CN112029706A - Method for improving performance of CHO (Chinese hamster ovary) producing cell strain based on gene means - Google Patents
Method for improving performance of CHO (Chinese hamster ovary) producing cell strain based on gene means Download PDFInfo
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Abstract
The invention discloses a method for improving the performance of a CHO (Chinese hamster ovary) producing cell strain based on a genetic means, which belongs to the technical field of biological cell genetic engineering and comprises the following steps: cell culture medium, cell monoclonal screening, monoclonal cell chromosome fluorescence in situ hybridization and analysis of factors for improving the performance of CHO production cell strains. The invention clearly observes that the performance of the CHO cell strain can reach higher cell density, and the cell density, the cell survival rate and the expressed protein are all improved, which shows that the culture medium of the invention is a stable and balanced whole, the components interact with each other to jointly promote the growth of Chinese hamster ovary cells, provide rich nutrition environment for the growth of the CHO cell strain cells, and avoid various defects of serum addition.
Description
Technical Field
The invention relates to a CHO cell strain, relates to the technical field of biological cell genetic engineering, and particularly relates to a method for improving the performance of a CHO production cell strain based on a genetic means.
Background
With the continuous development of genomics, molecular biology and modern medicine, the biomedical industry gradually exhibits its unique advantages and has rapidly developed in China in recent decades, wherein recombinant protein drugs, especially antibody drugs, have been developed most rapidly, a large number of enterprises and research institutions have been engaged in anti-tumor, cardiovascular and immune disease-oriented antibody development and research works in China, and a plurality of antibody drugs have been on the market.
Chinese hamster ovary Cells (CHO) are the first choice for expressing medicinal bioactive macromolecules, and the CHO rarely secretes endogenous proteins of the CHO, so that the protein purification is facilitated. Secretory expression is the main expression pathway of CHO, but the expression of many proteins (cytokines, etc.) is relatively low, and the ability to increase cellular secretion can significantly promote the expression of recombinant proteins.
During the expression of secretory proteins by CHO cells, a Signal peptide consisting of 15-30 hydrophobic amino acids is initially synthesized, which is then recognized by a Signal-Recognition Particle (SRP) in the cytoplasm, resulting in a pause in protein synthesis, then the ribosome binds to the endoplasmic reticulum membrane, the Signal peptide is inserted into the lumen of the endoplasmic reticulum membrane by virtue of its hydrophobicity, the SRP will leave after the ribosome binds to the endoplasmic reticulum membrane, and the protein synthesis in the pause state will resume. The signal peptide is then cleaved by a signal peptidase. An appropriate signal peptide helps to increase the expression level of the recombinant protein. However, when the signal peptide is linked to the N-terminus of the foreign protein, the cleavage site is shifted, and the cleavage of the amino acid of the target protein may occur.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for improving the performance of a CHO producing cell strain based on a gene means, so as to solve the problems in the background technology.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a method for improving the performance of a CHO producing cell strain based on a gene means comprises the following steps:
step 1, preparing a cell culture medium;
step 2, screening cell monoclonals;
3, performing fluorescence in situ hybridization on chromosomes of the monoclonal cells;
and 4, analyzing factors for improving the performance of the CHO producing cell strain.
Preferably, the preparation process of the cell culture medium in the step 1 is as follows:
a1, firstly, culturing a Chinese Hamster Ovary (CHO) cell line in a suspension manner, and taking the CHO-K1 cell line as a basal medium;
a2, adding glutamine, anti-caking agent, amino acid, soluble vitamin, inorganic salt, albumin and transferrin into the culture medium, adding a proper amount of water for injection to 75-80% of the total volume, and slightly and slowly stirring for dissolving;
and A3, continuously adding vitamin E and vitamin D, adding 0.22% polysorbate, and stirring to dissolve to obtain a prepared basal medium solution A.
Preferably, the solution A obtained in the step A3 is added with sodium bicarbonate and is slightly and slowly stirred to be dissolved, a proper amount of water for injection is added to be stirred to prepare a solution B, the pH value of the solution B is adjusted to 7.1-7.3 by using 0.9-1.2mol/L sodium hydroxide solution or 0.9-1.2mol/L hydrochloric acid solution to prepare a solution C, and the solution C is subjected to positive pressure filtration sterilization by using a 0.22 mu m filter membrane to obtain the cell culture medium.
Preferably, the monoclonal cell screening process in step 2 is as follows: selecting a signal peptide capable of highly secreting and expressing a protein in Chinese hamster ovary cells, and connecting the signal peptide to be selected in series with a foreign target protein to obtain a recombinant protein.
