CN112458059B - Stable cell strain for recognizing H3K18la rabbit monoclonal antibody and construction method thereof - Google Patents
Stable cell strain for recognizing H3K18la rabbit monoclonal antibody and construction method thereof Download PDFInfo
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Abstract
The invention provides a stable cell strain for recognizing a rabbit monoclonal antibody H3K18la and a construction method thereof. The invention constructs a CHO-S stably transformed cell engineering strain CHO-JJ-9 with an integration site of Bxb1 by phiC31 integrase, wherein the preservation number is CCTCC NO: c2020246; then the target gene is quickly and efficiently transferred into a CHO-JJ-9 stable transfer cell engineering strain by Bxb1 integrase to construct a CHO-S stable transfer cell strain which can be used for expressing the target gene; wherein, the antibody gene for recognizing H3K18la is transferred into engineering bacteria CHO-JJ-9 by Bxb1 integrase to obtain a stable cell strain for recognizing H3K18la rabbit monoclonal antibody, the preservation number is CCTCC NO: C2020245. the method provided by the invention greatly shortens the construction time of the stably transformed cell strain, improves the success rate of constructing the stably transformed cell strain, obtains the stably transformed cell strain with high copy number, can obviously shorten the period of supplying materials to industrial customers in large batch, and reduces the cost.
Description
Technical Field
The invention belongs to the field of antibody preparation, and particularly relates to a stable cell strain for recognizing an H3K18la rabbit monoclonal antibody and a construction method thereof.
Background
The traditional preparation method of the secretory monoclonal antibody is to use a rabbit immunization method, then take the immune cells of the rabbit and a rabbit osteoma cell fusion method, and comprises the processes of antigen immunization, B cell sorting, cell fusion, cell strain screening, cell bank construction and the like. The traditional prepared fusion cell strain is easy to generate the phenomena of chromosome loss, gene deletion and the like, the stability of different batches of antibody production is seriously influenced, and the product quality is influenced. In addition, the cell lines have problems such as low antibody production and difficulty in cell culture.
Then, antibody genes are cloned by recombinant technology and then transfected into cell strains, and most companies use a transient transfection system, which has the advantages of short cycle, high cost (mainly the cost of transfection reagents and plasmid extraction) and low expression level. Therefore, the stable transfected cell line is the best choice for supplying a large amount of antibody, however, the traditional CHO-S stable transfected cell line is constructed by electric shock transfection, then screening is carried out for about 1.5 months, and then subcloning is carried out, finally the stable transfected cell line is obtained, the whole period needs about 3-4 months, and in the method, the target gene is randomly integrated into the CHO-S genome, and the efficiency is very low. Therefore, a method for obtaining the CHO-S stable cell strain of the monoclonal antibody with higher stable transformation efficiency and shorter period is urgently needed.
Lactic acid was first isolated from milk in 1780 by the swedish chemist karl W hill (Carl Wilhelm Scheele), and for a long time it was considered to be only a waste product produced during glycolysis. In recent years, some new functions of lactic acid are successively discovered, for example, lactic acid can be absorbed by tumor cells and transported to mitochondria for oxidation to provide energy, and the lactic acid in the tumor microenvironment has an inhibitory effect on the killing function of immune cells, and the lactic acid is used for regulating and controlling natural immune signals.
Researchers such as Yingming ZHao and Lev Becker, university of Chicago, USA, found that histone lactonization modification can regulate gene expression. Nature published 24 months and 10 months in 2019 published the result. Researchers have found that lactate-derived lactosylation of histone lysine residues directly promotes chromatin gene transcription as an epigenetic modification. Researchers have identified 28 sites of lactate on the core histones of human and mouse cells. Hypoxia and bacterial stimulation induce the production of lactic acid by glycolysis and it serves as a precursor that promotes the lactatization of histones. Using M1 macrophages exposed to bacteria as a model system, researchers found that histone lactosylation and acetylation had different temporal dynamics. In the late phase of M1 macrophage polarization, lactic modification of histones was enhanced, thereby inducing homeostatic genes involved in wound healing, including Arg 1. Collectively, these results indicate that the endogenous "lactate clock" in bacterially challenged M1 macrophages turns on gene expression to promote homeostasis. Thus, histone lactogenesis offers an opportunity to improve understanding of the function of lactic acid and its role in various pathophysiological conditions, including infection and cancer.
