CN114478786A - Preparation method of PI3K nano antibody - Google Patents
Preparation method of PI3K nano antibody Download PDFInfo
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- C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/005—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies constructed by phage libraries
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- 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
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1037—Screening libraries presented on the surface of microorganisms, e.g. phage display, E. coli display
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- C12N15/09—Recombinant DNA-technology
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- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C40B30/00—Methods of screening libraries
- C40B30/04—Methods of screening libraries by measuring the ability to specifically bind a target molecule, e.g. antibody-antigen binding, receptor-ligand binding
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- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
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- C07K2317/00—Immunoglobulins specific features
- C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
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- C07—ORGANIC CHEMISTRY
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- C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
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- C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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- C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
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Abstract
The invention discloses a preparation method of a PI3K nano antibody, and relates to the technical field of nano antibody preparation. The preparation method comprises the following steps: and constructing a nano antibody phage display library, and then carrying out screening and prokaryotic expression on the PI3K nano antibody based on the nano antibody phage display library. Compared with the conventional antibody, the PI3K nano antibody prepared by the preparation method provided by the invention has the advantages of strong stability, good solubility, good antigen binding property, easiness in expression, easiness in mass preparation and the like.
Description
Technical Field
The invention relates to the technical field of preparation of nano antibodies, in particular to a preparation method of a PI3K nano antibody.
Background
The nano antibody (Nb) is a natural single-domain antibody, is the smallest known functional antigen specific binding fragment at present, and is easy to produce through genetic engineering. Nb has high specificity, high affinity, low immunogenicity, high solubility and high stability at room temperature, and strong tissue penetration ability, thereby improving the efficacy of drug delivery systems. Compared with the traditional antibody, Nb has advantages in the aspects of targeted drug delivery and tumor targeting, has huge clinical application potential, and can improve the performance of targeting deep tissue tumor in PDT.
Currently, methods for obtaining antibodies include monoclonal antibodies, ribosome display libraries, phage display libraries, etc., wherein the most widely used technique is phage display libraries. However, currently, bacterial transformation and phage packaging are required in the phage display process, and some display systems are also required to be subjected to transmembrane secretion, so that the capacity and molecular diversity of the constructed library are greatly limited. The number of different sequence molecules contained in commonly used phage display libraries is generally limited to 109. Once the phage display library is built, effective in vitro mutation and recombination are difficult to perform, so that the molecular genetic diversity in the library is limited, and an antibody sequence is lost.
PI3K is an intracellular phosphatidylinositol kinase, which is related to the products of oncogenes such as v.src and v.ras, and PI3K itself has serine/threonine (Ser/Thr) kinase activity and also phosphatidylinositol kinase activity. Is composed of regulatory subunit p85 and catalytic subunit p 110.
PI3K itself has serine/threonine (Ser/Thr) kinase activity, as well as phosphatidylinositol kinase activity. PI3K can be classified into 3 types, and its structure and function are different. The most widely studied of these is class I PI3K, which is a heterodimer of PI3K consisting of a regulatory subunit and a catalytic subunit. The regulatory subunit contains domains of SH2 and SH3 and interacts with a target protein containing a corresponding binding site. This subunit is commonly referred to as p85, referenced to the first discovered subtype (isotype). There are 4 catalytic subunits, p110 α, β, δ, γ, whereas δ, γ are restricted to leukocytes, the remainder being widely distributed in various cells. Is composed of regulatory subunit p85 and catalytic subunit p 110.
At present, the preparation of PI3K nano antibodies still exists in the preclinical development stage, and has the problems of poor affinity and poor stability.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a preparation method of a PI3K nano antibody to solve the technical problems.
The invention is realized by the following steps:
the invention provides a preparation method of a PI3K nano antibody, which comprises the following steps: constructing a nano antibody phage display library, and then carrying out screening and prokaryotic expression on the PI3K nano antibody based on the nano antibody phage display library;
the construction of the nano antibody phage display library comprises the steps of taking RNA of peripheral blood cells of an immune animal as a template, obtaining cDNA through reverse transcription, carrying out PCR amplification by taking a cDNA chain as a template to obtain a VHH fragment of the immune animal, and connecting the VHH fragment of the immune animal with a display carrier to construct the nano antibody display library; the PCR amplification was two rounds of PCR.
