Preparation method and application of oligopeptide / polypeptide substitution antibody

Abstract

The invention relates to a preparation method and application of an oligopeptide / polypeptide substitution antibody, and belongs to the technical field of antibody preparation. In order to solve the problems that an existing antibody preparation method is high in complexity, poor in stability and limited in platform applicability, the invention provides a preparation method of an oligopeptide / polypeptide substitution antibody, firstly, a random DNA sequence is synthesized and cloned to a two-hybrid system prey vector to construct a peptide library, an antigen protein is cloned to a two-hybrid system bait vector, and then the oligopeptide / polypeptide substitution antibody is obtained. Screening an antigen interaction peptide sequence through bait and prey library two-hybrid interaction, and performing prokaryotic expression purification or direct artificial synthesis on the antigen interaction peptide sequence to obtain a short peptide / polypeptide interacting with an antigen protein as an antibody substitute. According to the invention, ethical dispute and batch difference of animal immunity in traditional antibody preparation are avoided, period is shortened, cost is reduced, non-specific binding is less, sensitivity is greatly improved, plant endogenous non-tag protein can be specifically recognized, and the method is widely applicable to scenes of antibody substitution.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of antibody preparation, and particularly relates to a preparation method of short peptide / polypeptide substitute antibody and application thereof. BACKGROUND

[0002] In protein function research, the core role of antibody is to realize the positioning and detection, interaction research, function regulation and purification and enrichment of target protein by specific recognition and binding with target protein. The high specificity and diversity of antibody make it a core tool for protein function research. The preparation of traditional polyclonal or monoclonal antibody needs to go through animal immunization, or cell fusion and multiple screening, and the cycle is as long as several months. Moreover, there are significant ethical controversies and batch difference risks in the production process, and a large number of animals sacrifice their lives. For complex biological systems such as plants, endogenous secondary metabolites (such as phenols and terpenes) and high-concentration pigments often induce serious non-specific binding in immunodetection, which significantly reduces the reliability of the results.

[0003] In recent years, although certain progress has been made in antibody substitute technologies such as nanobodies and nucleic acid aptamers, their application still faces multiple limitations, including high preparation complexity, poor environmental stability and narrow applicable technology platform. For example, nanobodies still need animal immunization or complex in vitro display screening, and nucleic acid aptamers are easily enzymatically inactivated in plant crude extracts. These limitations make it difficult for existing antibody preparation methods to meet the needs of protein research, and restrict the progress of high-throughput and native state protein research. SUMMARY

[0004] To solve the problems of high complexity, poor environmental stability and narrow applicable technology platform of existing antibody preparation methods, the application provides a preparation method of short peptide / polypeptide substitute antibody and application thereof.

[0005] The technical scheme of the application is as follows:

[0006] A preparation method of short peptide / polypeptide substitute antibody, comprising the following steps:

[0007] Step one, synthesizing a random DNA sequence and cloning it into a prey vector of a two-hybrid system to form a random peptide prey library;

[0008] Step two, cloning an antigen protein into a bait vector of the two-hybrid system to construct a bait recombinant vector;

[0009] Step three, performing two-hybridization of the random peptide prey library in step one and the bait recombinant vector in step two to obtain positive clones;

[0010] Step four, screening and Sanger sequencing the positive clones in step three to deduce the sequence of short peptide / polypeptide.

[0011] Step five, the short peptide / polypeptide sequence predicted in step four is expressed in prokaryotes and purified to obtain a short peptide / polypeptide of the surrogate antibody, or the short peptide / polypeptide is directly synthesized artificially.

[0012] Further, the random DNA sequence in step one has a length of 15-100 bp.

[0013] Further, the double-hybrid system is a yeast double-hybrid system or a bacterial double-hybrid system.

[0014] Further, the antigen protein cloned into the bait vector in step two is a partial sequence or a complete sequence.

