Cell model for expressing acetylcholine receptor protein

Abstract

The invention relates to a cell model for expressing acetylcholine receptor protein, which comprises a lentiviral vector, and subunit genes of human AChR alpha 1, beta 1, Rapsn, delta and epsilon are stably expressed in the lentiviral vector. Alpha 1, beta 1, delta, epsilon and Rapsn of AChR are simultaneously expressed through the lentiviral vector, an AChR receptor model with complete functions is successfully constructed, exogenous genes can be stably integrated into a host cell genome through the lentiviral vector, and long-term stable expression is achieved. Compared with transient transfection, the method can still maintain efficient expression of AChR after multiple passages, and is suitable for long-term experiment and drug screening. Meanwhile, the optimized transfection condition improves the synchronous expression efficiency of multiple subunits, and avoids the problem of unbalanced subunit expression, thereby ensuring the correct assembly and function maintenance of AChR receptors.

Description

Technical Field

[0001] The invention relates to the technical field of cell models, and in particular to a cell model for expressing acetylcholine receptor protein. Background Art

[0002] Acetylcholine receptors (AChRs) are a type of ion channel receptor located on the cell membrane and are involved in the transmission of nerve signals. AChRs are particularly important at the neuromuscular junction, where they trigger muscle contraction by binding to acetylcholine. Abnormal expression or dysfunction of AChRs is associated with a variety of neuromuscular diseases, such as myasthenia gravis (MG). Therefore, establishing a cell model that stably expresses AChR protein is of great significance for studying the function of AChRs, disease mechanisms, and screening related drugs.

[0003] At present, cell models expressing AChR usually use electrotransfection or lentiviral transfection technology. Although these methods can introduce some subunits of AChR into cells, since AChR is a complex five-subunit structure (α, β, δ, ε and Rapsn subunit), existing cell models often lack the expression of certain key subunits, and thus cannot accurately simulate the natural AChR structure and function. In particular, the Rapsn (receptor aggregation protein) subunit plays a vital role in AChR aggregation and function maintenance. In the existing technology, this subunit is rarely introduced at the same time, resulting in an imperfect model and limiting the accuracy of the research.

[0004] In addition, the existing lentiviral transfection methods also have certain problems in terms of subunit ratio, transfection efficiency, and cell survival rate, making it difficult to obtain a cell model that efficiently, stably, and long-term expresses AChR. Therefore, how to construct a cell model containing all five AChR subunits to accurately simulate the expression of human AChR receptors has become an important technical issue in current research. Summary of the invention

[0005] Based on this, the present invention provides a cell model for expressing acetylcholine receptor protein to overcome the shortcomings of the prior art. The technical solution removes untransfected cells and debris through multiple gradient centrifugation steps to obtain a cell model with higher purity. Combined with optimized culture medium and culture conditions, the cell model can be passaged for a long time while maintaining stable AChR expression, providing a reliable platform for subsequent functional studies and drug screening.

[0006] In summary, this protocol successfully constructed a cell model that fully expressed the acetylcholine receptor protein by integrating five AChR subunits into the lentivirus, solving the problems of incomplete subunit expression, low transfection efficiency, and poor model stability in existing technologies, and provided innovative technical support for the study of AChR-related diseases.

[0007] One object of the present invention is to provide a cell model for expressing acetylcholine receptor protein, comprising a lentiviral vector, wherein the lentiviral vector stably expresses subunit genes of human AChRα1, β1, Rapsn, δ, and ε;

[0008] The method for preparing a cell model for expressing acetylcholine receptor protein comprises the following steps:

[0009] S1. Construction of lentivirus for human AChRα1, β1, Rapsn, δ, ε;

[0010] S2. Culture HEK 293T cells;

[0011] S3. HEK293T cells were transfected with human AChRα1, β1, Rapsn, δ, and ε lentivirus, cultured, gradient centrifuged, concentrated and collected, cultured and passaged to obtain a cell model for expressing acetylcholine receptor protein.

[0012] Furthermore, the concentration ratio of the human AChRα1, β1, Rapsn, δ, and ε lentivirus transfected HEK293T cells is 4-6:1-2.

[0013] Furthermore, the process of the gradient centrifugation includes three gradient centrifugations, and the conditions of the three gradient centrifugations are as follows:

[0014] First gradient centrifugation: 600-1000g, 8-20min;

[0015] Second gradient centrifugation: 2500-3500g, 15-30min;

[0016] The third gradient centrifugation: 9000-11000g, 30-60min.

[0017] Furthermore, the gradient centrifugation temperature is 4-8°C.

[0018] Furthermore, the HEK293T cells are cultured in a high-glucose medium.

