阅读说明：本技术 一种筛选稳定转染的克隆蛋白的方法 (Method for screening stably transfected clone protein ) 是由 高谋 于 2021-08-10 设计创作，主要内容包括：本发明公开了一种筛选稳定转染的克隆蛋白的方法,涉及克隆蛋白筛选技术领域,该筛选稳定转染的克隆蛋白的方法,包括细胞培养、传代培养、配制筛选培养基、转染、统计转染率、更换培养基、加药选择、换药培养、减药培养、停药培养、标记细胞群、孔板培养、鉴定前准备、准备采摘和鉴定。本发明通过将原代细胞经过培养分离并进行传代培养,形成多个对照组和实验组,同时通过筛选培养基进行筛选和转染,达到一定转染率之后,通过筛选培养基进行筛选培养,达到转染细胞群体并分离出转染效果较好的细胞群体,最后成功得到转染克隆蛋白的目的,在生物学和医学上具有重要意义。(The invention discloses a method for screening stably transfected cloned protein, which relates to the technical field of cloned protein screening and comprises the steps of cell culture, subculture, preparation of screening culture medium, transfection, counting of transfection rate, culture medium replacement, medicine adding selection, medicine changing culture, medicine reducing culture, medicine stopping culture, marking of cell groups, pore plate culture, preparation before identification, preparation before picking and identification. According to the invention, primary cells are cultured, separated and subcultured to form a plurality of control groups and experimental groups, and meanwhile, a screening culture medium is used for screening and transfecting to achieve a certain transfection rate, and then the screening culture medium is used for screening and culturing to achieve the purposes of transfecting cell populations and separating cell populations with good transfection effects, and finally, the purpose of successfully obtaining transfected clone protein is achieved, so that the method has important significance in biology and medicine.)

1. A method of screening for stably transfected cloned proteins, comprising: the method comprises the following steps:

s1, cell culture: performing primary cell culture by adopting an amino acid balanced saline medium;

s2, subculturing: when the cell confluence reaches a certain index, carrying out separation subculture;

s3, preparing a screening medium: mixing G418 and HEPES, dissolving with distilled water, filtering, sterilizing, and storing at low temperature;

s4, transfection: when the cell density rises by 50-70%, transfecting quantitative GFP, vector DNA and plasmid into the cells;

s5, statistical transfection efficiency: after 18 hours after transfection, carrying out transfection examination every 2 hours to obtain the transformation efficiency;

s6, replacing culture medium: removing the amino acid balanced saline medium and washing with PBS;

s7, medicine adding selection: adding a screening culture medium for cell selection;

s8, dressing change culture: replacing the screening culture medium regularly according to the color of the culture medium and the growth condition of cells;

s9, drug reduction culture: when the cell death rate is increased, the concentration of the screening culture medium is reduced to continue screening culture, and the screening culture medium is replaced periodically;

s10, stopping drug culture: after 2 weeks of screening, resistant clones appear, at which time the drug is stopped and the culture is continued;

s11, labeled cell population: selecting and labeling cell populations that directly look like a gray circle, almost filling the field of view under the microscope;

s12, well plate culture: circling out the marked colony, scraping the surrounding substances, picking the selected cell colony, and placing the cell colony on a pore plate added with a medium for continuous culture;

s13, preparation before identification: repeatedly adding PBS, and washing the medium;

s14, preparing picking: taking a small piece of quantitative filter paper soaked in trypsin, placing the small piece of quantitative filter paper on the top of a cell population, and picking the small piece of quantitative filter paper when the cells grow to four weeks under a microscope;

s15, identification: and (4) carrying out transfection and clone protein identification on the picked cell population.

2. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S1, before the cell culture, the dish needs to be washed repeatedly with 75% ethanol and distilled water, and then distilled water is used.

3. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S2, the degree of cell confluence is 80% to 100%.

4. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S2, the density of the cell subculture is 1:2 to 1: 20.

5. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S3, the screening medium is ready for use and when temporarily stored before use, the screening medium is stored at a temperature of not higher than-4 ℃.

6. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S8, the frequency of changing the screening medium is 2 to 5 days per time.

7. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S9, the concentration of the medium after replacement does not exceed 50%.

8. The method of claim 1, wherein the selection of stably transfected clonal proteins is performed by: in step S12, the medium contains all the antibiotics required for the growth of the cell.

Technical Field

The invention relates to the technical field of cloned protein screening, in particular to a method for screening stably transfected cloned protein.

Background

Transfection (transfection) is a process by which eukaryotic cells actively or passively introduce foreign DNA fragments under certain conditions to obtain a new phenotype, and in the prior art, recombinant fusion proteins are prepared by linking the encoded mouse CR2(complement receptor 2) sequence to the sequence encoding the extracellular domain of mouse Crry using residues 1-319 encoding the mature protein, and a linker encoding (GGGGS)2 is used to link CR2 to Crry. The transfected clone protein can be obtained only by screening and identifying through a certain process, and the clone protein has extremely important significance in both biology and medicine, so that the screening of the stable transfected clone protein is also significant, and the novel method for screening the stable transfected clone protein is provided.

Disclosure of Invention

Technical problem to be solved

Aiming at the defects of the prior art, the invention provides a method for screening stably transfected cloned protein, which solves the problem that the screening of stably transfected cloned protein has important significance in biology and medicine.

(II) technical scheme

In order to achieve the purpose, the invention provides the following technical scheme: a method of screening stably transfected clonal proteins comprising the steps of:

s1, cell culture: primary cell culture was performed using amino acid balanced saline medium.

S2, subculturing: when the cell confluence reaches a certain index, separation subculture is carried out.

S3, preparing a screening medium: mixing G418 and HEPES, dissolving with distilled water, filtering, sterilizing, and storing at low temperature.