The H3K18la antibody is an antibody for resisting L-lactonization of 18 th lysine of H3 histone, and the construction and recognition of the H3K18la rabbit monoclonal antibody stable cell strain for large-scale production of the H3K18la rabbit monoclonal antibody have important significance for research of diseases such as cancer, infection and the like. Therefore, a method for obtaining the stable cell strain of the H3K18la rabbit monoclonal antibody with higher stable transfer efficiency, shorter cycle and more stability is urgently needed.
Disclosure of Invention
In order to solve the problems, the invention provides an anti-H3K 18la stable transgenic cell strain with high anti-H3 histone 18 lysine lactate (H3K18la) antibody yield at the 18 th position, and a construction method and application thereof. According to the invention, a CHO-S stable transgenic cell engineering strain with an integration site of Bxb1 is constructed and obtained through phiC31 integrase; then the antibody gene is quickly and efficiently transferred into a CHO-S stably transferred cell engineering strain by Bxb1 integrase to construct and obtain the CHO-S stably transferred cell strain which can be used for producing the antibody; wherein, the anti-H3K 18la antibody gene is integrated into the CHO-JJ-9 engineering strain by Bxb1 integrase to obtain a stable cell strain for recognizing the H3K18la rabbit monoclonal antibody. The method provided by the invention greatly shortens the construction time of the stably transformed cell strain, improves the success rate of constructing the stably transformed cell strain, obtains the stably transformed cell strain with high copy number, can obviously shorten the period of supplying materials to industrial customers in large batch, and reduces the cost.
The research of the research group shows that the phiC31 integrase has similar sites of homologous recombination with some integrases ubiquitous in CHO-S cells.
It is not to say that the phiC31 integrase is possessed by CHO-S cells themselves, but that the integration site of phiC31 integrase is homologous to some genes in CHO-S cells. Therefore, the present invention creatively utilizes this site, and introduces a new integrase site with higher integration efficiency (Bxb1) into CHO-S cells by phiC31 integrase.
According to the invention, a Bxb1 integrase site with higher integration efficiency is introduced into a CHO-S cell through phiC31 integrase, then antibiotic screening and GFP (green fluorescent protein) integration experiments are screened, a CHO-S cell with more Bxb1 integrase site copy number is selected, and the position of the Bxb1 integration site in the CHO-S genome is obtained through sequencing and is used as an engineering strain.
When a stable transgenic cell strain is constructed next time, only the sites with integrase on both sides of a target gene are needed to be transfected by a chemical reagent, and under the action of the integrase, the homologous recombination is carried out on the same sites, so that the stable transgenic cell strain can be integrated into the Bxb1 integration site of the CHO-S cell engineering strain at fixed points, and a uniform stable transgenic cell strain library can be obtained within 1 month.
Compared with the traditional preparation method, the engineering strain can shorten the time for constructing the stably transformed cell strain, improve the success rate (about 70%) for constructing the stably transformed cell strain, and the obtained stably transformed cell strains are all high-copy, so that the large-batch delivery period can be shortened, and the cost is reduced; the traditional preparation method has random integration and uncertain integration sites, and the cell strain obtained by the method can obtain the position of the Bxb1 integration site in the CHO-S genome through sequencing, so that the integration site is clear and the quality is stable.
In one aspect, the invention provides an engineered strain of CHO-S stable transgenic cells, comprising an integration site for Bxb1 integrase and an integration site for phiC31 integrase.