[0015] Further, in step four, the positive clones with good growth and preferential blue color change are selected for high-concentration plasmid extraction and Sanger sequencing.

[0016] Further, in step four, the DNA sequence and the amino acid sequence of the short peptide / polypeptide are deduced.

[0017] The application of the preparation method of the short peptide / polypeptide surrogate antibody in protein function research, which includes protein immunolocalization, protein function mechanism analysis, disease diagnosis and treatment, drug development, and protein interaction research.

[0018] Further, the protein function research is a function research of plant proteins.

[0019] Advantages of the present application:

[0020] The present application breaks through the limitations of traditional antibody preparation and innovatively integrates the three-in-one technical route of "synthetic random peptide library-double-hybrid in vivo screening-short peptide / polypeptide purification". Through the double-hybrid system, the in situ interaction of antigen proteins and random short peptides / polypeptides is directly screened under physiological conditions, ensuring that the screened short peptides / polypeptides can accurately recognize the target proteins in the native conformation. Subsequently, the Sanger sequencing is used to analyze the peptide sequence, and the prokaryotic expression or artificial synthesis is used to quickly obtain high-specificity short peptides / polypeptides. This system not only avoids the ethical controversy and batch difference problems in traditional antibody preparation by animal immunization, but also shortens the research and development period from several months to two weeks, reduces the cost to the level of hundreds of yuan, and significantly improves the efficiency and economy.

[0021] The short peptide / polypeptide of the present application exhibits disruptive performance in the unlabeled detection of plant wild type samples. Compared with traditional antibodies, its molecular weight is smaller, which significantly reduces the steric hindrance, thereby improving the binding efficiency in experiments such as immunoprecipitation (IP), Western blotting (WB), etc., while reducing non-specific binding, and greatly improving sensitivity. In addition, this technology does not need to rely on a label protein, and can directly recognize endogenous natural proteins, and is compatible with multi-dimensional platforms such as co-immunoprecipitation (Co-IP), chromatin immunoprecipitation (ChIP), etc., providing a more reliable tool for plant protein interaction network and function research.

[0022] Based on the targeted design capability of the target amino acid sequence, the present application can optimize the short peptide / polypeptide within a specific functional domain, providing customized molecular tools for precise diagnosis and targeted treatment. Its application scenarios far exceed traditional antibodies, for example, it can be widely used in the field of plant science (such as analysis of disease resistance signal transduction and metabolic regulation mechanism), and has the potential for cross-species and multi-scenario applications, and can efficiently construct high-affinity recognition probes for low-abundance targets, complex spatial conformation or special modified proteins, promoting the development of protein function research and precise intervention strategies. In addition, the ethical friendliness and low cost of this method make it have broad potential in basic research and industrial application, and is expected to become a benchmark for antibody replacement technology, promoting the development of molecular biology research towards high efficiency and standardization. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 Figure 1 is a graph of the binding specificity verification results of the protein interaction short peptide / polypeptide, A is the self-activation detection of BpNAC90 protein in the yeast two-hybrid system, B is the specific interaction verification of the short peptide (AD-SPN-1 / 2 / 3) and the corresponding BpNAC90 protein truncated fragment (BD-NAC90-1 / 2 / 3) in the point-to-point yeast two-hybrid system, and C is the interaction verification of each truncated fragment of BpNAC90 (BD-NAC-n) and the short peptide (AD-SPN-m, m≠n) for other segments to combine the specificity;

[0024] Figure 2 Figure 2 is a graph of the short peptide / polypeptide as an antibody for protein IP purification detection, A is the immunoprecipitation of SPN1 short peptide on BpNAC90 prokaryotic expression protein, B is the immunoprecipitation of SPN1 short peptide on target protein BpNAC90 in plant total protein, and C is the Co-IP analysis of SPN1 short peptide as an antibody;

