CN115896036A

CN115896036A - Method for constructing HEK293 cell strain of high-expression recombinant protein

Info

Publication number: CN115896036A
Application number: CN202211692573.6A
Authority: CN
Inventors: 张帆; 魏迎东; 李海兰; 徐文正
Original assignee: Jiangsu Huakang Biotechnology Co ltd
Current assignee: Jiangsu Huakang Biotechnology Co ltd
Priority date: 2022-12-28
Filing date: 2022-12-28
Publication date: 2023-04-04

Abstract

The invention relates to the technical field of biology, and discloses a method for constructing a HEK293 cell strain of a high-expression recombinant protein, wherein the HEK293 cell strain of an eIF3i subunit is overexpressed through a genetic engineering method, so that the proliferation speed of cells is improved. The cell strain is applied to recombinant protein expression, so that the production rate of the recombinant protein is accelerated, and the expression quantity of the recombinant protein is greatly improved. HEK293 cells produced recombinant protein in an amount of 4.3mg/30ml, while HEK293-eIF3i cells produced 5.59mg/30ml. The expression level of the protein of HEK293-eIF3i-89 is highest, 2.18mg/10ml, while the expression level of the HEK293 cell is only 1.42mg/ml.

Description

Method for constructing HEK293 cell strain of high-expression recombinant protein

Technical Field

The invention relates to the technical field of biology, in particular to a method for constructing a HEK293 cell strain of high-expression recombinant protein.

Background

A considerable number of the biologics in the current new biomedical products are produced by recombinant cell expression, where most of the proteins are produced in mammalian expression systems. HEK293 cells and CHO cells are the most commonly used cells in eukaryotic protein expression systems, HEK293 as a totipotent cell plays a significant role in virus production and recombinant protein production, but in the research and development experiments at the present stage, the most commonly used biopharmaceutical protein preparation host is CHO because the host has extremely high productivity (0.1-1 g/L of batch culture and 1-10g/L of batch culture) and is suitable for large-scale industrial culture in a relatively short time. CHO cells are unable to carry out all types of human glycosylation because they lack certain glycosyltransferases, such as alpha (2-6) salivary glycosyltransferase and alpha (1-3/4) ribosyltransferase. HEK293 cells exhibit greater gamma-carboxylation of glutamate and sulfonation of tyrosine residues than CHO cells, these post-translational modifications being necessary for therapeutic glycoproteins. The production of protein by the HEK293 stable cell line was only one fifth of that of the CHO cell line, and the cell culture time was also slower than that of CHO cells (the HEK293 doubling time was 33h and CHO 14-17 h), resulting in the need for not only large quantities of transfection-grade plasmid DNA but also larger scale culture systems and longer culture times in recombinant protein production.

Eukaryotic translation factors (eIFs) refer to proteins involved in the process of eukaryotic translation initiation, and at least 12 different initiation factors have been discovered, the interaction of which with ribosomes, mrnas, and initiation trnas to accomplish eukaryotic translation initiation. Among them, eIF3 is also involved in the regulation of cell growth and cell cycle.

Disclosure of Invention

Technical problem to be solved

The invention relates to an HEK293 cell strain for stably expressing an eIF3i subunit gene of a human and a construction method and application thereof.

(II) technical scheme

A method for constructing a HEK293 cell strain with high expression of recombinant protein comprises the following steps:

s1: synthesizing the eIF3i subunit gene of human by gene and constructing plasmid; then the constructed plasmid and two packaging plasmids psPAX2 and pMD2.G of the lentivirus are simultaneously transferred into 293T cells to form the packaged lentivirus;

s2: after concentrating the lentivirus, the lentivirus is infected into HEK293 cells, and a polyclonal HEK293-eIF3i cell strain without drug screening is constructed.

S3: drug screening was performed with puromycin (puro) until all cells of the control HEK293 died, obtaining a polyclonal HEK293-eIF3i cell line.

S4: and detecting the proliferation speed of the polyclonal HEK293-eIF3i cell.

S5: the polyclonal HEK293-eIF3i cell and the HEK293 cell are respectively transfected with the same recombinant protein, and the expression amount of the recombinant protein is compared.

S6: and (3) monoclonal selection: a monoclonal HEK293-eIF3i cell strain (HEK 293-eIF3i 1-200) is obtained by selecting a monoclonal from a polyclonal HEK293-eIF3i cell by an infinite dilution method and culturing.