Furthermore, the sequence of the integration site of the phiC31 integrase is shown as Seq ID No.1 in the sequence table, the sequence of the integration site of the Bxb1 integrase is shown as Seq ID No.2 in the sequence table, and the preservation number of the engineering strain is CCTCC NO: C2020246. the engineering strain is named as CHO-JJ-9. The CHO-S stable cell engineering strain CHO-JJ-9 is preserved in China center for type culture Collection in 2020, 11 months and 20 days, with the preservation number of CCTCC NO: c2020246, classification name Chinese hamster ovary cell CHO-JJ-9, preservation address eight-way 299 of Wuchang district of Wuhan city, Hubei province, school of Wuhan university, postal code 430072.
In another aspect, the present invention provides a CHO-S stable transgenic cell line for expressing a gene of interest, the cell line comprising an integration site for Bxb1 integrase and an integration site for phiC31 integrase, and further comprising the gene of interest.
Further, the target gene is connected with an integration site of Bxb1 integrase, and the target gene is an antibody gene; wherein, the sequence of the integration site of the phiC31 integrase is shown as Seq ID No.1 in the sequence table, and the sequence of the integration site of the Bxb1 integrase is shown as Seq ID No.2 in the sequence table.
Further, the target genes are anti-H3K 18la heavy chain gene and light chain gene; wherein, the nucleotide sequence of the heavy chain gene resisting H3K18la is shown as Seq ID NO.3 in the sequence table, and the nucleotide sequence of the light chain gene resisting H3K18la is shown as Seq ID NO.4 in the sequence table; the preservation number of the CHO-S stable cell strain is CCTCC NO: C2020245. the CHO-S stable cell strain can be used for stably expressing an anti-H3K 18la gene and is named as CHO-JJ-9-H3K18 la. The CHO-S stable cell strain CHO-JJ-9-H3K18la is preserved in China center for type culture Collection in 20 months 11 in 2020 with the preservation number of CCTCC NO: c2020245, classification and designation of Chinese hamster ovary cell CHO-JJ-9-H3K181a, preservation address of Wuhan city Wuchang district eight-way 299, university of Wuhan, Hubei province, postal code 430072.
The gene fragment of the invention for resisting H3K18la is obtained by amplification from rabbit B lymphocytes through RT-PCR, and then the fragment is constructed into a vector pcDNA3.4 through homologous recombination. The obtained CHO-S stable cell strain can be used for expressing heavy chain genes and light chain genes of anti-H3K 18la, namely the stable cell strain for recognizing the rabbit monoclonal antibody of H3K18 la.
In another aspect, the invention provides a method for constructing a CHO-S stably transfected cell engineering strain, comprising the following steps: the integration site of Bxb1 integrase was inserted into the CHO-S cell line using phiC31 integrase.
Further, the method mainly comprises the step of transferring a plasmid containing a Bxb1 integrase site and a plasmid containing phiC31 integrase into CHO-S cells by a cotransformation method.
Further, the method specifically comprises:
a) cells were adjusted to 3X10 the day before transfection6Viable cells/ml, the cell density was readjusted to 6X10 the following day6Per ml, and plated in 6-well plates;
b) gently mix 8ul Expifeacmine CHO transfection reagent, dilute the transfection reagent into 100ul OptiPRO;
c) adding 1ug of plasmid containing phiC31 integrase and 1ug of plasmid containing Bxb1 integrase site into 100ul OptiPRO culture medium, and standing at room temperature for 5 min;
(d) the two were mixed, left to stand at room temperature for 10-20 minutes, and then the mixture was gently added dropwise to the cells.
Further, the method also comprises a step of screening resistance genes and selecting stable monoclonal strains and/or a step of selecting optimal expression quantity clones, wherein the sequence of the integration site of phiC31 integrase is shown as Seq ID No.1 in the sequence table, and the sequence of the integration site of Bxb1 integrase is shown as Seq ID No.2 in the sequence table.
Further, the steps of screening and selecting stable monoclonal strains by the resistance genes are as follows:
i) screening by using hygromycin to obtain a cell strain;
II) subcloning the cell line obtained in step I).
