 to SEQ ID NO.5) combined with Cre in 293T cells to generate EGFP reporter.

[0022] Figure 8The figure is a flow cytometric graph showing that five Cre-specific shark single-domain antibody proteins (SEQ ID NO. 1 to SEQ ID NO. 5) bind to Cre in 293T cells to generate EGFP reporter. DETAILED DESCRIPTION

[0023] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0024] The "include" or "comprising" described in the present invention is an open description, which contains the specified components or steps described, as well as other specified components or steps that do not substantially affect the technical effect. When it is used in this application to describe the sequence of a protein or nucleic acid, the protein or nucleic acid may be composed of the sequence, or may have additional amino acids or nucleotides at one or both ends of the protein or nucleic acid, but still have the activity described in the present invention.

[0025] The present inventors have conducted extensive and in-depth research and, after a large number of screenings, discovered a class of shark-derived single-domain antibodies specific to Cre recombinase, which can specifically recognize Cre recombinase.

[0026] Herein, the terms "single domain antibody", "anti-Cre single domain antibody", "Cre-specific single domain antibody" and "heavy chain antibody variable region domain" can be used interchangeably, and mean single domain antibodies that specifically recognize and bind to Cre recombinase. Single domain antibodies are the variable regions of heavy chain antibodies.

[0027] In order to enable those skilled in the art to better understand the technical solution of the present invention, the preferred embodiments of the present invention are described in detail below, but the following embodiments do not limit the protection scope of the present invention.

[0028] In the embodiments of the present invention, conventional experimental methods are used without further explanation. The processes involved in the embodiments without further explanation are all understandable and easily implementable by those skilled in the art based on the product instructions or basic knowledge in the field. The reagents or instruments used without indicating the manufacturer are conventional products that can be purchased commercially, and therefore will not be described in detail.

[0029] Example 1: Purification of antigens and immunization of bamboo sharks

[0030] 1. Construction of recombinant expression plasmid: The gene sequence (Gene ID: 2777477) was inserted into the expression vector pET-28a-TrxA-SUMO by double enzyme digestion to obtain the pET-28a-Cre recombinant plasmid, and then the recombinant plasmid was transformed into BL21Star (DE3) cells.

[0031] 2. Induced expression and purification of recombinant proteins

[0032] 1. Inoculate the recombinant expression bacteria BL21 Star (DE3)-pET-28a-Cre into LB medium containing Amp resistance at a ratio of 1:100, and culture at 37°C and 220 rpm for 4 hours.

[0033] 2. Add IPTG to make the final concentration of IPTG 0.1-0.5 mM, preferably 0.2 mM, and continue culturing at 37°C for 4-6 h (preferably 5 h) and 16°C for 14-16 h (preferably 16 h).

[0034] 3. After induction, centrifuge the bacterial pellet and resuspend it in 1×PBS (40ml PBS per 1L cells), and use a high-pressure cell disruptor to lyse it at a pressure of 800bar. After centrifugation, incubate the lysate supernatant with Ni-NTA beads at 4°C for 3 hours, wash 3 times with 50mM imidazole solution, and elute with 300mM imidazole solution. Add SUMO protease (Ulp1) and digest it at 30°C for 2h, and remove the digested solution using a Ni-NTA column. The SDS-PAGE test results of the purified Cre antigen are as follows: Figure 1 As shown, its purity is above 90%, which is suitable for subsequent immune experiments.

[0035] Cre antigen was diluted to 1 μg / ml and mixed with an equal volume of biphasic adjuvant to immunize bamboo sharks. The inoculation site was the intersection of the fins and abdomen on both sides. The control group used BSA to immunize sharks. Immunization was performed every two weeks, with the mass of the antigen being 100 μg each time, for a total of 8 times.