Preferably, the chromosome fluorescence in situ hybridization process of the monoclonal cell in the step 3 is as follows:
b1, culturing the cells to be cloned in a culture medium culture solution, and performing gene transfection after the cells are polymerized to 40-80%;
b2, cell growth recovery is carried out for 1 week after gene transfection, and then neomycin with the concentration capable of killing non-gene transfected cells is used for screening for 10-20 days;
b3, after B2 lethal screening, adding a culture solution containing growth factors under neomycin with the concentration of maintaining the growth of transgenic cells, carrying out cell culture amplification in the culture solution for 5-10 days, simultaneously, selecting signal peptides capable of highly secreting and expressing proteins in Chinese hamster ovary cells, and respectively connecting the signal peptides to be selected in series with exogenous target proteins to obtain recombinant proteins;
b4, scraping normal cells by using a cell scraper after the transgenic cells in the B3 grow to form clones, and reserving the transgenic cell clones;
b5, selecting and marking selected transgenic cell monoclonals, digesting the monoclonals by using 0.15-0.25% of pancreatin, quickly placing the digested monoclonals into a new culture vessel for culture and amplification, culturing and amplifying until 65-85% of cells are polymerized, digesting and suspending the cultured and amplified transgenic monoclonals by using 0.15-0.25% of pancreatin, adding culture solution and neomycin with the concentration lethal non-gene transfected cells, continuously culturing the cells, and killing residual non-gene transfected cells in the culture solution under the action of the neomycin with the concentration lethal non-gene transfected cells and the pancreatin;
b6, transplanting the monoclonal cell obtained from B5 into a new culture vessel for culture, and adding centrifugally collected culture solution containing growth factors in the logarithmic growth phase of the cell under the condition of neomycin with the concentration for maintaining the growth of the transgenic cell to obtain a large amount of purified transgenic monoclonal cells.
Preferably, the factors for improving the performance of the CHO producing cell line in the step 4 are as follows: according to the above process, since the number of the most cells of the chromosome is 21(2n =22), only 8 normal chromosomes are provided, and the rest 13 normal chromosomes are rearranged, 20 Chinese Hamster Ovary (CHO) derived CHO-K1 lines have chromosomes which can adhere to the wall or grow in suspension, under appropriate conditions, the clone formation rate can reach 88%, the cell doubling time is 11-13h, namely, the physical and chemical conditions of pH, temperature, oxygen, carbon dioxide and tension are the factors for improving the performance of the CHO producing cell line, artificial control can be performed according to actual needs, and meanwhile, the chemical, physical and biological factors can be applied as conditions for experimental observation, and the factors can also be under strict artificial control.
Due to the adoption of the technical scheme, compared with the prior art, the invention has the technical progress that:
the invention provides a method for improving the performance of a CHO producing cell strain based on a gene means, which provides a rich nutritional environment for the growth of Chinese hamster ovary cells and avoids various defects of serum addition.
Detailed Description
Based on the embodiments of the present invention, all other embodiments obtained by a person of ordinary skill in the art without any creative effort belong to the protection scope of the present invention, and the present invention is further described in detail with reference to the embodiments as follows:
the invention provides a method for improving the performance of a CHO producing cell strain based on a gene means, which comprises the following steps:
step 1, cell culture medium;
step 2, screening cell monoclonals;
3, performing fluorescence in situ hybridization on chromosomes of the monoclonal cells;
and 4, analyzing factors for improving the performance of the CHO producing cell strain.
Example 1
In this embodiment, the present invention provides a technical solution: the preparation process of the cell culture medium comprises the following steps:
a. firstly, a Chinese Hamster Ovary (CHO) cell line is cultured in a suspension way, and the CHO-K1 cell line is used as a basal medium; adding glutamine, an anti-caking agent, amino acid, soluble vitamin, inorganic salt, albumin and transferrin into a culture medium, adding a proper amount of water for injection to 75-80% of the total volume, and slightly and slowly stirring for dissolving; continuously adding vitamin E and vitamin D, adding 0.22% polysorbate, stirring and dissolving to obtain a prepared basal medium solution A;
b. adding sodium bicarbonate into the solution A, slightly and slowly stirring for dissolving, adding a proper amount of water for injection, stirring to prepare a solution B, adjusting the pH of the solution B to 7.2-7.4 by using 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution to prepare a solution C, filtering and sterilizing the solution C under positive pressure by using a 0.22 mu m filter membrane to obtain a cell culture medium, and freezing for storage.