Further, the steps of screening and selecting stable monoclonal strains by the resistance genes are as follows:
i) putting the transfected cells into a complete culture solution, culturing for 48h, then replacing the complete culture solution with a hygromycin screening culture medium containing 500-1000ug/ml, and replacing the culture solution once every 2-3 days, wherein the complete culture solution is DMEM + 10% FBS;
II) after 2-3 weeks, adjusting the cell density to 0.5 cell/well, spreading the cells in a 96-well plate, observing the condition of each well after 3-4 weeks, selecting the wells with obvious cell proliferation, and carrying out expanded culture.
Further, the step of selecting the clone with the optimal expression amount is as follows:
co-transfecting a plasmid containing GFP and a plasmid containing Bxb1 integrase to the cell strain selected in the step 2);
and secondly, screening antibiotics, determining the optimal expression amount to clone according to the fluorescence intensity and the number of the stable transgenic cells, and using the optimal expression amount as a CHO-S stable transgenic cell engineering strain.
Further, the step of selecting the clone with the optimal expression amount is as follows:
preparing a plasmid containing GFP, and cotransfecting the plasmid and a Bxb1 integrase plasmid into the CHO-S cell selected in the step II);
and secondly, screening antibiotics hygromycin and puromycin on the transfected CHO-S cells to obtain stable transgenic cell strains, and determining the optimal clone according to the fluorescence intensity and the number of the stable transgenic cells to serve as the CHO-S stable transgenic cell engineering strains.
Furthermore, 10 or more than 10 CHO-S cells are selected in step II).
The higher the fluorescence intensity, the higher the expression level, and the higher the number of cells, the more stable the expression.
In another aspect, the invention provides a method for constructing a CHO-S stably transfected cell line for expressing a target gene, which mainly adopts the method, and further comprises the steps of co-transfecting the target gene and a plasmid containing Bxb1 integrase to the CHO-S stably transfected cell engineering line, and establishing the CHO-S stably transfected cell line for expressing the target gene.
Further, the method also comprises a resistance selection of the CHO-S stable transfused cell strain for expressing the target gene.
Further, the specific method for resistance screening is as follows: and transferring the transfected cells into a culture solution for culture, replacing the complete culture solution with a screening culture solution containing 10ug/ml puromycin and 800ug/ml hygromycin after 48 hours, and replacing the screening culture solution every 3-4 days until the cells are stably increased.
Further, the final concentrations of puromycin and hygromycin in the selection medium were 10ug/ml and 800ug/ml, respectively.
Further, the cells in the selection medium were inoculated into the complete medium and cultured for 2 to 3 days, and after inoculation, the cell density was 5X105cells/ml。
Further, the target gene is an antibody gene.
Further, the antibody genes are heavy chain genes and light chain genes of the antibody.
The stable cell strains obtained by the method provided by the invention are all stable cell strains with a large copy number, and the stable cell strains for producing the antibody can be obtained within 1 month only by carrying out transient transfection on plasmids containing antibody heavy chain genes and antibody light chain genes.
Furthermore, the target gene is a heavy chain gene and a light chain gene of anti-H3K 18la, wherein the nucleotide sequence of the heavy chain gene of anti-H3K 18la is shown as Seq ID No.3 in the sequence table, and the nucleotide sequence of the light chain gene of anti-H3K 18la is shown as Seq ID No.4 in the sequence table.
In still another aspect, the present invention provides a method for producing an anti-H3K 18la antibody by recognizing a stable cell strain of rabbit monoclonal antibody H3K18la, comprising the steps of:
firstly, the stably transformed cell strain for identifying the H3K18la antibody is transferred into a shaking flask and subcultured in an environment of 37 ℃;
the method includes the following steps of (1) carrying out enlarged culture;
waiting for the cell density to reach 1x106Then, the temperature is adjusted to 32 ℃ for culture;
the culture was continued for 6 to 15 days, the culture solution was centrifuged, and the cell culture supernatant was obtained by filtration with a filter, and purified and eluted to obtain H3K18la antibody.
