CN113621577B - Culture medium additive for expressing recombinant protein by humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method - Google Patents

Abstract

The invention belongs to the technical field of gene recombinant vaccines, and particularly relates to a culture medium additive for expressing recombinant protein by a humanized HEK293 cell line, a recombinant hepatitis B vaccine and an expression method. The invention provides a culture medium additive for a humanized HEK293 cell line to express recombinant protein, wherein sodium butyrate and hydrocinnamic acid are added in the suspension serum-free culture process of HEK293 cells transfected with a recombinant expression vector, and preferably, the addition amount of the sodium butyrate is 1.0 to 3.0mol/L, and the addition amount of the hydrocinnamic acid is 0.2 to 1.0mol/L, so that the synergistic effect is realized, and the expression amount of the recombinant protein is increased. Experiments show that the culture medium additive provided by the invention is used for culturing at low temperature, and the expression level of a hepatitis B surface antigen (HBsAg) target gene in HEK293 cells is improved.

Description

Culture medium additive for expressing recombinant protein by humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method

Technical Field

The invention belongs to the technical field of gene recombinant vaccines, and particularly relates to a culture medium additive for expressing recombinant protein by a humanized HEK293 cell line, a recombinant hepatitis B vaccine and a recombinant hepatitis B vaccine expression method.

Background

Hepatitis b virus infection is one of the major public health problems to be solved urgently in the world today, and it is estimated that about 60% of people in china have been infected with hepatitis b virus, and although the world health organization suggests that people want to perform effective hepatitis b vaccination in the infant stage, 3.5 million people in the world still receive chronic Hepatitis B Virus (HBV) infection and are at high risk of developing liver decompensation, cirrhosis and even liver cancer. At present, the virus is difficult to be effectively eliminated in clinical antiviral treatment, and the most economic and effective prevention and control measure is to inject hepatitis B surface antigen (HBsAg), namely hepatitis B vaccine.

Currently commercialized hepatitis B vaccines are derived from Chinese hamster ovary Cells (CHO) and yeast expression systems, and recombinant proteins produced in non-human mammalian cells are modified into two human-like glycan structures different from human cells, namely N-hydroxymethylneuraminic acid (Neu 5 Gc) and galactose-alpha-1, 3-galactose groups. This difference in glycosylation pattern may be highly immunogenic in humans, since antibodies to both glycan structures are sometimes produced and may rapidly clear the recombinant protein from the human circulation.

This immunogenicity can be avoided by producing recombinant proteins in humanized HEK293 cells, which have molecular structures, physicochemical properties and biological functions consistent with those of the humanized proteins. Furthermore, HEK293 may rapidly produce recombinant proteins by transient expression. Although the HEK293 cell has some advantages in the production of recombinant therapeutic proteins, the problem of low protein yield is still to be solved, and it is important to further improve the expression level of recombinant proteins in the HEK293 cell expression system.

Disclosure of Invention

The invention aims to provide a culture medium additive for expressing recombinant protein by a humanized HEK293 cell line, wherein after a target protein expression vector is transfected into a HEK293 cell, sodium butyrate and hydrocinnamic acid are simultaneously added into the culture medium, so that the expression quantity of the target protein is improved.

The second purpose of the invention is to provide a recombinant hepatitis B vaccine, construct an expression vector which uses CAG as a promoter and comprises a hepatitis B surface antigen gene sequence, transfect humanized HEK293 cells, and culture the cells under the action of the culture medium additive of the invention so as to improve the expression quantity of recombinant proteins.

Meanwhile, the invention also provides an expression method of the recombinant hepatitis B vaccine, which utilizes the culture medium additive to culture under the low-temperature condition and improves the expression quantity of the recombinant protein.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the humanized HEK293 cell line expresses a culture medium additive for recombinant proteins, and comprises sodium butyrate and hydrocinnamic acid.

Preferably, the recombinant protein expression vector transfects HEK293 cells, and the culture medium additive is added in the suspension culture process; wherein the addition amount of the sodium butyrate is 1.0-3.0 mol/L, and the addition amount of the hydrocinnamic acid is 0.2-1.0 mol/L. Furthermore, serum-free medium is adopted for suspension culture.

Furthermore, the recombinant protein vector transfects HEK293 cells, and the culture medium additive is added after suspension culture for 24 hours.

The recombinant hepatitis B vaccine expression method includes transfecting HEK293 cell with expression vector containing hepatitis B surface antigen sequence, and adding the culture medium additive during suspension culture. Preferably, the addition amount of sodium butyrate is 1.0 to 3.0mol/L, and the addition amount of hydrocinnamic acid is 0.2 to 1.0mol/L. Furthermore, serum-free medium is adopted for suspension culture.