S7: and respectively transfecting the monoclonal cell strains with recombinant proteins, and comparing the recombinant proteins to obtain the expression quantity so as to obtain the monoclonal cell strain with the highest expression quantity.

Preferably, the base sequence of eIF3i is genetically synthesized in S1 and inserted into a polyclonal cleavage site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-eIF3i-IRES-Puro.

Preferably, the amino acid sequence of eIF3i in S1 is:

MKPILLQGHERSITQIKYNREGDLLFTVAKDPIVNVWYSVNGERLGTYMGHTGAVWCVDADWDTKHVLTGSADNSCRLWDCETGKQLALLKTNSAVRTCGFDFGGNIIMFSTDKQMGYQCFVSFFDLRDPSQIDNNEPYMKIPCNDSKITSAVWGPLGECIIAGHESGELNQYSAKSGEVLVNVKEHSRQINDIQLSRDMTMFVTASKDNTAKLFDSTTLEHQKTFRTERPVNSAALSPNYDHVVLGGGQEAMDVTTTSTRIGKFEARFFHLAFEEEFGRVKGHFGPINSVAFHPDGKSYSSGGEDGYVRIHYFDPQYFEFEFEA。

preferably, the method for infecting HEK293 by the virus in S2 comprises the following steps: HEK293 cells cultured to logarithmic growth phase were adjusted to density of 0.7X 10 ⁶ cells/mL, 24-well plate, 500 uL/well, MOI =5, virus titer 2.38E +7, required virus amount 63uL, and polybrene 6ug/mL per well. Changing the culture medium 24h after infection, and culturing with complete culture mediumTwo days, an unscreened cell line of polyclonal HEK293-eIF3i was obtained.

Preferably, the method for obtaining the stably transformed cell strain of the polyclonal HEK293-eIF3i by the S3 traditional Chinese medicine sieve comprises the following steps: cell lines of the non-drug-screened polyclonal HEK293-eIF3i were cultured to logarithmic growth phase and the cell density was adjusted to 0.7X 10 ⁶ cells/mL, 5ug/mL puro added, 2mL to six well plates, 150rmp,37 ℃,5% CO ₂ Two days of culture, viable cells were counted (trypan blue), cells were passaged, centrifuged and the culture was continued with complete medium containing 5ug/mL puro until all negative controls (HEK 293 not infected with virus) died. And (4) counting the activity rate of the HEK293-eIF3i to obtain a stable polyclonal HEK293-eIF3i cell strain.

Preferably, the culture method of the monoclonal HEK293-eIF3i cell strain in S6 comprises the following steps: culturing the polyclonal HEK293-eIF3i cell strain to a logarithmic growth phase, centrifuging, then re-suspending with a fresh culture medium, counting cells, performing gradient dilution, diluting the cells to a 96-well plate and paving four plates, observing the growth condition of the cells by a microscope after culturing, removing the polyclonal wells, adding the fresh culture medium into each well after culturing for culturing, sucking the cells out when the cells exceed 1/2 of the area of the bottom in the 96-well plate, culturing to a 24-well plate, and continuing to perform expanded culture to obtain the monoclonal HEK293-eIF3i cell strain.

(III) advantageous technical effects

Compared with CHO cells, HEK293 cells have better posttranslational glycosylation modification capacity and can express some recombinant proteins which cannot be expressed by the CHO cells, but because the cell proliferation speed and the protein expression level of the HEK293 cells are lower than those of the CHO cells, the application of the HEK293 biological pharmaceutical protein in preparation is lower than that of the CHO cells. Over-expression of the subunit of eIF3i influences the whole protein translation level, so that the proliferation speed of cells and the protein expression rate in the cells are improved.

The HEK293 cell strain for over-expressing the eIF3i subunit is obtained by a genetic engineering method, so that the proliferation speed of the cell is improved. The cell strain is applied to recombinant protein expression, so that the production rate of the recombinant protein is accelerated, and the expression quantity of the recombinant protein is greatly improved.

As can be seen from FIG. 4 of the specification, the amount of recombinant protein produced by HEK293 cells was 4.3mg/30ml, while the amount produced by HEK293-eIF3i cells was 5.59mg/30ml.

As seen in the attached figure 5 of the specification, the expression level of the protein of HEK293-eIF3i-89 is the highest, 2.18mg/10ml, while the expression level of the HEK293 cell is only 1.42mg/ml.