Further, the method specifically comprises the following steps:
first, the H3K18la antibody-stabilized cell line was transferred to a 250ml shake flask containing 50ml of a medium, and the flask was placed at 37 ℃ and 8% CO2Subculturing at 95rpm in the environment of (1);
second, after the subculture is completed, the cells are transferred to a larger volumeThe initial density of the cells after inoculation in the shake flask of (3X 10)5cells/ml; in order to obtain better dissolved oxygen in the culture, the volume in the flask should not exceed 1/5 of the total volume;
waiting for the cell density to reach 1x106Then, the temperature is adjusted to 32 ℃ for culture;
fourthly, the culture is continued for 6-15 days, the culture solution is collected, centrifuged and filtered through a 0.45um filter membrane to obtain cell culture supernatant, and the cell culture supernatant is purified and eluted to obtain the anti-H3K 18la antibody.
Further, in step (c), at least 2 passages are performed, and the cell density during the subculture process cannot exceed 2 × 106。
The yield (H3K18la antibody) of the CHO-S recognizing rabbit monoclonal antibody H3K18la can reach 2.5g/L, which is 8 times of transient expression.
In another aspect, the invention provides the use of Bxb1 integrase for constructing a CHO-S stable transgenic cell line, wherein the integration site of Bxb1 integrase is inserted into the CHO-S cell line through phiC31 integrase, and then the target gene and Bxb1 integrase are co-transfected into the CHO-S cell line, thereby constructing the CHO-S stable transgenic cell line.
Further, the target gene is an antibody gene.
Further, the antibody genes are heavy chain genes and light chain genes of the antibody.
Further, the CHO-S stable transgenic cell line is a CHO-S stable transgenic cell line for antibody production.
Further, the CHO-S stable cell strain for producing the antibody is a stable cell strain for recognizing H3K18la rabbit monoclonal antibody, and the heavy chain gene and the light chain gene of the antibody are the heavy chain gene and the light chain gene of anti-H3K 18 la.
Compared with the prior art, the invention has the beneficial effects that:
1. the traditional method needs at least 4 months for constructing the stable transfer cell strain, and the stable transfer efficiency is only 5 percent; the invention provides a CHO-S stable cell engineering strain, which can obtain the CHO-S stable cell strain in one month, and the stable cell efficiency can reach about 70 percent;
2. the traditional method is random integration, and the integration site is uncertain, so that the method provided by the invention can obtain the position of the Bxb1 integration site in the CHO-S genome through sequencing, the integration site is clear, and the quality is stable.
3. The CHO-S stable transgenic cell strain provided by the invention can form a stable transgenic cell pool without subcloning, and the expression quantity of antibodies in the cell pool is higher;
4. the antibody expression quantity of the CHO-S stable transgenic cell strain for producing the antibody provided by the invention is 8 times of that of transient expression, the antibody yield is stable among different production batches, and the antibody quality is high.
5. The obtained stable cell strains are all high-copy, so that the period of supplying materials to industrial customers in large quantities can be obviously shortened, and the cost is reduced.
Drawings
FIG. 1 is a flow chart of the construction method of the CHO-S stably transfected cell engineering strain in example 1
FIG. 2 is a schematic flow chart of the construction method of the CHO-S stably transfected cell engineering strain in example 1
FIG. 3 is a graph comparing cell proliferation during the resistance selection in example 1
FIG. 4 is a graph showing a comparison between the expression levels of transfected GFP proteins in example 1
FIG. 5 is a graph showing comparison of the construction method of CHO-S stably transfected cell line in example 2 and the FC detection of GFP transfection of cell lines obtained by the conventional method
FIG. 6 is a comparison of the SDS-PAGE results of the cell line culture supernatants obtained by the construction method of the CHO-S stable transfected cell line in example 4 and the conventional method
FIG. 7 is a WB detection result of the purified H3K18la antibody obtained in example 5
FIG. 8 is a graph showing the results of antibody specificity of Dot detection of the purified H3K18la antibody obtained in example 5
FIG. 9 is a graph showing the results of measuring the antibody specificity of the IHC antibody IHC obtained in example 5 by purifying H3K18la
Detailed Description
The present invention is described in further detail below with reference to examples, which are intended to facilitate the understanding of the present invention without limiting it in any way. The examples were carried out under the conventional conditions or conditions recommended by the manufacturer, and the reagents or instruments used therefor were not indicated by the manufacturer, and they were all conventional products commercially available.
