Nano antibody aiming at green fluorescent protein, application and GFP immunoaffinity adsorption material
阅读说明：本技术 一种针对绿色荧光蛋白的纳米抗体、应用和gfp免疫亲和吸附材料 (Nano antibody aiming at green fluorescent protein, application and GFP immunoaffinity adsorption material ) 是由 董春明 于 2019-11-08 设计创作，主要内容包括：本发明涉及一种针对绿色荧光蛋白的纳米抗体,所述抗体具有SEQ ID NO.1所示的氨基酸序列。本纳米抗体是一种可以与GFP特异性结合的单域抗体重链抗体(即纳米抗体),可以用于GFP及GFP融合蛋白的检测及纯化,例如用于制备检测和纯化GFP的试剂和工具等。(The invention relates to a nano antibody aiming at green fluorescent protein, which has an amino acid sequence shown in SEQ ID NO. 1. The nano antibody is a single-domain antibody heavy chain antibody (namely, the nano antibody) which can be specifically combined with GFP, and can be used for detecting and purifying GFP and GFP fusion protein, such as reagents and tools for preparing and detecting and purifying GFP.)
1. A nanobody against green fluorescent protein, characterized in that: the antibody has an amino acid sequence shown in SEQ ID NO. 1.
2. The nanobody against green fluorescent protein according to claim 1, characterized in that: the amino acid sequence of the antibody can be divided into four framework regions and three complementarity determining regions.
3. A nucleic acid molecule encoding the amino acid sequence of claim 1 or 2.
4. The nucleic acid molecule of claim 3, wherein: the sequence of the nucleic acid molecule is SEQ ID NO. 2.
5. A vector comprising the nucleic acid molecule of claim 3 or 4.
6. A host cell comprising the vector of claim 5.
7. The use of the nanobody against green fluorescent protein of claim 1 or 2 in immunodetection, or enrichment, detection and purification of green fluorescent protein and green fluorescent protein fusion protein.
8. The nanobody against green fluorescent protein of claim 1 or 2, which is an antibody capable of specifically binding to GFP, obtained by modification by random or site-directed mutagenesis techniques.
9. The GFP immunoaffinity adsorption material prepared by using the nanobody aiming at the green fluorescent protein as claimed in claim 1 or 2, wherein: the method comprises the following steps:
the method adopts agarose microspheres as a carrier and couples an anti-GFP nano antibody, and is specifically prepared as follows:
washing dry glue activated by CNBr with 0.1M HCl for 10 times, and balancing for 5min each time; washing for 10 times by using a coupling buffer solution which is a NA2HPO4 solution with the pH value of 7.2 and the concentration of 10mM, adding the anti-GFP label nano antibody and 2 mg/g of dry glue, and reacting for 3.5 hours at room temperature to covalently couple the anti-GFP label nano antibody and the CNBr activated dry glue;
after washing 3 times with a coupling buffer solution, the coupling buffer solution is a NA2HPO4 solution with the pH value of 7.2 and the concentration of 10mM, and the coupling buffer solution is added into a blocking solution for reaction for 2 hours at a room temperature to block unreacted active groups;
alternately washing the mixture for 3 times by using phosphate buffer solution and acetic acid buffer solution with 6 times of glue volume to obtain the immunoaffinity adsorption material covalently coupled with the anti-GFP label nano antibody;
wherein the phosphate buffer solution is 10mM and pH7.2, and the acetate buffer solution is 0.1M and pH4.5.
10. The GFP immunoaffinity adsorption material prepared from the nanobody against green fluorescent protein of claim 9, wherein: the dry glue is agarose gel microspheres, silica spheres or nano magnetic beads.
Technical Field
The invention belongs to the technical field of single-domain heavy chain antibodies (also called nano antibody technology) and genetic engineering antibodies, and particularly relates to a nano antibody aiming at Green Fluorescent Protein (GFP), application and a GFP immunoaffinity adsorption material.
Background
GFP is a protein consisting of about 238 amino acids, which is excited by blue light to ultraviolet light and emits green fluorescence. GFP is widely used in the fields of immunological detection, cell imaging, affinity purification, protein engineering and the like.
At present, most of monoclonal or polyclonal antibodies aiming at GFP are used for detection in the market, but the research and development and production processes of the monoclonal antibodies are extremely complicated and complicated, the stability of the antibodies is poor, the production cost is high, and the source of the polyclonal antibodies is limited. Since conventional antibodies contain an Fc fragment, there is a tendency for nonspecific binding or contamination. In contrast, the nano antibody is composed of only one structural domain, has the advantages of acid and alkali resistance, high temperature resistance, high specificity, small molecular weight, large-scale production and the like, and has no non-specific combination or pollution of heavy chains and light chains of common antibodies. The purification medium prepared by using the nano antibody as the ligand has the advantages of low cost, reusability and the like, and has wide application prospect.
