CN116789795A - Polypeptide for anchoring exogenous over-expressed protein on cell membrane and application - Google Patents

Abstract

The invention relates to the technical field of biology, in particular to a polypeptide for anchoring exogenous over-expressed protein on a cell membrane and application thereof. The mini-protein anchoring the over-expressed protein to the cell membrane of the present invention is (a) or (b): (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1; (b) The amino acid sequence shown in SEQ ID No.1 is substituted and/or deleted and/or added by one or more amino acid residues and has a polypeptide anchoring the over-expressed protein to a cell membrane. The polypeptide effectively reduces the risk of false negative in CBA antibody detection of non-membrane proteins such as GFAP and LGI1, provides a foundation for improving the performance of CBA antibody detection methods, reducing the cost and facilitating the operation, and lays a foundation for the development of the fields of disease detection diagnosis, vaccine and pharmacy development in the biological medicine industry.

Description

Polypeptide for anchoring exogenous over-expressed protein on cell membrane and application

Technical Field

The invention relates to the technical field of biology, in particular to a polypeptide for anchoring exogenous over-expressed protein on a cell membrane and application thereof.

Background

Autoimmune (autoimmune) refers to the phenomenon in which the immune system of the body responds to self-antigens by producing autoantibodies and/or autoimmune sensitized lymphocytes. Autoimmune diseases are diseases caused by autoimmune diseases in which autoantibodies react with the body to cause damage to the tissues. Recently, new findings have been made of antibodies and target antigens related to immune diseases, such as Homer3 autoantibodies for autoimmune encephalitis, PLA2R, nell-1 autoantibodies for specific for membrane nephritis, AQP4 and MOG autoantibodies for specific for diseases of the neuromyelitis spectrum, and the like. Autoimmune diseases can be diagnosed by detecting the presence or absence of specific IgG antibodies in a patient's body fluid. There are many methods for detecting antibodies, and the methods with wider application are: WB, ELISA, chemiluminescent, indirect fluorescent, flow-through liquid chip technology, etc. The Cell-based indirect fluorescence (CBA) antibody detection method was listed by neurology specialists at home and abroad as consensus and recommendation for diagnosing autoimmune diseases such as neuromyelitis optica (neuromyelitis) lineage diseases, autoimmune encephalomyelitis, and the like during 2018-2020.

The CBA antibody detection method is a method for using cells as a solid phase matrix, over-expressing target antigens on the surfaces of the cells, and qualitatively identifying specific antibodies in a sample through IgG secondary antibodies coupled with fluorescent groups. Compared with WB, ELISA and chemiluminescence methods, the CBA method has simple process, convenient operation and specificity and sensitivity, but for a multimeric target with a complex structure, an intracellular expressed target or a secreted protein, an over-expressed target antigen cannot be positioned on a cell membrane, so that the false negative and false positive risks of CBA method detection are increased. How to anchor the specific antigen that is overexpressed on the membrane for expression is therefore a matter of urgent need.

Disclosure of Invention

In order to overcome the defects of the technology, the invention provides a polypeptide which anchors exogenous over-expressed protein on a cell membrane, and fusion expression is carried out on the polypeptide and exogenous target protein, so that the target protein is successfully positioned on the cell membrane, and a foundation is provided for improving the performance, reducing the cost and facilitating the operation of a CBA antibody detection method.

To achieve the above object, the present invention provides a polypeptide as described in (a) or (b);

(a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1;

(b) A derivative polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 and has an anchor membrane function.

There are two types of transmembrane structures of proteins: alpha-helical and beta-barres. The alpha-helical structural protein is present in the inner membrane of bacterial cells or in the plasma membrane of eukaryotic cells, and sometimes also in the outer membrane of eukaryotic cells. alpha-helical typically has a signaling or transport channel function in cells, with transmembrane proteins in cells being the predominant type. alpha-helical is about 20 to 25 amino acid residues. Fusion expression of a polypeptide with an alpha-helical structure with a target antigen facilitates anchoring of the exogenous target to the cell membrane. Small integral membrane protein 1 (SMIM 1) is a single transmembrane polypeptide encoded by ENST00000444870.7 that is expressed on the cell membrane as a membrane structural protein. SMIM1 comprises 78 amino acids, the 47 th to 67 th AA are transmembrane regions with alpha-helical structure, the 1 st to 46 th AA are intracellular segments, and the 68 th to 78 th AA are extracellular regions. The SMIM1 polypeptide sequence is any one of the 1 st to 46 th AA sequences and/or any one of the 68 th to 78 th AA sequences deleted, which is helpful for driving fusion expressed target proteins to anchor on cell membranes and enhancing the effectiveness of anchoring exogenous target proteins on the cell membranes.