EXAMPLE 1 construction of CHO-S stably transfected cell engineering Strain
The method for constructing the CHO-S stable cell engineering strain provided by the embodiment is shown in a flow chart (figure 1) and a flow chart (figure 2), and comprises the following steps:
step 1) inserting the integration site of Bxb1 integrase into CHO-S cell line by using phiC31 integrase
a) Cells were adjusted to 3X10 the day before transfection6Viable cells/ml, the cell density was readjusted to 6X10 the following day6Per ml, and plated in 6-well plates;
b) gently mix 8ul Expifeacmine CHO transfection reagent, dilute the transfection reagent into 100ul OptiPRO;
c) synthesizing a plasmid containing integration sites of phiC31 integrase and Bxb1 integrase by gene, extracting the plasmid according to an extraction method of a kit extracted in Omega, adding 1ug of the plasmid containing phiC31 integrase and 1ug of the plasmid containing Bxb1 integrase sites into 100ul of OptiPRO culture medium by an expiCHO-S transfection method, and standing for 5 minutes at room temperature;
(d) the two were mixed, left to stand at room temperature for 10-20 minutes, and then the mixture was gently added dropwise to the cells.
Step 2) screening resistance genes and selecting stable monoclonal strains
I) putting the transfected cells into a complete culture solution, culturing for 48h, then replacing the complete culture solution with a hygromycin screening culture medium containing 500-1000ug/ml of hygromycin, preferably the complete culture solution plus 800ug/ml of hygromycin, and replacing the culture solution once every 2-3 days, wherein the complete culture solution is DMEM plus 10% FBS;
II) after 2-3 weeks, the cell proliferation is shown in FIG. 3, wherein the left picture of FIG. 3 shows that the cells have certain proliferation, the right picture of FIG. 3 shows that the cells have obvious proliferation, the adherent positive CHO cells are digested by pancreatin, the cell density is adjusted to 0.5 cell/well by using a limiting dilution method, the cells are paved into a 96-well plate, the condition of each well is observed after 3-4 weeks, 10 clones with faster cell growth are selected for expanding culture and frozen storage, and the 10 clones are named as CHO-JJ-1 to CHO-JJ-10.
Step 3) transfection of GFP protein to select optimum expression amount clone
Preparing a plasmid containing GFP, and co-transfecting the plasmid and a Bxb1 integrase plasmid into 10 CHO-S cells selected in the step 2) according to an expi CHO-S transfection method;
secondly, screening antibiotics hygromycin and puromycin on the transfected CHO-S cells to obtain stable transfected cell strains, respectively placing 10 clones under a microscope for observation, wherein the result is shown in figure 4, determining the optimal clone according to the fluorescence intensity and the number of stable transfected cells, selecting the clone with high expression level, finding that the fluorescence intensity and the expression level of the clone with the number of CHO-JJ-9 are optimal, and the CHO-JJ-9 is shown in the picture on the left side of figure 4, wherein the picture on the right side of figure 4 is the other clones with lower expression level. CHO-JJ-9 is selected as a CHO-S stable cell engineering strain, and cell preservation is carried out, wherein the preservation number is C2020246.
Example 2 comparison of the construction method of CHO-S stably transfected cell line provided by the present invention with the conventional method
In this example, CHO-JJ-9 was obtained according to the construction method using phiC31 and Bxb1 integrase provided in example 1, and cell lines constructed by random transient transfection of GFP-containing plasmids without phiC31 and Bxb1 integrase were subjected to FC (flow cytometry) detection after 2 weeks of culture, and the results are shown in FIG. 5, in which the left panel is a negative control, the middle panel is the detection result using CHO-JJ-9 provided in example 1, the transfection expression rate is 96.1%, and the right panel is the detection result using transient transfection, and the transfection expression rate is 36.7%. The transfection expression rate was divided by the total number of cells before the initial selection to obtain the stable transformation efficiency, which was 75% using the method of example 1 and only 4.5% using random transient transfection.