[0036] Example 2: Screening of Cre-specific shark-derived single domain antibodies by next generation sequencing (NGS)

[0037] The spleen and PBMC of the sharks were taken after immunization, and RNA from the spleen and PBMC of the sharks was extracted for RT-PCR to obtain cDNA libraries. The cDNA libraries were constructed by high-throughput sequencing (NGS) using the VNAR library construction primers. The sequencing libraries were prepared using the method recommended by the manufacturer. Ultra TMII DNA library preparation kit (NEB, USA). Indexes were added to both ends of each library sample for subsequent differentiation. The library was quantified by qPCR (concentration of 1.5 nM), and only qualified libraries were submitted to the Illumina platform for sequencing using the PE250 strategy based on the required effective library concentration and the required data volume.

[0038] The data from the machine were analyzed and processed, and the complete shark VNAR sequence obtained by each group was finally spliced ​​together ( Figure 2 ). About 5000-7000 full-length VNAR sequences were screened from the Cre and BSA immunization groups, and the classification of these VNARs revealed that most of them belonged to the IgNAR1 type. NGS can quickly obtain the sequence and abundance information of single-domain antibodies produced by sharks after immunization, greatly saving time and cost.

[0039] Example 3: Screening of Cre-specific shark-derived single domain antibodies by phage display

[0040] The VNAR library for phage display was prepared using a two-round PCR method using VNAR library construction primers. PCR products of 500 to 600 bp were recovered in the first PCR reaction, and products of 200 to 400 bp were recovered in the second PCR reaction. The recovered PCR products were digested with Sfi I and connected to the p3RdV vector, and then transformed into SS320 cells. M13KO7 was used to assist phage packaging in SS320. The resulting phage library was screened and enriched for 3-4 rounds using Cre antigen. It can be seen from phage ELISA that the library showed significant enrichment from the first round of panning, and the degree of enrichment of the library tended to be stable from the second to the third round of panning ( Figure 3 ). A total of 88 single clones were selected from the third and fourth round panning plates with good panning enrichment for IPTG induction, and the supernatant after induction was tested by ELISA to detect the absorbance value of OD450. The red dotted line is the average absorbance value of all clones, and the green dots are the absorbance values ​​of the wells with absorbance values ​​higher than the average of the whole plate ( Figure 4 ).

[0041] Example 4: Detection of Cre-specific shark-derived single-domain antibody peptides from immunized shark serum using mass spectrometry

[0042] Serum was separated from shark blood, incubated with Avi-labeled TrxA-SUMO protein at room temperature for 2 hours, and then streptavidin magnetic beads were added for overnight incubation at 4°C. The next day, the magnetic beads were removed by a magnetic rack to remove the IgNAR bound to the TrxA-SUMO tag. The Avi-labeled TrxA-SUMO-Cre antigen was added to the supernatant and incubated at room temperature for 2 hours, then streptavidin magnetic beads were added again and incubated overnight at 4°C. The magnetic beads were washed 3-4 times with 1×PBS (pH 7.4) containing 0.05% Tween-20, the bound IgNAR was eluted, and mass spectrometry was performed after digestion. Subsequently, the obtained peptides were compared with the high-throughput sequencing results to find the corresponding complete IgNAR antibody sequence. The results are as follows. Figure 5 As shown in the figure, the diversity of VNAR is mainly composed of CDR1, HV2, HV4 and CDR3, and the sequences of the remaining peptides are relatively conservative. Among them, the diversity of the sequence at CDR3 is the richest, and it contributes the most to the types of VNAR.

[0043] Based on the above results, this study screened and obtained 5 better Cre-specific shark-derived single domain antibodies, which were labeled as Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5, and the amino acid sequences of their variable regions VNAR are shown in SEQ ID NO.1 to SEQ ID NO.5, respectively.

[0044] Example 5: Construction of single domain antibody eukaryotic expression vector

[0045] The VNAR sequences obtained by screening were optimized according to human codons (the optimized nucleotide sequences of Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5 are shown in SEQ ID NO.6 to SEQ ID NO.10, respectively), and the optimized DNA sequences were synthesized. The synthesized DNA was cloned into the expression vector pcNDA3.1-VP64 using a seamless cloning kit, and transformed into DH5α cells to pick clones and sequence. Subsequently, an endotoxin-free extraction kit was used to extract the endotoxin-free plasmid for cell transfection.