The invention also provides a gene for encoding the polypeptide in the scheme, and the nucleotide sequence of the gene is (a), (b) or (c);

(a) A nucleotide sequence as shown in SEQ ID NO. 2;

(b) A nucleotide sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO.2 and codes for a protein with anchor membrane function;

(c) Has more than 80 percent of homology with the nucleotide sequence shown in SEQ ID NO.2 and codes for a nucleotide sequence.

The invention also provides a recombinant fusion protein containing the polypeptide.

The recombinant fusion protein provided by the invention contains the polypeptide anchoring the over-expressed protein to a cell membrane. The specific target antigen gene and the polypeptide of the invention are fused and expressed, so that the expressed antigen is anchored on a cell membrane, and when the antibody is detected, the antibody can be combined without penetrating the cell membrane, thus reducing the CBA antibody detection operation step and simultaneously reducing the risk of false negative.

The invention also provides a recombinant expression vector, recombinant bacteria or transgenic cell line of the gene.

The invention also provides application of the polypeptide in anchoring target protein to cell membrane.

The invention also provides application of the recombinant fusion protein in anchoring target protein to cell membrane.

The invention also provides a method for anchoring target protein to cell membrane, wherein the polypeptide is used as an anchor membrane structure to anchor target protein to cell membrane.

Further, the method specifically comprises the following steps:

s1, introducing a fusion gene into a target cell, so that a target protein is anchored on a cell membrane of the target cell; the fusion gene sequentially comprises a polypeptide encoding gene sequence and a target protein encoding gene sequence from upstream to downstream;

s2, introducing the fusion gene into a target cell, so that a target protein is anchored on a cell membrane of the target cell; the fusion gene sequentially comprises a gene sequence for encoding a target protein and a gene sequence for encoding a polypeptide from upstream to downstream.

The beneficial effects are that: the polypeptide not only provides a directional expression method for the exogenous protein, but also provides a tool for positioning and expressing the exogenous protein on the cell surface for bioconversion; the method for anchoring the specific targets GFAP and LGI1 to the cell membrane effectively reduces the risk of false negative in CBA antibody detection, provides a basis for improving the performance, reducing the cost and facilitating the operation of the CBA antibody detection method, and lays a foundation for the development of the fields of disease detection diagnosis, vaccine and pharmacy development in the biological medicine industry.

Drawings

FIG. 1 is a fluorescence micrograph of EGFP protein of example 1.

FIG. 2 is a diagram showing the expression of plasmids and the localization of LGI1 in example 2.

Detailed Description

The present invention will be described in detail with reference to specific embodiments thereof, so that those skilled in the art can better understand the technical solutions of the present invention. The experimental procedures, which do not address the specific conditions in the examples below, are generally carried out under conventional conditions or under conditions recommended by the manufacturer. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. Percentages and parts are by weight unless otherwise indicated. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only. EXAMPLE 1 EGFP protein fusion expressed by SMIM1 protein is anchored on cell membrane

1. Construction of SMIM1-EGFP fusion expression plasmid

1.1 designing a fusion expressed gene sequence and synthesizing the gene fragment: according to the structural characteristics of SMIM1 protein, designing a fusion expressed gene fragment SMIM1-EGFP, wherein the fusion expressed gene fragment comprises a SIMI1 part of polypeptide fragment followed by an EGFP full-length gene, the gene sequence is entrusted to the company for synthesis, and the nucleotide sequence of the SMIM1-EGFP is SEQ ID NO.3.