[0025] Figure 3Figure for Western blotting detection of short peptide / polypeptide as antibody to protein expression, A is SPN1 short peptide for detecting overexpressed BpNAC90-Flag tagged protein in transgenic plants, B is SPN1 short peptide for detecting endogenous protein BpNAC90 in wild type plants, C is SPN1 short peptide for IP and subsequent WB detection of endogenous protein BpNAC90;

[0026] Figure 4 Figure for ChIP-Seq analysis of short peptide / polypeptide as antibody, A is SPN1 short peptide and Flag antibody ChIP-seq quality comparison, B is ChIP-seq analysis of wild type white birch using SPN1 short peptide antibody;

[0027] Figure 5 Figure for mechanism of short peptide / polypeptide recognizing protein, A is conserved motif of SPN1 short peptide recognizing protein, B is interaction of SPN1 and motif "VSFYLH" and BpNAC90 lacking the motif. DETAILED DESCRIPTION

[0028] The technical solutions of the present application are further described below in conjunction with examples, but are not limited thereto, any modification or equivalent replacement to the technical solutions of the present application without departing from the spirit and scope of the technical solutions of the present application shall be encompassed in the protection scope of the present application. The process equipment or device not specifically mentioned in the following examples all adopts the conventional equipment or device in the art, and the raw materials used in the examples of the present application are all commercially available if not specifically mentioned; and the technical means used in the examples of the present application are all conventional means well known to those skilled in the art if not specifically mentioned.

[0029] Example 1

[0030] The present embodiment provides a preparation method of short peptide / polypeptide replacing antibody, taking BpNAC90 protein as target protein, and preparing short peptide / polypeptide antibody recognizing the same.

[0031] The specific operation steps of the present embodiment are as follows:

[0032] Step one, a DNA sequence encoding a short peptide / polypeptide with a length of 90 nt of a random base sequence is synthesized by Shengong Bioengineering (Shanghai) Co., Ltd., a homologous arm of a yeast two-hybrid vector pGADT7-Rec is introduced into the DNA sequence encoding the short peptide / polypeptide by using conventional PCR method in the art, and a DNA sequence encoding the short peptide / polypeptide with a homologous arm is obtained. The DNA sequence encoding the short peptide / polypeptide with a homologous arm is constructed into the yeast two-hybrid vector pGADT7-Rec by using conventional homologous recombination method in the art, and an AD-short peptide / polypeptide library is obtained.

[0033] Step two, BpNAC90 protein as antigen protein, its sequence into three segments, using the conventional homologous recombination method in the art, the different segment sequences are constructed into the yeast two-hybrid vector pGBKT7, get three BD clone vector BD-NAC90-1, BD-NAC90-2, BD-NAC90-3.

[0034] Step three, the AD-short peptide / polypeptide library obtained in step one and the three BD clone vectors obtained in step two are subjected to yeast two-hybrid.

[0035] The method of yeast two-hybrid of the BD clone vector and the AD-short peptide / polypeptide library in this embodiment is as follows:

[0036] (1) Saccharomyces cerevisiae strain Y2HGold is selected as a model organism for yeast two-hybrid.

[0037] (2) Prepare the standard library transformation premix, take 2520 μL of the premix and add it to a pre-cooled 50 mL centrifuge tube, mix with 1000 μL of Y2HGold competent cell precipitate (OD600≈1.5). Mix well with a pipette.

[0038] The formula of the standard library transformation premix used is: 1680 μL PEG Solution + 252 μL LiAc Solution (10×) + 40 μL Carrier DNA + 10 μL BD clone vector + 38 μL AD-short peptide / polypeptide library + 500 μL sterile deionized water.

[0039] (3) Place the resuspension in a constant temperature water bath, incubate at 30±0.5℃ for 50 minutes. To ensure transformation efficiency, take it out every 10 minutes, invert to mix well to maintain uniformity of the system.

[0040] (4) After incubation, add 160 μL DMSO to the transformation system, immediately transfer to a precisely temperature-controlled 42±0.2℃ water bath for heat shock for 30 minutes, mix well at a frequency of inverting every 10 minutes. After heat shock, centrifuge at 3000 rpm for 5 minutes, carefully discard the supernatant.