Drawings

Figure 1 is a schematic diagram of the construction of a plasmid that overexpresses eIF3 i.

FIG. 2 is the amplification curves of internal reference primer CT1 and WPRE primer CT2

FIG. 3 is a proliferation curve of HEK293 and HEK293-elF3 i.

FIG. 4 shows the expression levels of recombinant proteins produced by HEK293 and HEK293-elF3i

FIG. 5 shows the expression level of recombinant protein produced by HEK293-eIF3i monoclonal cell line

Detailed Description

(1) Construction of plasmids overexpressing eIF3 i: the base sequence of eIF3i is synthesized by gene and inserted into the polyclonal enzyme cutting site of plasmid plvx-CMV-MCS-IRES-Puro to obtain expression plasmid vector plvx-CMV-eIF3i-IRES-Puro.

The amino acid sequence of eIF3i is as follows:

MKPILLQGHERSITQIKYNREGDLLFTVAKDPIVNVWYSVNGERLGTYMGHTGAVWCVDADWDTKHVLTGSADNSCRLWDCETGKQLALLKTNSAVRTCGFDFGGNIIMFSTDKQMGYQCFVSFFDLRDPSQIDNNEPYMKIPCNDSKITSAVWGPLGECIIAGHESGELNQYSAKSGEVLVNVKEHSRQINDIQLSRDMTMFVTASKDNTAKLFDSTTLEHQKTFRTERPVNSAALSPNYDHVVLGGGQEAMDVTTTSTRIGKFEARFFHLAFEEEFGRVKGHFGPINSVAFHPDGKSYSSGGEDGYVRIHYFDPQYFEFEFEA

(2) And (3) slow virus packaging:

one day before packaging, cells were plated: when the cell confluence rate reached 85, the supernatant was discarded, and then 5ml PBS was added for washing, and PBS was discarded after washing. Adding 1mL of trypsin prepared in advance, slightly rotating the culture dish to ensure that the whole culture dish is covered, standing and digesting for 30s, discarding a supernatant, adding 7mL of culture medium into the culture dish to stop digestion after standing and digesting for about 30s, blowing and beating the cells into single cells for a plurality of times by using a pipette gun. The cell suspension was diluted with fresh medium as 1:4 (i.e., 40mL culture in a 10cm dish)Diluted), 10uL of the solution was added to a hemocytometer for counting. Diluting the cell suspension again according to the counting result, and diluting the packaging cells to 0.6X 10 ⁶ cells/mL. 10mL of the cell suspension was seeded into a 10cm dish. Shaking the culture dish gently, adding 5% CO ₂ And cultured in an incubator at 37 ℃.

After the virus packaging cells are passaged for 24h, the transfection operation can be carried out when the cell confluence rate is observed to reach 75%, the liquid is changed before transfection, the old culture medium is discarded, and the same amount of fresh culture medium is added. The transfection solution is prepared by the following method according to a 10cm example:

preparing a transfection liquid culture medium which is a serum-free culture medium, diluting plasmids and PEI respectively by using the culture medium, standing for 5min, adding a PEI tube into the plasmid tube, oscillating and uniformly mixing for about 15s, standing for 10min, then slightly adding the diluted plasmids and PEI into a culture dish, marking on the culture dish, uniformly mixing, and putting the mixture into an incubator for continuous culture. The medium was changed 18h after transfection, the old medium was discarded and an equal amount of fresh toxigenic medium was added. Placing into an incubator, and continuing to culture. And collecting cell supernatant after 48h of liquid change in a 15mL centrifugal tube, centrifuging for 3min at 200Xg, taking the supernatant, filtering the supernatant through a 0.22 mu m filter membrane, and collecting filtered liquid, namely virus supernatant.

And (3) virus concentration: the viral supernatant obtained above was transferred to a 100KD concentration tube, centrifuged at 1000Xg for 10min at 4 ℃ and a volume of 250uL.