In the embodiment, a rich nutritional environment is provided for the growth of Chinese hamster ovary cells, various defects caused by serum addition are avoided, and the components of the culture medium of the invention act together, so that harmful metabolites generated by the growth and metabolism of the Chinese hamster ovary cells are reduced, and the expression of functional proteins of the Chinese hamster ovary cells can be promoted under the condition of not adding a supplementary culture medium.
Example 2
On the basis of embodiment 1, the invention provides a technical scheme that: culturing of the expressed protein:
a. test subjects: the CHO cell culture medium prepared in example 1;
b. the test mode is as follows: performing fed-batch culture (adding a supplemented medium) at 37 deg.C in an environment of 5% carbon dioxide and 95% oxygen, wherein the supplemented medium is a fetal calf serum supplemented medium;
c. the process of shake flask culture: taking out the frozen cell culture medium, respectively adding the taken out frozen cell culture medium into four 125ml shake flasks, and after inoculation, ensuring that the total volume of the cells is 30ml, wherein the cell density is as follows: 0.5X 106/ml. Feeding is carried out when the cells grow to the middle stage of the logarithmic growth phase, about 750 mu l of concentrated corresponding feeding culture medium is added, the volume of cell culture liquid is not changed greatly (a little volume is lost during sampling and evaporation in the culture process and can be ignored), and the cells start to be cooled after entering the platform phase, so that the cells are converted into expression protein from the growth state.
Example 3
On the basis of the embodiment 1 and the embodiment 2, the invention provides a technical scheme that: preferably, the CHO cell strain monoclonal cell screening process comprises the following steps: culturing cells to be cloned in a culture medium culture solution, and performing gene transfection after the cells are polymerized to 40-80%; cell growth recovery was performed for 1 week after gene transfection, followed by 10-20 days of selection with neomycin at a concentration lethal to non-gene transfected cells; after lethal screening, adding a culture solution containing growth factors under neomycin with the concentration of maintaining growth of transgenic cells, carrying out cell culture amplification for 5-10 days in the culture solution, simultaneously selecting signal peptides capable of highly secreting and expressing proteins in Chinese hamster ovary cells, and respectively connecting the signal peptides to be selected in series with exogenous target proteins as signal peptides to be selected to obtain recombinant proteins; after the transgenic cells grow to form clones, scraping normal cells by using a cell scraper, and keeping the transgenic cell clones; selecting and marking selected transgenic cell monoclone, digesting the monoclone cell with 0.15-0.25% pancreatin, culturing and amplifying the digested monoclone cell in a new culture vessel to 65-85% cell polymerization, digesting and suspending the cultured and amplified transgenic monoclone cell with 0.15-0.25% pancreatin, adding culture solution and neomycin with the concentration lethal non-gene transfected cell, continuing to culture the cell, and lethal remaining non-gene transfected cell in the culture solution under the action of the neomycin with the concentration lethal non-gene transfected cell and the pancreatin; the obtained monoclonal cells are implanted into a new culture vessel for culture, under the condition of neomycin with the concentration of maintaining the growth of the transgenic cells, a large number of purified transgenic monoclonal cells can be obtained by adding centrifugally collected culture solution containing growth factors in the logarithmic growth phase of the cells, then the amino acid sequence of the recombinant protein is analyzed by a signal peptide analysis tool, the signal peptide on the recombinant protein sequence with accurate analysis of the cleavage site is selected, namely the signal peptide monoclonal cell chromosome fluorescence in-situ hybridization suitable for the exocrine expression of the exogenous target protein is obtained, according to the process, the physicochemical conditions of pH, temperature, oxygen, carbon dioxide and tension are main factors for improving the performance of a CHO production cell strain, the artificial control can be carried out according to the actual needs, and meanwhile, the chemical, physical and biological factors can be applied as conditions for experimental observation, these factors can also be under strict artificial control, so the observation from the above example that CHO producer cell performance can be improved by genetic means.
The following is a description of the beneficial effects of the method for improving the performance of the CHO producing cell strain based on the gene means.
Through the above examples 1, 2 and 3, it can be clearly observed that the performance of the CHO cell line can reach higher cell density, and the cell density, the cell survival rate and the protein titer are all improved, which indicates that the culture medium of the present invention is a stable and balanced whole, the components interact with each other to jointly promote the growth of the chinese hamster ovary cells, provide a rich nutritional environment for the growth of the chinese hamster ovary cells, and avoid various disadvantages of serum addition.
The present invention has been described in general terms in the foregoing, but it will be apparent to those skilled in the art that modifications and improvements can be made thereto based on the present invention. Therefore, modifications or improvements are within the scope of the invention without departing from the spirit of the inventive concept.