Through searching, the following two patent publications related to the patent application of the invention are found:
1. the coding gene of the green fluorescent protein nano antibody, the preparation method and the application thereof (CN108753792A) construct a GFP nano antibody library. Four nanobodies specifically binding to GFP were screened from the antibody library by phage display technology and named A12, E6, D5 and B9, respectively. The nucleotide sequences of the four nano antibody genes are obtained by sequencing, and are shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO. 3 and SEQ ID NO. 4, and the corresponding amino acid sequences are shown as SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7 and SEQ ID NO. 8. Cloning the A12 gene into a modified expression vector pADL-10b-His, and introducing into an SS320 strain; e6, D5 and B9 genes are respectively cloned into a modified expression vector pBAD24-Flag-His and are respectively introduced into a TOP10 strain, so that prokaryotic expression vectors and strains of four nano antibodies are obtained. The invention expresses and purifies four nano antibodies and proves that the four GFP nano antibodies can be specifically combined with GFP and can be applied to GFP detection in basic research.
2. A nano antibody aiming at green fluorescent protein and a coding sequence (CN108484764A) thereof, wherein the antibody comprises a
By contrast, the present patent application is substantially different from the above patent publications.
Disclosure of Invention
The invention aims to overcome the defects in the prior art and provides a nano antibody aiming at Green Fluorescent Protein (GFP), application and a GFP immunoaffinity adsorption material, wherein the nano antibody is a single-domain antibody heavy chain antibody (namely the nano antibody) which can be specifically combined with GFP, and can be used for detecting and purifying GFP and GFP fusion protein, such as reagents and tools for preparing and detecting and purifying GFP and the like.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a nano antibody aiming at green fluorescent protein has an amino acid sequence shown in SEQ ID NO. 1.
Furthermore, the amino acid sequence of the antibody may be divided into four framework regions and three complementarity determining regions.
A nucleic acid molecule encoding an amino acid sequence as described above.
Moreover, the sequence of the nucleic acid molecule is SEQ ID NO. 2.
A vector comprising a nucleic acid molecule as described above.
A host cell comprising a vector as described above.
The nano antibody aiming at the green fluorescent protein is applied to immunodetection, or enrichment, detection and purification of the green fluorescent protein and the green fluorescent protein fusion protein.
The nanobody aiming at the green fluorescent protein is an antibody which is obtained by modifying through random or site-directed mutagenesis technology and can be specifically combined with GFP.
The GFP immunoaffinity adsorption material prepared by the nano antibody aiming at the green fluorescent protein comprises the following steps:
the method adopts agarose microspheres as a carrier, couples an anti-GFP nano antibody (namely the nano antibody aiming at green fluorescent protein) and specifically comprises the following steps:
washing dry glue activated by CNBr with 0.1M HCl for 10 times, and balancing for 5min each time; washing for 10 times by using a coupling buffer solution which is a NA2HPO4 solution with the pH value of 7.2 and the concentration of 10mM, adding an anti-GFP label nano antibody, 2 mg/g of dry glue, and reacting for 3.5 hours at room temperature to covalently couple the anti-GFP label nano antibody (namely the nano antibody aiming at the green fluorescent protein) and the dry glue activated by CNBr;
after washing 3 times with a coupling buffer solution, the coupling buffer solution is a NA2HPO4 solution with the pH value of 7.2 and the concentration of 10mM, and the coupling buffer solution is added into a blocking solution for reaction for 2 hours at a room temperature to block unreacted active groups;
alternately washing the mixture for 3 times by using phosphate buffer solution and acetic acid buffer solution with 6 times of glue volume to obtain the immunoaffinity adsorption material covalently coupled with the anti-GFP label nano antibody;
wherein the phosphate buffer solution is 10mM and pH7.2, and the acetate buffer solution is 0.1M and pH4.5.
Moreover, the dry glue is agarose gel microspheres, silica spheres or nano magnetic beads.
The invention has the advantages and positive effects that:
1. the nano antibody is a single-domain antibody heavy chain antibody (namely, the nano antibody) which can be specifically combined with GFP, and can be used for detecting and purifying GFP and GFP fusion protein, such as reagents and tools for preparing and detecting and purifying GFP.
2. Through the gene sequence and the host cell of the nano antibody disclosed by the invention, the nano antibody can be efficiently expressed in escherichia coli, the production process is simple, the cost is low, and the yield is high.
3. The nano antibody only consists of one structural domain, has the advantages of acid and alkali resistance, high temperature resistance, high specificity, small molecular weight, large-scale production and the like, and has no nonspecific combination or pollution of heavy chains and light chains of common antibodies.
Drawings
FIG. 1 is a gene electrophoresis diagram of a nanobody of the present invention; wherein,
FIG. 2 is a diagram showing the colony PCR electrophoresis performed on the constructed GFP-specific single domain antibody library of the present invention; wherein,
FIG. 3 is a schematic diagram of screening specific single positive clones by phage enzyme-linked immunosorbent assay (ELISA) in accordance with the present invention; wherein, 1 is to couple the apolipoprotein on the enzyme label plate, 2 is a nano antibody, 3 is a mouse anti-HA antibody, 4 is an antibody marked by goat anti-mouse alkaline phosphatase, and 5 is an alkaline phosphatase developing solution;
FIG. 4 is an electrophoresis chart of SDS-PAGE of GFP nanobodies expressed in the present invention after purification by nickel column resin gel affinity chromatography; wherein,
Detailed Description
The following detailed description of the embodiments of the present invention is provided for the purpose of illustration and not limitation, and should not be construed as limiting the scope of the invention.