[0041] (5) Resuspend the pellet with 3 mL of YPD Plus recovery medium, transfer to a 50 mL sterile centrifuge tube, incubate in a constant temperature shaker at 30℃, 220 rpm for 120 minutes to activate the expression of resistance genes, and centrifuge at 3000 rpm for 5 minutes at room temperature after recovery to collect the bacterial cells.

[0042] (6) Resuspend the pellet with 10 mL of sterile deionized water or 0.9% saline, evenly spread 200 μL of the bacterial solution per plate on agar plates (diameter 150 mm, containing 2% agar).

[0043] Step four, the fusion protein after yeast two-hybrid in step three was cultured, and positive single colonies with rapid growth and rapid blue change were selected. The plasmid was extracted for Sanger sequencing, and the short peptide / polypeptide sequence after sequencing was screened to obtain the short peptide / polypeptide sequence interacting with BpNAC90. The short peptide / polypeptide was cloned by using the conventional PCR method in the art.

[0044] The culture conditions of the yeast two-hybrid fusion protein in this example are as follows:

[0045] According to the complexity requirement of the library, it is usually necessary to coat ≥40 plates to achieve library coverage. Invert culture at 30°C for 72-96 hours until single colonies appear, and the colony density is 200-300 / plate.

[0046] The screening method of positive clones is as follows:

[0047] The yeast colonies grown on the transformation plate were transferred to the SD / -Trp / -His / -Leu+X-α-gal quadruple deficiency screening medium, and after 72-96 hours of inverted culture at 30°C, single colonies with significant blue change and good growth were selected.

[0048] The screening medium used is: 6.7 g / L Yeast Nitrogen Base+20 g / L glucose+20 mg / L 13 kinds of amino acids except Try / His / Leu and uracil+40 mg / L X-α-gal stain (dissolved in DMF solvent)+20 g / L agar powder, pH 5.8±0.1.

[0049] The primer used for Sanger sequencing in this example is AD-Specific Sequencing Primer:

[0050] The F end is shown as SEQ ID No: 1:

[0051] 5'-CTATTCGATGATGAAGATACCCCACCAAACCC-3';

[0052] The R end is shown as SEQ ID No: 2:

[0053] 3'-GTGAACTTGCGGGGTTTTTCAGTATCTACGATT-5'.

[0054] After sequencing, the vector sequence was removed using the NCBI online software, the length of the short peptide / polypeptide sequence was compared, the short peptide / polypeptide with a length of 90 bp was selected, the coding region was analyzed, and cloning was performed.

[0055] Step five, directly synthesizing the short peptide / polypeptide. Or, the short peptide / polypeptide sequence obtained in step four is expressed in prokaryotes. In this embodiment, the prokaryotic expression pMAL-C5x vector containing maltose binding protein (MBP) is selected for prokaryotic expression. The short peptide / polypeptide is constructed into the pMAL-C5x vector, and the E. coli ER2523 competent strain is transformed. The E. coli ER2523 strain carrying the short peptide / polypeptide expression vector is inoculated in LB liquid medium containing ampicillin (Amp) for culture. When the OD600 value of the bacterial solution reaches 0.6, 0.1 mM of isopropyl-β-D-thiogalactoside (IPTG) is added to induce the expression of the fusion gene, and the culture is continued at 37°C for 2 hours. The purified protein obtained by using the MBP-containing agarose beads is the short peptide / polypeptide antibody against BD-BpNAC90.