Claims (6)
1. A method for improving the performance of a CHO producing cell strain based on a gene means is characterized in that: the method comprises the following steps:
step 1, preparing a cell culture medium;
step 2, screening cell monoclonals;
3, performing fluorescence in situ hybridization on chromosomes of the monoclonal cells;
and 4, analyzing factors for improving the performance of the CHO producing cell strain.
2. The method for improving the performance of the CHO producing cell strain based on the genetic means as claimed in claim 1, wherein the method comprises the following steps: the preparation process of the cell culture medium in the step 1 comprises the following steps:
a1, firstly, culturing Chinese Hamster Ovary (CHO) cell line in suspension, and preparing based on the CHO-K1 cell line;
a2, adding glutamine, anti-caking agent, amino acid, soluble vitamin, inorganic salt, albumin and transferrin into the culture medium, adding a proper amount of water for injection to 75-80% of the total volume, and slightly and slowly stirring for dissolving;
and A3, continuously adding vitamin E and vitamin D, adding 0.22% polysorbate, and stirring to dissolve to obtain a prepared basal medium solution A.
3. The method for improving the performance of the CHO producing cell strain based on the gene means as claimed in claim 2, wherein the method comprises the following steps: and B, adding sodium bicarbonate into the solution A obtained in the step A3, slightly and slowly stirring for dissolving, adding a proper amount of water for injection, stirring to prepare a solution B, adjusting the pH of the solution B to 7.2-7.4 by using 1mol/L sodium hydroxide solution or 1mol/L hydrochloric acid solution to prepare a solution C, and filtering and sterilizing the solution C under positive pressure by using a 0.22-micron filter membrane to obtain the cell culture medium.
4. The method for improving the performance of the CHO producing cell strain based on the genetic means as claimed in claim 1, wherein the method comprises the following steps: the monoclonal cell screening process in the step 2 comprises the following steps:
b1, culturing the cells to be cloned in a culture medium culture solution, and performing gene transfection after the cells are polymerized to 40-80%;
b2, cell growth recovery is carried out for 1 week after gene transfection, and then neomycin with the concentration capable of killing non-gene transfected cells is used for screening for 10-20 days;
b3, after B2 lethal screening, adding a culture solution containing growth factors under neomycin with the concentration of maintaining the growth of transgenic cells, carrying out cell culture amplification in the culture solution for 5-10 days, simultaneously, selecting signal peptides capable of highly secreting and expressing proteins in Chinese hamster ovary cells, and respectively connecting the signal peptides to be selected in series with exogenous target proteins to obtain recombinant proteins;
b4, scraping normal cells by using a cell scraper after the transgenic cells in the B3 grow to form clones, and reserving the transgenic cell clones;
b5, selecting and marking selected transgenic cell monoclonals, digesting the monoclonals by using 0.15-0.25% of pancreatin, quickly placing the digested monoclonals into a new culture vessel for culture and amplification, culturing and amplifying until 65-85% of cells are polymerized, digesting and suspending the cultured and amplified transgenic monoclonals by using 0.15-0.25% of pancreatin, adding culture solution and neomycin with the concentration lethal non-gene transfected cells, continuously culturing the cells, and killing residual non-gene transfected cells in the culture solution under the action of the neomycin with the concentration lethal non-gene transfected cells and the pancreatin;
b6, transplanting the monoclonal cell obtained from B5 into a new culture vessel for culture, and adding centrifugally collected culture solution containing growth factors in the logarithmic growth phase of the cell under the condition of neomycin with the concentration for maintaining the growth of the transgenic cell to obtain a large amount of purified transgenic monoclonal cells.
5. The method for improving the performance of the CHO producing cell strain based on the genetic means as claimed in claim 1, wherein the method comprises the following steps: the fluorescence in situ hybridization process of the chromosome of the monoclonal cell in the step 3 comprises the following steps: analyzing the amino acid sequence of the recombinant protein by a signal peptide analysis tool, selecting a cutting site to analyze the signal peptide on the recombinant protein sequence accurately, namely, performing fluorescence in situ hybridization on the chromosome of a signal peptide monoclonal cell suitable for the exocrine expression of the exogenous target protein.
6. The method for improving the performance of the CHO producing cell strain based on the genetic means as claimed in claim 1, wherein the method comprises the following steps: the factors for improving the performance of the CHO producing cell strain in the step 4 are as follows: according to the process, the physical and chemical conditions of pH, temperature, oxygen, carbon dioxide and tension are main factors for improving the performance of the CHO producing cell strain, and can be artificially controlled according to actual needs, and meanwhile, the chemical, physical and biological factors can be applied as conditions for experimental observation, and the factors can also be under strict artificial control.
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