Compared with the conventional antibody, the PI3K nano antibody prepared by the preparation method provided by the invention has the advantages of strong stability, good solubility, good antigen binding property, easy expression and easier mass preparation.
In a preferred embodiment of the invention, the immunized animal is selected from the group consisting of camelids.
In an alternative embodiment, the camelid is selected from a camel or an alpaca.
In a preferred embodiment of the present invention, the display vector is selected from phage display vectors.
The phage display technology has many advantages, unifies the genotype and phenotype of protein/polypeptide, and establishes direct physical connection between protein and its genetic gene; the required antibody can be obtained by rapid screening; by the technology, not only specific antibodies of general antigens can be screened, but also antibodies of non-immunogenic or toxic antigens can be generated, and human antibodies with recognition functions, such as scFv, Fab, bifunctional antibodies, nanobodies, trivalent quadrivalent antibodies and the like can be generated. The obtained antibody can be Fab, scFv or nano antibody, can also be subjected to antibody engineering transformation and converted into other forms of small molecule antibodies, immunotoxins, targeted cytokines or full antibodies and the like, and is widely applied; compared with the production of the monoclonal antibody by immune animals, the production of the monoclonal antibody by using the phage display technology has the advantages of rapid production, simple and convenient operation, higher efficiency, capability of producing humanized antibody and higher utilization value.
In an alternative embodiment, the phage display vehicle is selected from the group consisting of the M13 phage display system, the lambda phage display system, the T4 phage display system, and the T7 phage display system.
In an alternative embodiment, the phage display vector is selected from the group consisting of pComb3X phage display vectors.
In an alternative embodiment, the vector after the VHH fragment of the immunized animal is linked to the display vector is competent for conversion, and the nanobody library is prepared by infecting the nanobody library with the helper phage VCSM 13.
The competence is selected from competent cells of Escherichia coli, for example E.coli XL-1 Blue. The transformation method is electric transformation or heat shock transformation.
In an alternative embodiment, PCR identification is included after the nanobody library is prepared, and it is determined whether the strain contains VHH fragments or not by identification, and the VHH fragment insertion rate is judged.
In a preferred embodiment of the present invention, the screening of the PI3K nanobody includes: coating a solid phase carrier with hyaluronic acid, adding a nano antibody display library, analyzing and monitoring an enrichment index through phage ELISA, and collecting phage particles after elutriation; infecting the collected phage particles with competent cells, performing IPTG induced expression, taking fermentation supernatant, and performing ELISA analysis to obtain the high-affinity nano antibody strain.
In a preferred embodiment of the invention, the prokaryotic expression comprises transferring the high-affinity nano antibody strain into an escherichia coli competent cell, and inducing prokaryotic expression by IPTG. The E.coli competent cells may be selected from TOP 10.
In a preferred embodiment of the present invention, the method for preparing the PI3K nanobody further comprises purifying the PI3K nanobody after prokaryotic expression. And purifying to remove other cell impurities or culture solution impurities.
In a preferred embodiment of the present invention, the purification is performed by affinity chromatography or ion exchange chromatography. The purification process used in the invention is convenient to operate, has wide universality, does not need to depend on expensive experimental instruments, has low cost and is suitable for large-scale popularization and application.
In a preferred embodiment of the present invention, the affinity chromatography treatment comprises using Ni-agotose affinity chromatography;
in an alternative embodiment, the affinity chromatography process comprises the use of His-tag mediated Ni-Agorose affinity chromatography. His-tag mediation can improve the efficiency of purification.
In a preferred embodiment of the present invention, the ion exchange chromatography comprises chromatography using DEAE anion exchange medium and/or hydroxyapatite. The use of hydroxyapatite can remove components such as foreign proteins.
In a preferred embodiment of the present invention, the ion exchange chromatography is followed by gel filtration chromatography, which comprises first purifying with DEAE anion exchange medium and then performing a second purification with superdex200 gel medium.
The nano antibody prepared by the purification process has weak cross reaction with other immunoglobulin antibodies, and has strong antibody specificity and good immune effect.