[0056] I. Detection of BpNAC90 transcriptional activation activity and verification of specificity of short peptide / polypeptide binding by yeast two-hybrid

[0057] To evaluate the transcriptional activity of BpNAC90 and determine its transactivation domain, the BpNAC90 gene is cloned into the pGBKT7 vector to construct pGBKT7-BpNAC90. The constructed plasmid pGBKT7-BpNAC90 and the empty vector pGBKT7 are transformed into yeast Y2H cells, respectively. The yeast transformant carrying pGBKT7-BpNAC90 can grow normally on SD / -Trp medium, but cannot grow normally on SD / -Trp / -His / -Leu / X-α-Gal medium, indicating that BpNAC90 does not have transcriptional activation activity, as shown in the results of Figure 1 A.

[0058] To find the short peptide / polypeptide that specifically recognizes BpNAC90, the BpNAC90 protein is divided into three segments in this embodiment, and each segment is subjected to yeast two-hybrid screening for interacting peptides (BD-NAC90-1, BD-NAC90-2, and BD-NAC90-3) with a random peptide library. After 3 days of culture at 30°C, single colonies that grow normally and turn blue are selected for Sanger sequencing. Then, according to the sequencing results, the possible peptide sequences are deduced, and primers are synthesized according to the deduced sequences. The primers are re-cloned into the pGADT7-Rec vector to obtain the corresponding vectors AD-SPN1, AD-SPN2, and AD-SPN3 (interacting with the three truncated sequences of BpNAC90, respectively). Then, the point-to-point yeast two-hybrid interaction verification is performed again with BD-NAC90-1, BD-NAC90-2, and BD-NAC90-3. The results show that the above truncated proteins can interact with the corresponding short peptide / polypeptide, as shown in Figure 1B. And when the truncated protein sequence was interacted with other two screened short peptide / polypeptide sequences of the same protein, respectively, it was found that they did not interact, as shown in Figure 1 C, indicating that the interaction of short peptide / polypeptide and protein truncated sequence is specific.

[0059] II. Application of short peptide / polypeptide as antibody

[0060] To verify the antibody function of short peptide / polypeptide, the screened short peptide / polypeptide SPN1, SPN2 and SPN3 were first fused with Strep tagII tag for prokaryotic expression, and MBP-BpNAC90 recombinant protein was constructed, and MBP tag antibody and different short peptide / polypeptide combinations (SPN1, SPN2, SPN3, SPN1+2+3) were used for immunization / binding precipitation. The results showed that all short peptide / polypeptides could specifically capture BpNAC90. The non-specific band of short peptide / polypeptide IP product was significantly reduced, and the purification efficiency of different short peptide / polypeptide combinations had no significant difference, as shown in Figure 2 A. Further verification in plant system: total protein of overexpressing Flag-BpNAC90 white birch was extracted, and anti-Flag antibody and SPN1 short peptide / polypeptide were used for IP. Western blotting (WB) detection showed that the target band of short peptide / polypeptide IP product was more specific, and the non-specific background was significantly lower than that of tag antibody, as shown in Figure 2 B.

[0061] To further detect the effect of short peptide / polypeptide as antibody for CoIP, the interaction protein BpSRK2A of BpNAC90 was found, His-SPN1 was used as antibody, Strep tagII-BpSRK2A and Flag-BpNAC90 were co-transformed into white birch, and the interaction complex was precipitated by His magnetic beads. WB detection (anti-Flag / anti-Strep tagII antibody) confirmed that BpNAC90 interacted with BpSRK2A, as shown in Figure 2 C, the results were consistent with the control group of tag antibody, indicating that short peptide / polypeptide could replace antibody to complete CoIP detection.

[0062] To test whether short peptide / polypeptide can be used as antibody for WB detection, SPN1-Strep tagII was used to detect total protein of overexpressing Flag-BpNAC90 white birch, and the target band was consistent with anti-Flag antibody, but the non-specific band was reduced, as shown in Figure 3 A. The key breakthrough is the detection of wild type white birch (without tag): short peptide / polypeptide SPN1 successfully detected endogenous BpNAC90 protein, while tag antibody had no signal, as shown in Figure 3As shown in B. Further IP-WB serial experiments were performed on birch nucleus protein using SPN1 to confirm its ability to directly bind to the endogenous protein BpNAC90, as shown in Figure B. Figure 3 As shown in C. This not only demonstrates that short peptides / polypeptides can be used as antibodies for Western blotting detection, with significantly improved specificity compared to conventional labeled antibodies, but also provides a new method for the extraction and detection of label-free proteins.