Example 3 construction of stably transfected cell lines recognizing rabbit monoclonal antibody H3K18la
In this example, the CHO-JJ-9 engineered strain obtained in example 1 was used to obtain a stable cell strain recognizing rabbit monoclonal antibody against H3K18la by co-transfection of gene against H3K18la and Bxb1 integrase, which specifically comprises the following steps,
(1) an anti-H3K 18la heavy chain fragment and light chain fragment plasmid and a Bxb1 integrase-containing plasmid are co-transfected into an engineering strain CHO-JJ-9, wherein the anti-H3K 18la heavy chain nucleotide sequence is shown as SEQ ID No.3, and the anti-H3K 18la light chain nucleotide sequence is shown as SEQ ID No. 4. The plasmid was prepared according to the Omega transfection plasmid extraction kit and the plasmid transfection procedure was performed according to the expi CHO-S transfection kit instructions.
(2) After 48 hours, the culture medium is changed to contain puromycin of 10ug/ml and hygromycin of 800ug/ml for culture, the culture medium is changed once every 2 to 3 days, and after 2 to 3 weeks, a stable cell strain pool can be obtained.
(3) Using trypsinization, the cells were transferred to a 125ml shake flask containing 30ml CHO-S expression medium for approximately one week of stationary culture.
(4) Then according to the cell density of 0.5x106And (3) replacing the cells into a fresh 125ml shake flask containing 30ml of culture solution, culturing for 1-2 weeks to obtain a stable cell strain CHO-JJ-9-H3K18la which can recognize the H3K18la rabbit monoclonal antibody, and freezing.
Example 4 comparison of construction method for identifying H3K18la Rabbit monoclonal antibody stably transfected cell line with conventional method
In this example, according to the construction method of the rabbit monoclonal antibody stable cell strain recognizing H3K18la provided in example 3, and only plasmids containing heavy chain fragments and light chain fragments of anti-H3K 18la were randomly and transiently transfected without using phiC31 and Bxb1 integrase to construct, the culture supernatants of the obtained cell strains were subjected to SDS-PAGE electrophoresis, and the results are shown in fig. 6, wherein the target antibodies were 55KD and 25KD, channels 5 and 6 are supernatants of the rabbit monoclonal antibody stable cell strains recognizing H3K18la constructed according to the method provided in example 3, and channels 1, 2, 3, 4, 7 and 8 are supernatants of the constructed cell strains randomly and transiently transfected with heavy chain fragments and light chain fragments containing anti-H3K 18 la. The OD280 value was measured by a spectrophotometer (Nanodropp) to determine the antibody content in the supernatant of each channel cell line, and the results are shown in Table 1.
TABLE 1 content of antibody in cell strain supernatant constructed by the method provided in example 3 and conventional method
As can be seen from table 1, the antibody content in the supernatants of channels 5 and 6 (recognizing H3K18la rabbit monoclonal antibody stably transfected cell lines provided in example 3) was significantly higher than that of the cell lines of the other channels; in the other channels, the highest antibody content was 7 and 8, the average value of channels 1, 2, 3, 4, 7, 8 was about 240ug/ml, and the average value of channel 5 and 6 was about 8 times that of channel 6, which indicates that the antibody expression level of the supernatant of the stably transfected cell line obtained by the construction method for identifying the H3K18la rabbit monoclonal antibody stably transfected cell line provided in example 3 was 8 times or more higher than that of the supernatant of the cell line obtained by the conventional method.