Measuring the titer of the virus: 293T cells were plated one day before infection at a cell density of 0.2X 10 ⁶ cells/mL,500 uL/well, plated in 24-well plates. The confluency of cells on the day of infection was about 30%. 10uL of the concentrated lentivirus was added to the wells and mixed by slow shaking. The genome of the cells was extracted 48h after infection. The titer of lentivirus was obtained by qPCR detection, and the extracted genome was diluted 10-fold, with the following ligands:

qPCR taq mix	10uL
		PrimerF	0.8uL
PrimerR	0.8uL
		genome	2uL
H ₂ O	6.4uL

And (3) taking the rpp gene as an internal reference gene, detecting the gene WPRE carried in the lentivirus, and relatively quantitatively calculating the titer of the virus. The virus titer calculation method was as follows: titers (transduction Units per mL, TU/mL): TU/mL = (2) ^(Ct1-Ct2) X N × 2 × 1000)/V, CT1= CT value of amplification of internal reference primer, CT2= CT value of amplification of WPRE primer, N = number of cells at infection (2 × 10) ⁵ cells), V = volume of virus fluid (uL) at the time of addition

The gene rpp30 is used as an internal reference gene, the gene WPRE brought in by the lentivirus is detected, and the titer of the virus is calculated relatively and quantitatively.

The virus titer calculation method was as follows:

titers (transduction Units per mL, TU/mL): TU/mL = (2) ^(Ct1-Ct2) ×N×2×1000)/V

CT1= amplification of CT value by internal reference primer

CT2= WPRE primer amplification CT value

N = number of cells at infection (approximately equal to 2 × 10) ⁵ cells)

V = volume of virus solution added (uL)

Virus titer =2.38E +7

(3) Viral infection suspension HEK293:

HEK293 cells were infected with appropriate virus amounts (MOI = 5) depending on the virus titer.

HEK293 cells cultured to logarithmic growth phase were adjusted to density of 0.7X 10 ⁶ cells/mL, 24-well plate, 500 uL/well. MOI =5, virus titer 2.38E +7, required virus amount 63uL, 6ug/mL polybrene per well. And (3) changing the culture solution 24h after infection, and culturing for two days by using a complete culture medium to obtain the polyclonal HEK293-eIF3i cell strain without drug screening.

(4) And (3) obtaining a cell strain of the stably transformed polyclonal HEK293-eIF3i by drug screening:

cell lines of the non-drug-screened polyclonal HEK293-eIF3i were cultured to logarithmic growth phase and the cell density was adjusted to 0.7X 10 ⁶ cells/mL, 5ug/mL puro added, 2mL to six well plates, 150rmp,37 ℃,5% CO ₂ Two days of culture, viable cells were counted (trypan blue), cells were passaged, centrifuged and the medium was replaced, and culture was continued with complete medium containing 5ug/mL puro until all negative controls (HEK 293 not infected with virus) died. The activity rate of the HEK293-eIF3i is calculated to be more than 80 percent, and a stable polyclonal HEK293-eIF3i cell strain is obtained.

(5) Monoclonal culture:

culturing stable polyclonal HEK293-eIF3i cell strain to logarithmic phase, centrifuging to obtain 2mL, centrifuging to obtain 200xg3min, resuspending with 1mL fresh culture medium, counting cells to 2 × 10 ⁶ cells/mL, diluted to 0.5cell/120 uL/well in a gradient, 96-well plate four plates. After four days of culture, the growth of the cells was observed microscopically, and the wells for polyclonals were excluded. On the seventh day of culture, add liquid, add 100uL of fresh medium per well. After 14 days of culture, the cells were almost 1/2 of the area of the bottom of the 96-well plate, and the cells were aspirated and cultured in a 24-well plate. The expansion culture was continued to obtain a monoclonal HEK293-eIF3i cell line (1-200).

(6) Testing the proliferation rate of HEK293 cells overexpressing eIF3 i:

polyclonal HEK293-eIF3i cells and HEK293 at 0.5X 10 ⁶ cells/mL Density inoculates 30mL in 150mL shake flasksIn, with serum-free medium at 37 ℃,5% ₂ After 7 days of culture, cell counts were counted at intervals to compare the proliferation rates of HEK293 cells overexpressing eIF3i and HEK293 cells.

(7) Testing the expression quantity of the HEK293-eIF3i produced recombinant protein:

HEK293-eIF3i cells and HEK293 at 2X 10 ⁶ cells/mL were inoculated into 30mL in 150mL shake flasks, plasmids expressing recombinant proteins were transfected, cell supernatants were collected on the sixth day after transfection, proteins were purified, the amount of protein expression was detected, and the amounts of recombinant proteins produced by eIF3 i-overexpressed HEK293 cells and HEK293 cells were compared.

HEK293 cells produced recombinant protein in an amount of 4.3mg/30mL, while HEK293-eIF3i cells produced 5.59mg/30mL.