[0063] ChIP-Seq comparisons showed that the peak coverage depth of the short peptide / polypeptide antibody (SPN1) in Flag-BpNAC90 transgenic plants was significantly higher than that of the Flag antibody. Figure 4 As shown in A. Furthermore, tag-free ChIP-Seq was achieved in wild-type birch, with Peaks enrichment significantly higher than the Input control, such as... Figure 4 As shown in B, this provides a new tool for studying natural protein interactions.

[0064] III. Mechanisms by which short peptides / peptides recognize protein sequences

[0065] To verify whether short peptide / peptide recognition specifically binds to a particular segment of a protein, SPN1 was used as a bait and subjected to yeast two-hybrid assay with an AD-Birch cDNA library. The obtained single clones were sequenced and analyzed for conserved domains. It was found that all proteins recognized by SPN1 contained the conserved domain "VSFYLH". The yeast two-hybrid results confirmed the interaction between SPN1 and VSFYLH. However, after removing the short peptide / peptide recognition sequence from BpNAC90, it was found that the SPN1 short peptide / peptide could not interact with the BpNAC90 protein. This indicates that the short peptide / peptide specifically recognizes a specific amino acid sequence of a protein, as shown in the results. Figure 5 As shown.

Claims

1. A method for producing a short peptide / polypeptide surrogate antibody, characterized by, The method comprises the following steps: Step 1: synthesizing a random DNA sequence and cloning the random DNA sequence into a prey vector of a double-hybrid system to form a random peptide prey library; Step 2: cloning an antigen protein into a bait vector of the double-hybrid system to construct a bait recombinant vector; Step 3: performing double-hybridization between the random peptide prey library of step 1 and the bait recombinant vector of step 2 to obtain positive clones; Step 4: screening and Sanger sequencing the positive clones in step 3 to deduce the sequence of a short peptide / polypeptide; Step 5: performing prokaryotic expression and purification on the sequence of the short peptide / polypeptide deduced in step 4 to obtain a short peptide / polypeptide replacing an antibody, or directly synthesizing the short peptide / polypeptide.

2. The method of claim 1, wherein the short peptide / polypeptide is selected from the group consisting of SEQ ID NOs: 1 to 6. The random DNA sequence in step 1 has a length of 15-100 bp.

3. The method for preparing a short peptide / polypeptide surrogate antibody according to claim 1 or 2, characterized in that, The double-hybrid system is a yeast double-hybrid system or a bacterial double-hybrid system.

4. The method of claim 3, wherein the short peptide / polypeptide is selected from the group consisting of SEQ ID NOs: 1 to 6. The antigen protein cloned into the bait vector in step 2 is a partial sequence or a complete sequence.

5. The method of claim 4, wherein the short peptide / polypeptide is selected from the group consisting of SEQ ID NOs: 1 to 6. In step 4, the positive clones with good growth and priority to turn blue are selected to extract high-concentration plasmids and perform Sanger sequencing.

6. The method for preparing a short peptide / polypeptide substitute antibody according to claim 5, characterized in that, In step 4, the DNA sequence and the amino acid sequence of the short peptide / polypeptide are deduced.

7. Use of a method for the production of a short peptide / polypeptide surrogate antibody according to any one of claims 1 to 6 in the study of protein function, characterized in that, The protein research includes protein immunolocalization, protein function mechanism analysis, disease diagnosis and treatment, drug development and protein interaction research.

8. The use of the method for preparing a short peptide / polypeptide surrogate antibody according to claim 7 in the study of protein function, characterized in that, The protein function research is a function research of plant proteins.

Images