Example 5 recognition of H3K18la Rabbit monoclonal antibody stably transfected cell line production anti-H3K 18la antibody
In this embodiment, the anti-H3K 18la antibody is further obtained by using the stable cell strain of the rabbit monoclonal antibody recognizing H3K18la obtained in example 3, and the specific method is as follows:
(1) the H3K18la antibody-transfected cell line obtained in example 3 was transferred to a 250ml shake flask containing 50ml of medium and placed at 37 ℃ in 8% CO2Subculturing at 95rpm in the environment of (1); subculturing the cell strain CHO-S every 2-3 days, wherein at least more than 2 passages are needed, and excessive cell density is avoided in the process of the passages;
(2) after subculture, cells were transferred to larger volume flasks with initial densities greater than 3X10 after cell inoculation5cells/ml; in order to obtain better dissolved oxygen in the culture, the volume in the flask should not exceed 1/5 of the total volume;
(3) after culturing for 48h, the cell density in the culture solution was examined by microscope to find whether it reached 1X106cells/ml, until the cell density reaches 1x106cellAfter s/ml, the temperature of the shaker was adjusted to 32 ℃ for cultivation.
(4) Continuously culturing for 6-15 days (the cell density in the culture solution can reach 7-8x10 to the maximum6cells/ml), collecting 100ml of culture solution, centrifuging at 8000rpm for 15min, filtering with a 0.45um filter membrane to obtain cell culture supernatant, purifying and eluting the cell supernatant by using a GE AKTA GO protein separation and purification system to obtain eluent with the volume of 20ml, obtaining the antibody against H3K18la, and determining the content of the antibody to be 10mg/ml and the total mass of the antibody to be 200mg by using Nanodropp.
The purified H3K18la antibody was detected by WB (Western Blot Western blotting), and the detection results are shown in FIG. 7, wherein the left channel is H3K18la antibody for detecting HeLa cell lysate, and the right channel is H3K18la antibody for detecting cell lysate prepared after the addition of sodium lactate to HeLa cells for induction. The 18 th lactic acid modification of histone has low level of modification in HeLa cell, and when adding inducer sodium lactate, HeLa cell is induced to produce more lactic acid modification of histone. As can be seen from FIG. 7, the size of this band is around 15kd, indicating that the antibody recognizes histone H3, while the right band is significantly stronger than the left band, indicating that the antibody recognizes the lactate of histone H3.
The specificity of the H3K18la antibody is detected by Dot blot (Dot blot hybridization), the detection result is shown in FIG. 8, wherein the polypeptides represented by channels 1-14 are shown in Table 2, and the antibody only recognizes the polypeptide of H3K18la (namely, the 18 th lysine lactate modification of histone H3) and has almost no cross with other polypeptides.
TABLE 2 respective channels and corresponding polypeptides in Dot blot detection
The specificity of the H3K18la antibody was tested using IHC, and the results are shown in fig. 9, where a is a 2 hour incubation with a non-lactonized modified H3 polypeptide and H3K18la antibody, and B is a 2 hour incubation with a specific peptide (H3K18la) and H3K18la antibody. It can be seen that the antibody has obvious nuclear staining after the incubation of the non-lactic acid modified H3 polypeptide, and no nuclear staining is seen after the specific blocking of the H3K18la polypeptide, so that the H3K18la antibody obtained by the embodiment is indeed the H3K18la antibody, and the specificity is very good.
Although the present invention is disclosed above, the present invention is not limited thereto. Various changes and modifications may be effected therein by one skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
<110> Hangzhou Jingjie Biotechnology Ltd
<120> stable cell strain for recognizing H3K18la rabbit monoclonal antibody and construction method thereof
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Claims (1)
1. A CHO-S stable cell strain is characterized in that the target genes are anti-H3K 18la heavy chain gene and light chain gene; wherein, the nucleotide sequence of the heavy chain gene resisting H3K18la is shown as Seq ID NO.3 in the sequence table, and the nucleotide sequence of the light chain gene resisting H3K18la is shown as Seq ID NO.4 in the sequence table; the preservation number of the CHO-S stable cell strain is CCTCC NO: C2020245.
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