1.2 ligation vector of synthetic EGFP-SMIM1 sequence: the pcDNA3.1 vector was supplied by Gene synthesis and the synthesized SMIM1-EGFP fusion fragment was ligated into the vector multiple cloning site, designated pcDNA3.1-SMIM1-EGFP plasmid.

1.3 construction of control plasmids: the pcDNA3.1 vector was supplied by Gene synthesis and EGFP gene fragment was ligated into the vector multiple cloning site, designated pcDNA3.1-EGFP plasmid.

2. Localization of fusion proteins in 293 cells

The experimental group plasmid pcDNA3.1-SMIM1-EGFP and the control group plasmid pcDNA3.1-EGFP are used for transfecting 293 cells, and the expression condition and the position of green fluorescence of the EGFP are observed to detect the over-expression of the fusion protein and the positioning thereof. The specific method comprises the following steps:

culture of 2.1293 cells: the cultured 293 cells are digested into single cells by 0.25% pancreatin, resuspended to a cell concentration of 5×106cells/mL by using DMEM medium containing 5% fetal bovine serum, and then inoculated into 12-well cell culture plates, 1 mL/well, and transfected when cultured for 18-20 h under 5% co2 at 37 ℃ until the cell wall attachment confluency is about 70% -80%.

2.2 preparation of DNA/PEI mixture: diluting each 2. Mu.g of the recombinant plasmids pcDNA3.1-SMIM1-EGFP and pcDNA3.1-EGFP to 125. Mu.L; then, 12. Mu.L of 1mg/mL PEI (polyetherimide) solution was diluted to 125. Mu.L with opti-MEM medium, and after 5 minutes of standing at room temperature, the two plasmid mixtures and the PEI mixture solution were mixed, respectively, and then left at room temperature for 20 minutes.

2.3 transfection: adding the incubated DNA/PEI mixture into each well of a 12-well cell culture plate according to the amount of 250 mu L per well, gently mixing, and continuously culturing under the conditions of 37 ℃ and 5% CO 2; cells were fixed after 16 h.

2.4 observation of results: the localization of the experimental group pcDNA3.1-SMIM1-EGFP and the control group pcDNA3.1-EGFP was observed under a fluorescence microscope, and the result is shown in FIG. 1. The results in FIG. 1A show that EGFP green fluorescent protein is expressed in whole cells, and FIG. 1B shows that the fusion protein SMIM1-EGFP is mainly expressed on the cell membranes of 293 cells.

EXAMPLE 2 fusion expression of SMIM1 protein the autologous brain target antigen LGI1 was anchored on the cell membrane 1. Construction of SMIM1-EGFP fusion expression plasmid

1.1 designing a fusion expressed gene sequence and synthesizing the gene fragment: according to the structural characteristics of SMIM1 protein, designing a fusion expressed gene fragment SMIM1-LGI1, wherein the fusion expressed gene fragment comprises a part of SIMI1 polypeptide fragment and then LGI1 full-length gene, the gene sequence is entrusted to the company for synthesis, and the nucleotide sequence of the SMIM1-EGFP is SEQ ID NO.4.

1.2 ligation of synthetic SMIM1-LGI1 sequences to vectors: pIRES-EGFP vector was supplied by Gene synthesis and the synthesized SMIM1-LGI1 fusion fragment was ligated into vector multiple cloning sites, designated pIRES-SMIM1-LGI1 plasmid.

1.3 construction of control plasmids: the pIRES-EGFP vector is provided by gene synthesis company, and the LGI1 gene fragment is connected to the vector polyclonal site and named pIRES-LGI1 plasmid; the nucleotide sequence is SEQ ID No.5.

2. Expression and localization of fusion proteins in 293 cells

The 293 cells are transfected with the experimental group plasmid pIRES-SMIM1-LGI1 and the control group plasmid pIRES-LGI1, the successful overexpression of the fusion protein is detected by observing the green fluorescent expression condition of the pIRES-EGFP vector, and the localization of the LGI1 protein in the 293 cells is detected by the LGI1 specific antibody. The specific method comprises the following steps:

culture of 2.1293 cells: the cultured 293 cells are digested into single cells by 0.25% pancreatin, resuspended to a cell concentration of 4×106cells/mL by using DMEM medium containing 5% fetal bovine serum, and then inoculated into 12-well cell culture plates, 1 mL/well, and transfected when cultured for 18-20 h under 5% co2 at 37 ℃ until the cell wall attachment confluency is about 70% -80%.