The raw materials used in the invention are conventional commercial products unless otherwise specified; the methods used in the present invention are conventional in the art unless otherwise specified.
A nano antibody aiming at green fluorescent protein has an amino acid sequence shown in SEQ ID NO. 1.
Preferably, the amino acid sequence of the antibody can be divided into four framework regions and three complementarity determining regions.
A nucleic acid molecule encoding an amino acid sequence as described above.
Preferably, the sequence of the nucleic acid molecule is SEQ ID NO. 2.
A vector comprising a nucleic acid molecule as described above.
A host cell comprising a vector as described above.
The nano antibody aiming at the green fluorescent protein is applied to immunodetection, or enrichment, detection and purification of the green fluorescent protein and the green fluorescent protein fusion protein.
The nanobody aiming at the green fluorescent protein is an antibody which is obtained by modifying through random or site-directed mutagenesis technology and can be specifically combined with GFP.
The GFP immunoaffinity adsorption material prepared by the nano antibody aiming at the green fluorescent protein comprises the following steps:
the method adopts agarose microspheres as a carrier, couples an anti-GFP nano antibody (namely the nano antibody aiming at green fluorescent protein) and specifically comprises the following steps:
washing dry glue activated by CNBr with 0.1M HCl for 10 times, and balancing for 5min each time; washing for 10 times by using a coupling buffer solution which is a NA2HPO4 solution with the pH value of 7.2 and the concentration of 10mM, adding an anti-GFP label nano antibody, 2 mg/g of dry glue, and reacting for 3.5 hours at room temperature to covalently couple the anti-GFP label nano antibody (namely the nano antibody aiming at the green fluorescent protein) and the dry glue activated by CNBr;
after washing 3 times with a coupling buffer solution, the coupling buffer solution is a NA2HPO4 solution with the pH value of 7.2 and the concentration of 10mM, and the coupling buffer solution is added into a blocking solution for reaction for 2 hours at a room temperature to block unreacted active groups;
alternately washing the mixture for 3 times by using phosphate buffer solution and acetic acid buffer solution with 6 times of glue volume to obtain the immunoaffinity adsorption material covalently coupled with the anti-GFP label nano antibody;
wherein the phosphate buffer solution is 10mM and pH7.2, and the acetate buffer solution is 0.1M and pH4.5.
Preferably, the dry gel is agarose gel microspheres, silica spheres or nano magnetic beads.
The invention provides a nano antibody aiming at GFP, which has an amino acid sequence shown in SEQ ID NO. 1. The amino acid sequence thereof can be divided into four Framework Regions (FR) and three Complementary Determining Regions (CDR).
The invention also provides a nucleic acid molecule, which codes SEQ ID NO.2, and the specific sequence of the nucleic acid molecule can be obtained at any time through genetic codons.
The nucleic acid sequences provided by the invention or at least part of the sequences can be expressed by means of a suitable expression system to obtain the corresponding proteins or polypeptides. These expression systems include bacteria, yeast. Filamentous fungi, animal cells, plant cells, insect cells, or cell-free expression systems.
The invention also provides a vector comprising the nucleic acid sequence. Due to the degeneracy of the genetic code, the nucleic acid sequence can vary from one applicator to another.
The invention also provides a host cell comprising the protein or the expression vector.
The amino acid sequence provided by the invention can be used as a precursor, and is transformed by a random or site-directed mutagenesis technology to obtain a mutant with better properties (water solubility, stability, affinity, specificity and the like), and the mutant can be specifically combined with GFP.
The invention also relates to the application of the nano antibody aiming at GFP in immunodetection, enrichment and purification.
The invention also relates to an immunoaffinity adsorption material aiming at GFP, which can take a nano antibody aiming at GFP as a ligand, wherein the nano antibody aiming at GFP has an amino acid sequence shown in SEQ ID NO. 1. The carrier material is not limited to agarose gel, and silicon spheres, nano magnetic beads and the like can also be selected.
The preparation and detection of the invention are as follows:
according to the invention, Green Fluorescent Protein (GFP) is firstly used for immunizing a bactrian camel in Xinjiang, and after 4 times of immunization, the bactrian camel peripheral blood lymphocytes are extracted and a GFP specific nano antibody library is constructed. GFP is coupled on an enzyme label plate to display the correct spatial structure of protein, so that the epitope of GFP is exposed, and the antigen in the form is used for screening a GFP immune nano antibody gene library (camel heavy chain antibody phage display gene library) by utilizing a phage display technology, so that a nano antibody strain capable of being efficiently expressed in escherichia coli is obtained.
The invention will be further illustrated with reference to the following specific examples.