S4, transfection: when the cell density rises 50% -70%, GFP, vector DNA and plasmid are transfected into the cells quantitatively.

S5, statistical transfection efficiency: 18 hours after transfection, transfection examination was performed every 2 hours to obtain transformation efficiency.

S6, replacing culture medium: the amino acid-balanced saline medium was removed and washed with PBS.

S7, medicine adding selection: and adding a screening medium for cell selection.

S8, dressing change culture: the screening medium was changed periodically depending on the color of the medium and the growth of the cells.

S9, drug reduction culture: when the cell death rate increases, the concentration of the screening medium is reduced to continue the screening culture, and the medium is periodically replaced.

S10, stopping drug culture: after 2 weeks of selection, resistant clones appeared, at which time drug withdrawal continued to culture.

S11, labeled cell population: cell populations that directly look like a gray circle, almost filling the field of view under the microscope, were selected and labeled.

S12, well plate culture: and (4) circling out the marked population, scraping surrounding substances, picking the selected cell population, and placing the cell population on a well plate added with a medium for continuous culture.

S13, preparation before identification: PBS was repeatedly added to the reaction mixture to wash the medium.

S14, preparing picking: a small piece of quantitative filter paper soaked in trypsin is taken and placed on the top of a cell population, and the cells can be picked up when the cells grow to four weeks on the quantitative filter paper under a microscope.

S15, identification: and (4) carrying out transfection and clone protein identification on the picked cell population.

Preferably, in step S1, the culture dish needs to be repeatedly washed with 75% ethanol and distilled water before cell culture, and then distilled water is used.

Preferably, in step S2, the degree of cell confluence is 80% to 100%.

Preferably, in step S2, the cell is subcultured at a density of 1:2 to 1: 20.

Preferably, in step S3, the selection medium is ready for use and when temporarily stored prior to use, the selection medium is stored at a temperature of no more than-4 ℃.

Preferably, in step S8, the frequency of replacing the selection medium is 2 to 5 days per time.

Preferably, in step S9, the concentration of the medium after replacement is not more than 50%.

Preferably, in step S12, the medium contains all the antibiotics required for the growth of the cell.

(III) advantageous effects

The invention provides a method for screening stably transfected cloned protein, which has the following beneficial effects:

according to the invention, primary cells are cultured, separated and subcultured to form a plurality of control groups and experimental groups, and meanwhile, a screening culture medium is used for screening and transfecting to achieve a certain transfection rate, and then the screening culture medium is used for screening and culturing to achieve the purposes of transfecting cell populations and separating cell populations with good transfection effects, and finally, the purpose of successfully obtaining transfected clone protein is achieved, so that the method has important significance in biology and medicine.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The invention provides a technical scheme that: a method of screening stably transfected clonal proteins comprising the steps of:

s1, cell culture: primary cell culture was performed using amino acid balanced saline medium.

S2, subculturing: when the cell confluence reaches a certain index, separation subculture is carried out.

S3, preparing a screening medium: mixing G418(Geneticin, an aminoglycoside antibiotic) and HEPES (4-hydroxyethyl piperazine ethanesulfonic acid, a hydrogen ion buffer, capable of controlling a constant pH range for a long time), dissolving with distilled water, wherein the concentration of HEPES is 1M, filtering, sterilizing, and storing at low temperature.

S4, transfection: when the cell density rises by 50-70%, quantitative GFP (Green Fluorescent Protein), vector DNA and plasmid are transfected into the cell.

S5, statistical transfection efficiency: 18 hours after transfection, transfection examination was performed every 2 hours to obtain transformation efficiency.

S6, replacing culture medium: the amino acid-balanced saline medium was removed and washed with PBS (phosphate buffered saline, typically as a solvent).

S7, medicine adding selection: and adding a screening medium for cell selection.

S8, dressing change culture: the selection medium is changed periodically, not too frequently, or not too long, depending on the color of the medium and the growth of the cells.

S9, drug reduction culture: when the cell death rate increases, the concentration of the screening medium is reduced to continue the screening culture, and the medium is periodically replaced.

S10, stopping drug culture: after 2 weeks of selection, resistant clones appeared, at which time drug withdrawal continued to culture.

S11, labeled cell population: cell populations that directly look like a gray circle, almost filling the field of view under the microscope, were selected and labeled.

S12, well plate culture: and (4) circling out the marked population, scraping surrounding substances, picking the selected cell population, and placing the cell population on a well plate added with a medium for continuous culture.

S13, preparation before identification: PBS was repeatedly added to the reaction mixture to wash the medium.

S14, preparing picking: a small piece of quantitative filter paper soaked in trypsin is taken and placed on the top of a cell population, and the cells can be picked up when the cells grow to four weeks on the quantitative filter paper under a microscope.

S15, identification: and (4) carrying out transfection and clone protein identification on the picked cell population.

In step S1, the culture dish needs to be repeatedly washed with 75% ethanol and distilled water before cell culture, and then distilled water is used.

In step S2, the confluency of cells is 80% to 100%.

As a technical optimization scheme of the invention, in step S2, the density of cell subculture is 1: 2-1: 20.

As a technical optimization scheme of the invention, in step S3, the screening culture medium is used as it is, and when the screening culture medium is temporarily stored before use, the storage temperature of the screening culture medium is not higher than-4 ℃.

As a technical optimization scheme of the invention, in step S8, the frequency of replacing the screening medium is 2-5 days/time.

As a technical optimization of the present invention, in step S9, the concentration of the culture medium after replacement does not exceed 50%.

As a technical optimization of the invention, in step S12, the medium contains all the antibiotics required for the growth of the cell.

It is noted that, herein, relational terms such as first and second, and the like may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus.

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.