(8) Testing the expression quantity of the HEK293-eIF3i monoclonal cell strain for producing recombinant protein:

single clone HEK293-eIF3i (1-200) cells and HEK293 at 2X 10 ⁶ cells/mL are inoculated with 10mL in a 50mL shake tube at the density, plasmids expressing recombinant proteins are transfected, cell supernatants are collected on the sixth day after transfection, proteins are purified, and the expression amount of the proteins is detected.

Claims

1. A method for constructing a HEK293 cell line of high expression recombinant protein is characterized in that: the construction method comprises the following steps:

s1: synthesizing the eIF3i subunit gene of human and constructing a plasmid; then the constructed plasmid and two packaging plasmids psPAX2 and pMD2.G of the lentivirus are simultaneously transferred into 293T cells to form the packaged lentivirus;

s2: concentrating lentivirus, infecting HEK293 cells, and constructing a polyclonal HEK293-eIF3i cell strain without drug screening;

s3: performing drug screening by using puromycin until all cells of a control HEK293 die to obtain a polyclonal HEK293-eIF3i cell strain;

s4: detecting the proliferation speed of the polyclonal HEK293-eIF3i cell;

s5: respectively transfecting the same recombinant protein by using a polyclonal HEK293-eIF3i cell and a HEK293 cell, and comparing the expression quantity of the recombinant protein;

s6: and (3) monoclonal selection: selecting a monoclonal from a polyclonal HEK293-eIF3i cell by an infinite dilution method, and culturing to obtain a monoclonal HEK293-eIF3i cell strain;

s7: and respectively transfecting the recombinant proteins by the monoclonal cell strains, and comparing the recombinant proteins to obtain the expression quantity to obtain the monoclonal cell strain with the highest expression quantity.

2. The method for constructing the HEK293 cell line highly expressing recombinant proteins as claimed in claim 1, wherein the method comprises the following steps: the base sequence of eIF3i is synthesized by the gene in S1 and is inserted into a polyclonal enzyme cutting site of a plasmid plvx-CMV-MCS-IRES-Puro to obtain an expression plasmid vector plvx-CMV-eIF3i-IRES-Puro.

3. The method for constructing the HEK293 cell line with high recombinant protein expression as claimed in claim 1, which is characterized in that: the amino acid sequence of eIF3i in S1 is as follows:

4. the method for constructing the HEK293 cell line highly expressing recombinant proteins as claimed in claim 1, wherein the method comprises the following steps: the method for infecting HEK293 by the virus in S2 comprises the following steps: and (3) culturing the HEK293 cells cultured to the logarithmic growth phase, adjusting the cell density, culturing in a 24-pore plate, adjusting the virus titer and the required virus amount, adding polybrene to each pore, changing the solution after infection, and culturing with a complete culture medium to obtain the polyclonal HEK293-eIF3i cell strain without drug screening.

5. The method for constructing the HEK293 cell line highly expressing recombinant proteins as claimed in claim 1, wherein the method comprises the following steps: the method for obtaining the stably-transformed polyclonal cell strain HEK293-eIF3i by the S3 traditional Chinese medicine sieve comprises the following steps: culturing a polyclonal HEK293-eIF3i cell strain without drug screening to a logarithmic growth phase, adjusting cell density, adding puromycin, culturing in a six-well plate, counting living cells, carrying out passage on the cells, centrifuging, changing liquid, continuously culturing by using a complete culture medium of puromycin until all negative controls die, and counting the activity rate of the HEK293-eIF3i to obtain a stable polyclonal HEK293-eIF3i cell strain.

6. The method for constructing the HEK293 cell line with high recombinant protein expression as claimed in claim 1, which is characterized in that: the culture method of the monoclonal HEK293-eIF3i cell strain in the S6 comprises the following steps: culturing the polyclonal HEK293-eIF3i cell strain to a logarithmic growth phase, centrifuging, then re-suspending with a fresh culture medium, counting cells, performing gradient dilution, diluting into a 96-well plate and laying in four plates, observing the growth condition of the cells under a microscope after culture, removing polyclonal holes, adding the fresh culture medium into each hole after culture for culture, sucking the cells out when the area of the cells exceeds 1/2 of the area of the bottom in the 96-well plate, culturing to a 24-well plate, and continuing expanding culture to obtain the monoclonal HEK293-eIF3i cell strain.