[0019] Furthermore, the culture and subculture conditions are culture in a carbon dioxide incubator at a temperature of 35-37°C.

[0020] Furthermore, the culture medium is a culture medium containing 4-6 μg / mL of puromycin and blasticidin.

[0021] Furthermore, the cell aggregation degree during the culture passage is 70-90%.

[0022] Specifically, the cell model maintains stable AChR expression over multiple generations, which is highly reproducible and ensures the reliability of the experimental results. The stability and applicability of the model are further guaranteed by controlling the cell aggregation and optimizing the culture conditions.

[0023] The present invention has the following beneficial effects:

[0024] The present invention simultaneously expresses α1, β1, δ, ε and Rapsn of AChR through a lentiviral vector, and successfully constructs a functionally complete AChR receptor model. The lentiviral vector can stably integrate exogenous genes into the host cell genome to achieve long-term stable expression. At the same time, the optimized transfection conditions improve the synchronous expression efficiency of multiple subunits, avoid the problem of uneven subunit expression, thereby ensuring the correct assembly and functional maintenance of the AChR receptor. In addition, the present invention effectively ensures the good growth state of cells and the continuous expression of AChR protein through optimized culture conditions, including high-sugar culture medium and suitable culture temperature and environment. The cell model maintains stable expression in multiple generations, and has good repeatability and stability. Through the control of cell polymerization and the optimization of generation conditions, the reliability of the model in long-term experiments is further guaranteed, and it is suitable for in-depth functional research. DETAILED DESCRIPTION

[0025] In order to more clearly illustrate the technical solution of the present invention, the following examples are listed. Unless otherwise stated, the raw materials, reactions and post-treatment methods shown in the examples are common raw materials on the market and technical methods well known to those skilled in the art.

[0026] The words "preferred", "preferably", "more preferably", etc. in the present invention refer to embodiments of the present invention that can provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0027] It should be understood that, except in any operating examples, or where otherwise indicated, all numbers expressing, for example, the amounts of ingredients used in the specification and claims should be understood to be modified in all cases by the term "about". Therefore, unless indicated to the contrary, the numerical parameters set forth in the following specification and the appended claims are approximate values ​​that vary depending on the desired properties to be obtained by the present invention.

[0028] DMEM high glucose medium was purchased from Corning Incorporated, USA.

[0029] Example 1

[0030] A cell model for expressing acetylcholine receptor protein, comprising a lentiviral vector, wherein the lentiviral vector stably expresses subunit genes of human AChRα1, β1, Rapsn, δ, and ε;

[0031] The method for preparing a cell model for expressing acetylcholine receptor protein comprises the following steps:

[0032] S1. Construct human AChRα1, β1, Rapsn, δ, and ε sequences into CMV-MCS-PGK-Puro plasmid, and sequence to identify the sequences; amplify the target fragment, perform double restriction digestion on the recovered target gene, and connect the overexpression vector part with the target fragment; construct the lentivirus;

[0033] S2. HEK-293T cells were cultured with fetal bovine serum and DMEM high glucose medium;

[0034] S3-1. HEK293T cells were transfected with human AChRα1, β1, Rapsn, δ, and ε lentivirus for 24 h (the concentration of human AChRα1, β1, Rapsn, δ, and ε lentivirus and HEK293T cells was 2:1). After 48 h of transfection, fresh culture medium was replaced and cultured for another 48 h. The cell supernatant was then collected and cultured. The cells were then subjected to the first gradient centrifugation at 8000 g for 15 min, the second gradient centrifugation at 2000 g for 20 min, and the third gradient centrifugation at 10000 g for 40 min. The concentrated solution was collected and concentrated.

[0035] S3-2. When the cell aggregation degree in the concentrate is about 80%, transfer it to a T25 culture flask; add culture medium containing 5 μg / mL puromycin and blasticidin to culture the cell lines stably expressing human AChRα1, β1, Rapsn, δ, and ε for 2 weeks; after culturing at 37°C in a carbon dioxide incubator for 72 hours, the transfected cells are separated at a ratio of 1:10 to obtain a cell model for expressing acetylcholine receptor protein.