The invention has the following beneficial effects:
compared with the conventional antibody, the PI3K nano antibody prepared by the preparation method provided by the invention has the advantages of strong stability, good solubility, good antigen binding property, easiness in expression, easiness in mass preparation and the like.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a PCR-verified electropherogram;
FIG. 2 is an SDS PAGE pattern of nanobodies;
fig. 3 is a standard graph of nanobody 1.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
Example 1
And preparing a nano antibody phage display library.
Collecting monomodal camel peripheral blood, centrifuging at 2000rp for 5min, collecting peripheral blood cells (PMBC), and adding Trizol to extract total RNA. Total RNA was used as a template in accordance with InvitrogenTMSuperScriptTMReverse transcription is carried out by a reverse transcriptase kit, and a cDNA first chain is prepared by RT-PCR. Two rounds of PCR were performed using the first strand of cDNA as a template with the following primers.
First round PCR amplification primers:
an upstream primer: 5'-CTGCTTCTTCTACAAGGCTCC-3'
A downstream primer: 5'-TGCTGTTGGACCACTGTCCC-3'
Second round PCR amplification primers:
VHH FR1：5’
-ACTGGCCCAGGCGGCCGATGTGCAGCTGCAGGAGTCTGGAGGAGG-3’；
VHH FR4：5’
-ACTGGCCGGCCTGGCCTGAGGAGACGGTGACCAGGGTC-3’。
two rounds of PCR amplification yielded a camelid VHH fragment of 400-500 bp. And connecting the VHH fragment obtained by amplification with a pComb3X phage display vector, and electrically transforming E.coli XL-1Blue competence to prepare a nano antibody library. The nano antibody library capacity is measured and is about 2 multiplied by 109. Randomly selecting 12 colonies, and identifying by PCR that 12 colonies all contain VHH fragments with VHH fragment insertion rate of 100%(FIG. 1). And further infecting an E.coli XL-1Blue nano antibody library by using a helper phage VCSM13 to prepare a phage display library.
Example 2
In this example, PI3K nanometer antibody was screened and prokaryotic expression was performed.
Coating a 96-well plate with 100 mu g/mL hyaluronic acid, washing with PBS, adding the nano-antibody phage display library (phage particles) in example 1, analyzing and monitoring the enrichment index through phage ELISA, washing for 3 rounds, infecting the washed and collected phage particles with E.coli XL-1Blue, performing IPTG induction expression, taking fermentation supernatant, and analyzing through HRP-coupled anti-HA-monoclonal antibody ELISA to obtain 10 high-affinity nano-antibody strains. Further, 10 plasmids were transferred to e.coli TOP10, prokaryotic expression was induced by IPTG, and subjected to His-tag mediated Ni-agarose affinity chromatography, followed by primary purification using DEAE anion exchange medium, and secondary purification using superdex200 gel medium to obtain 2 high-expression PI3K nanobodies (fig. 2).
Experimental example 1
Activity and specificity detection
Purpose of the experiment: the invention carries out specificity detection on the nano antibody prepared in the example 1.
And (2) evaluating the specificity of the PI3K nano antibody 1 and the specificity of the PI3K nano antibody 2 by respectively using VII type collagen, nestin, endothelin, leucosin, laminin and heparin sulfate as controls, detecting by using an ELISA method after developing by using an HRP coupled anti-HA monoclonal antibody as a detection antibody, and measuring the absorbance at 450 nm. The results show that: the control antigens of collagen VII, nestin, endothelin, leucosin, laminin and heparin sulfate are all negative, and 2 high-expression PI3K nano antibodies 1 and PI3K nano antibody 2 have high specificity to hyaluronic acid.
Experimental example 2
And (3) measuring the affinity of the nano antibody and the concentration of hyaluronic acid by using an indirect competition method ELISA.