2.2 preparation of DNA/PEI mixture: diluting each 2. Mu.g of the recombinant plasmids pcDNA3.1-SMIM1-EGFP and pcDNA3.1-EGFP to 125. Mu.L; then, 12. Mu.L of 1mg/mL PEI (polyetherimide) solution was diluted to 125. Mu.L with opti-MEM medium, and after 5 minutes of standing at room temperature, the two plasmid mixtures and the PEI mixture solution were mixed, respectively, and then left at room temperature for 20 minutes.

2.3 transfection: adding the incubated DNA/PEI mixture into each well of a 12-well cell culture plate according to the amount of 250 mu L per well, gently mixing, and continuously culturing under the conditions of 37 ℃ and 5% CO 2; cells were fixed after 20 h.

2.4 incubation of antibodies: the fixed cells were washed 2 times with PBS, incubated with a sample containing LGI1 specific antibody for 30min, washed 2 times with PBS, incubated with red fluorescent-labeled goat anti-human IgG (H+L) secondary antibody for 30min, washed 2 times with PBS, and 1mL of PBS was added to each well.

2.5 observation of results: the control and experimental groups were observed under a fluorescence microscope for plasmid expression and LGI1 localization. Both the experimental group (see FIG. 2D) and the control group (see FIG. 2A) showed green fluorescence, indicating successful expression of the recombinant plasmid. Wherein the control group has green fluorescence, and no red fluorescence after detection by the LGI1 specific antibody, which indicates that the LGI1 is expressed but not located on the cell membrane (see FIG. 2B and FIG. 2C), while the experimental group has red fluorescence after detection by the LGI specific antibody, which indicates that the SMIM1-LGI1 fusion protein is located on the cell membrane (see FIG. 2E and FIG. 2F); experiments show that SMIM1 can effectively locate target proteins on cell membranes through fusion expression.

Finally, it should be noted that the above description is only a preferred embodiment of the present invention, and that many similar changes can be made by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Claims (8)

1. A polypeptide that anchors an exogenous over-expressed protein to a cell membrane, characterized in that: the polypeptide is (a) or (b);

(a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO. 1;

(b) A derivative polypeptide which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in SEQ ID NO.1 and has an anchor membrane function.

2. A gene encoding the polypeptide of claim 1, wherein the nucleotide sequence of the gene is (a), (b) or (c);

(a) A nucleotide sequence as shown in SEQ ID NO. 2;

(b) A nucleotide sequence which hybridizes with the nucleotide sequence shown in SEQ ID NO.2 and codes for a protein with anchor membrane function;

(c) Has more than 80 percent of homology with the nucleotide sequence shown in SEQ ID NO.2 and codes for a nucleotide sequence.

3. A recombinant fusion protein comprising the polypeptide of claim 1.

4. A recombinant expression vector, recombinant bacterium or transgenic cell line comprising the gene of claim 2.

5. Use of the polypeptide of claim 1 for anchoring a protein of interest to a cell membrane.

6. Use of the recombinant fusion protein of claim 3 for anchoring a protein of interest to a cell membrane.

7. A method for anchoring a target protein to a cell membrane, wherein the polypeptide of claim 1 is used as an anchor membrane structure to anchor the target protein to the cell membrane.

8. The method according to claim 7, characterized in that it comprises in particular the following steps:

s1, introducing a fusion gene into a target cell, so that a target protein is anchored on a cell membrane of the target cell; the fusion gene sequentially comprises a polypeptide encoding gene sequence and a target protein encoding gene sequence from upstream to downstream;

s2, introducing the fusion gene into a target cell, so that a target protein is anchored on a cell membrane of the target cell; the fusion gene sequentially comprises a gene sequence for encoding a target protein and a gene sequence for encoding a polypeptide from upstream to downstream.