[0036] Example 2

[0037] A cell model for expressing acetylcholine receptor protein, comprising a lentiviral vector, wherein the lentiviral vector stably expresses subunit genes of human AChRα1, β1, Rapsn, δ, and ε;

[0038] The method for preparing a cell model for expressing acetylcholine receptor protein comprises the following steps:

[0039] S1. Construct human AChRα1, β1, Rapsn, δ, and ε sequences into CMV-MCS-PGK-Puro plasmid, and sequence to identify the sequences; amplify the target fragment, perform double restriction digestion on the recovered target gene, and connect the overexpression vector part with the target fragment; construct the lentivirus;

[0040] S2. HEK-293T cells were cultured with fetal bovine serum and DMEM high glucose medium;

[0041] S3-1. HEK293T cells were transfected with human AChRα1, β1, Rapsn, δ, and ε lentivirus for 24 h (the concentration of human AChRα1, β1, Rapsn, δ, and ε lentivirus and HEK293T cells was 2:1). After 48 h of transfection, fresh culture medium was replaced and cultured for another 48 h. The cell supernatant was then collected and cultured. The cells were then subjected to the first gradient centrifugation at 8000 g for 15 min, the second gradient centrifugation at 2000 g for 20 min, and the third gradient centrifugation at 10000 g for 40 min. The concentrated solution was collected and concentrated.

[0042] S3-2. When the degree of cell aggregation in the concentrate is about 80%, transfer it to a T25 culture flask; add culture medium containing 4 μg / mL puromycin and blasticidin to culture the cell lines stably expressing human AChRα1, β1, Rapsn, δ, and ε for 2 weeks; after culturing at 37°C in a carbon dioxide incubator for 72 hours, the transfected cells are separated at a ratio of 1:10 to obtain a cell model for expressing acetylcholine receptor protein.

[0043] Example 3

[0044] A cell model for expressing acetylcholine receptor protein, comprising a lentiviral vector, wherein the lentiviral vector stably expresses subunit genes of human AChRα1, β1, Rapsn, δ, and ε;

[0045] The method for preparing a cell model for expressing acetylcholine receptor protein comprises the following steps:

[0046] S1. Construct human AChRα1, β1, Rapsn, δ, and ε sequences into CMV-MCS-PGK-Puro plasmid, and sequence to identify the sequences; amplify the target fragment, perform double restriction digestion on the recovered target gene, and connect the overexpression vector part with the target fragment; construct the lentivirus;

[0047] S2. HEK-293T cells were cultured with fetal bovine serum and DMEM high glucose medium;

[0048] S3-1. HEK293T cells were transfected with human AChRα1, β1, Rapsn, δ, and ε lentivirus for 24 h (the concentration of human AChRα1, β1, Rapsn, δ, and ε lentivirus and HEK293T cells was 2:1). After 48 h of transfection, fresh culture medium was replaced and cultured for another 48 h. The cell supernatant was then collected and cultured. The cells were then subjected to the first gradient centrifugation at 8000 g for 15 min, the second gradient centrifugation at 2000 g for 20 min, and the third gradient centrifugation at 10000 g for 40 min. The concentrated solution was collected and concentrated.

[0049] S3-2. When the degree of cell aggregation in the concentrate is about 80%, transfer it to a T25 culture flask; add culture medium containing 5 μg / mL puromycin and blasticidin to culture the cell lines stably expressing human AChRα1, β1, Rapsn, δ, and ε for 2 weeks; after culturing at 37°C in a carbon dioxide incubator for 72 hours, the transfected cells are separated at a ratio of 1.5:8 to obtain a cell model for expressing acetylcholine receptor protein.

[0050] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0051] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A cell model for expressing acetylcholine receptor protein, characterized in that: It comprises a lentiviral vector, wherein the lentiviral vector stably expresses subunit genes of human AChRα1, β1, Rapsn, δ, and ε; The method for preparing a cell model for expressing acetylcholine receptor protein comprises the following steps: S1. Construction of lentivirus for human AChRα1, β1, Rapsn, δ, ε; S2. Culture HEK 293T cells; S3. HEK293T cells were transfected with human AChRα1, β1, Rapsn, δ, and ε lentivirus, cultured, gradient centrifuged, concentrated and collected, cultured and passaged to obtain a cell model for expressing acetylcholine receptor protein.

2. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The concentration ratio of the human AChRα1, β1, Rapsn, δ, and ε lentivirus transfected HEK293T cells is 4-6:1-2.

3. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The process of the gradient centrifugation includes three gradient centrifugations, and the conditions of the three gradient centrifugations are as follows: First gradient centrifugation: 600-1000g, 8-20min; Second gradient centrifugation: 2500-3500g, 15-30min; The third gradient centrifugation: 9000-11000g, 30-60min.

4. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The gradient centrifugation temperature is 4-8°C.

5. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The HEK293T cells were cultured in a high-glucose medium.

6. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The culture and subculture conditions are as follows: culturing in a carbon dioxide incubator at a temperature of 35-37°C.

7. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The culture medium is a culture medium containing 4-6 μg / mL of puromycin and blasticidin.

8. The cell model for expressing acetylcholine receptor protein according to claim 1, characterized in that: The cell aggregation degree during the culture passage is 70-90%.