[0046] Example 6: Verification of the ability of the screened single domain antibodies to bind to Cre protein in mammalian cells

[0047] The single domain antibodies 1 to 5 obtained by the above expression screening were fused with VP64 and co-transfected with Cre and dLox-EGFP plasmids into 293T cells. Cre can bind to the dLox site. If the VNAR obtained by the screening can bind to the Cre protein, VP64 will activate the EGFP reporter. From the Western-Blot results ( Figure 6) It can be seen that all VNARs can be well expressed in cells, providing a basis for their subsequent functional activity in cells. From the fluorescence detection graph 48h after transfection ( Figure 7 ) As can be seen, VNAR can bind to Cre and activate the EGFP reporter, which is further tested by flow cytometry ( Figure 8 ), the VNAR of SEQ ID NO.2 has the best binding and reporting ability to Cre. The above results show that the VNAR obtained by screening can specifically bind to Cre.

[0048] It should be noted that the inventors have also conducted comparative studies on the current monoclonal antibody against Cre (ab92452), and found that its expression in the above-mentioned cells is relatively difficult (monoclonal antibodies are relatively large and difficult to operate), and when the heavy chain thereof was used for the same experiment, it was found that it did not have a very good application effect (not as obvious as the effects of the 5 single-domain antibodies disclosed in the present invention), which may be due to its insufficient sensitivity and insufficient binding specificity.

[0049] In addition, it should be noted that the Cre-specific shark-derived single-domain antibodies 1 to Cre-specific shark-derived single-domain antibodies 5 screened by the present invention can be used in a variety of scenarios. In Western-Blot, these 5 strains of Cre-specific shark-derived single-domain antibodies can be used as primary antibodies to detect the expression of Cre protein, and they have good sensitivity and specificity. In experiments such as flow cytometry or immunofluorescence, after penetrating the cells, these 5 strains of Cre-specific shark-derived single-domain antibodies can achieve specific labeling detection of Cre protein, and they have good sensitivity and specificity. In ELISA experiments, these 5 strains of Cre-specific shark-derived single-domain antibodies can detect Cre protein in coated samples, which is also reflected in Example 3. The five Cre-specific shark-derived single-domain antibodies expressed in prokaryotes can be used as primary antibodies to bind to Cre protein. During expression, the five Cre-specific shark-derived single-domain antibodies are fused with the Flag tag and can be bound to VNAR through the HRP-Anti-Flag secondary antibody, and then developed with TMB colorimetric solution to read the OD450 value, which has good sensitivity and specificity.

[0050] Finally, it should be noted that the present invention is not limited to the above embodiments, and there are many variations. All variations that can be directly derived or associated with the content disclosed by ordinary technicians in this field should be considered as the protection scope of the present invention.

Claims

1. A Cre-specific shark-derived single-domain antibody, characterized in that: The Cre-specific shark-derived single domain antibodies include Cre-specific shark-derived single domain antibodies 1 to Cre-specific shark-derived single domain antibodies 5, wherein the amino acid sequences of the variable regions VNAR of Cre-specific shark-derived single domain antibodies 1 to Cre-specific shark-derived single domain antibodies 5 are shown as SEQ ID NO.1 to SEQ ID NO.5, respectively.

2. The single domain antibody according to claim 1, characterized in that The nucleotide sequences encoding the variable regions VNAR of Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5 are shown as SEQ ID NO.6 to SEQ ID NO.10, respectively.

3. A recombinant plasmid, characterized in that: The recombinant plasmid contains any one of the nucleotide sequences described in SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 and SEQ ID NO.

10.

4. Use of the Cre-specific shark-derived single domain antibody 1 to Cre-specific shark-derived single domain antibody 5 as claimed in claim 1 in the detection of Cre protein.

5. The use according to claim 4, characterized in that: The Cre protein detection is Western-Blot detection of Cre protein.

6. The use according to claim 4, characterized in that: The Cre protein detection is flow cytometry or immunofluorescence detection of Cre protein.

7. The use according to claim 4, characterized in that: The Cre protein detection is ELISA detection of Cre protein.

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