The working concentrations of PI3K nano antibody 1 and PI3K nano antibody 2 are determined by optimizing hyaluronic acid coating concentration and selecting proper confining liquid, and HRP coupled anti-HA monoclonal antibody is used asDetecting the antibody, and establishing an indirect competitive ELISA analysis method for quantitatively determining the concentration of the hyaluronic acid. According to the procedure of indirect competitive ELISA, a standard curve of indirect competitive ELISA for hyaluronic acid was prepared, and the linear range was 0.01-1000ng/ml, with the lowest detection limit being 0.01ng/ml (see FIG. 3). The affinity (Kd) of PI3K nanobody 1 and PI3K nanobody 2, respectively, was 4.1 × 10 as determined by indirect competition ELISA method-12And 8.5X 10-11mol/L。
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A preparation method of a PI3K nano antibody is characterized by comprising the following steps: constructing a nano antibody phage display library, and then carrying out screening and prokaryotic expression on the PI3K nano antibody based on the nano antibody phage display library;
the construction of the nano antibody phage display library comprises the steps of taking RNA of peripheral blood cells of an immune animal as a template, obtaining cDNA through reverse transcription, carrying out PCR amplification by taking a cDNA chain as a template to obtain a VHH fragment of the immune animal, and connecting the VHH fragment of the immune animal with a display carrier to construct the nano antibody display library;
the PCR amplification was two rounds of PCR.
2. The method of claim 1, wherein the immunized animal is selected from the group consisting of camelids;
preferably, the camelid is selected from a camel or an alpaca.
3. The method for producing the PI3K nanobody of claim 1 or 2, wherein the display vector is selected from a phage display vector;
preferably, the phage display vehicle is selected from the group consisting of an M13 phage display system, a lambda phage display system, a T4 phage display system, or a T7 phage display system;
preferably, the phage display vector is selected from the group consisting of pComb3X phage display vectors;
preferably, the vector obtained by connecting the VHH fragment of the immune animal with the display vector is subjected to competence transformation to prepare a nano antibody library, and the nano antibody library is infected by using a helper phage VCSM13 to prepare the nano antibody display library.
4. The method for preparing PI3K nanobody of claim 1 or 2, wherein the screening of PI3K nanobody comprises: adding hyaluronic acid coated solid phase carrier into the nano antibody display library, monitoring enrichment index through phage ELISA analysis, and collecting phage particles after elutriation; infecting the collected phage particles with competent cells, performing IPTG induced expression, taking fermentation supernatant, and performing ELISA analysis to obtain the high-affinity nano antibody strain.
5. The method for preparing the PI3K nanobody of claim 4, wherein the prokaryotic expression comprises transferring the high-affinity nanobody strain into an Escherichia coli competent cell, and inducing prokaryotic expression by IPTG.
6. The method for preparing the PI3K nanobody of claim 5, wherein the PI3K nanobody is further purified after prokaryotic expression of the PI3K nanobody.
7. The method for preparing the PI3K nanobody of claim 5, wherein the purification is performed by affinity chromatography and ion exchange chromatography.
8. The method of claim 7, wherein the affinity chromatography comprises affinity chromatography using Ni-agotose;
preferably, the affinity chromatography treatment comprises the use of His-tag mediated Ni-Agorose affinity chromatography.
9. The method for preparing the PI3K nanobody of claim 7, wherein the ion exchange chromatography comprises chromatography using DEAE anion exchange medium and/or hydroxyapatite.
10. The method for preparing the PI3K nanobody of claim 9, wherein the ion exchange chromatography is followed by gel filtration chromatography, which comprises a first purification using DEAE anion exchange medium and a second purification using Superdex200 gel medium.
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| CN106854246A (en) * | 2015-12-09 | 2017-06-16 | 南京东极生物科技有限公司 | A kind of nano antibody and its clinical practice for PI3K |
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| US20210002370A1 (en) * | 2019-07-05 | 2021-01-07 | Shihezi University | Pd-l1 nanobodies, preparation methods and uses thereof |
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| CN106854246A (en) * | 2015-12-09 | 2017-06-16 | 南京东极生物科技有限公司 | A kind of nano antibody and its clinical practice for PI3K |
| CN108484773A (en) * | 2018-03-12 | 2018-09-04 | 安徽合创健康生物技术有限公司 | A kind of hyaluronic acid nanometer antibody, its coded sequence and application thereof |
| CN108659129A (en) * | 2018-05-17 | 2018-10-16 | 新疆大学 | A kind of nano antibody and its preparation method and application of resisting GPC 3 albumen |
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