Furthermore, the recombinant hepatitis B vaccine expression method comprises the steps of transfecting HEK293 cells with an expression vector containing a hepatitis B surface antigen sequence, performing suspension culture for 24 hours, and adding the culture medium additive.

Further, the expression vector containing the hepatitis B surface antigen sequence is transfected into a DMEM high-sugar medium dissolved with a transfection reagent to culture HEK293 cells for 48 hours, then the cells are transferred to be subjected to suspension culture, and the culture medium additive is added after the cells are subjected to suspension culture for 24 hours.

Preferably, the expression method of the recombinant hepatitis B vaccine comprises the steps of transfecting HEK293 cells with an expression vector containing a hepatitis B surface antigen sequence, carrying out suspension culture at 37 ℃ for 24 hours, and then cooling to 33 ℃ for low-temperature culture.

In order to further improve the expression amount of the recombinant hepatitis B vaccine, preferably, the promoter of the expression vector containing the hepatitis B surface antigen sequence is a CAG promoter.

The recombinant hepatitis B vaccine is prepared by adopting the expression method; wherein the hepatitis B surface antigen sequence is shown in SEQ ID NO. 1.

The invention has the following beneficial effects:

the invention provides a culture medium additive for expressing recombinant protein by a humanized HEK293 cell line, wherein sodium butyrate and hydrocinnamic acid are added in the suspension serum-free culture process of HEK293 cells transfected with a recombinant expression vector, and preferably, the addition amount of the sodium butyrate is 1.0-3.0 mol/L, and the addition amount of the hydrocinnamic acid is 0.2-1.0 mol/L, so that the synergistic effect is realized, and the expression amount of the recombinant protein is improved. Experiments show that the culture medium additive provided by the invention is used for improving the expression level of a hepatitis B surface antigen (HBsAg) target gene in HEK293 cells by culturing at low temperature.

Drawings

FIG. 1 is a schematic diagram of an expression vector according to example 3 of the present invention;

FIG. 2 is a graph showing the comparison of the fluorescence analysis results of the inverted fluorescence microscope of the HBsAg expression vector transfected HEK293 cells constructed by different promoters in example 5 of the present invention;

FIG. 3 is a graph showing the comparison of the results of the transfection efficiency of HEK293 cells transfected by HBsAg expression vectors constructed by different promoters in example 5 of the present invention;

FIG. 4 is a graph showing the comparison of the average fluorescence intensity of HEK293 cells transfected with HBsAg expression vectors constructed by different promoters in example 5 of the present invention.

Detailed Description

The invention will be further described with reference to specific embodiments, but the scope of the invention is not limited thereto.

Various media, reagents, E.coli (E.coli JM 109), HEK293 cell line reagents, pIRES-Neo plasmid, pEGFP-C1 plasmid, tool enzymes, suspension serum-free medium and the like used in the following examples and test examples are commercially available. The procedures in the examples and experimental examples are not specifically indicated, but are generally performed by conventional techniques in the art, for example, by referring to molecular cloning, a laboratory manual, compiled by Sambrook et al (Sambrook J & Russell DW. Molecular cloning: a laboratory Manual.2001), or by instructions provided by manufacturers of products.

Example 1 optimized design of HBsAg Gene

The amino acid sequence disclosed by Genbank No. AAS20191.1 is subjected to codon optimization to obtain an optimized hepatitis B surface antigen gene (HBsAg gene), and the sequence of the optimized hepatitis B surface antigen gene is shown as SEQ ID NO. 1.

Example 2 construction of EGFP recombinant expression vector pIRES-EGFP

The embodiment provides a construction method of engineering bacteria containing a recombinant expression vector, which comprises the following steps:

1) PCR amplification of EGFP Gene

Primers P1 and P2 (used for amplifying 720bp EGFP gene DNA) are designed according to the Enhanced Green Fluorescent Protein (EGFP) gene sequence (GenBank: U55763.1, 613-1332 bases) of the pEGFP-C1 vector, ecoRV and NheI enzyme cutting sites are respectively introduced into the 5' ends of the primers, and the primer sequences are shown as follows (enzyme cutting sites are underlined):

P1：5′-CCGGATATCATGGTGAGCAAAGGGCGAGGAG-3'; as shown in SEQ ID NO. 2;

P2：5′-CTAACCGGTGGACTTGTACAGCTCGTCCCATGC-3'; shown as SEQ ID NO. 3.

EGFP gene was amplified using pEGFP-C1 plasmid (available from Clontech, USA) as a template and primers P1, P2, and the reaction system is shown in Table 1 below.

TABLE 1 PCR amplification System

Reaction procedure: 95 ℃ for 3min,94 ℃ for 40s, 56-60 ℃ for 30s,72 ℃ for 40s, 4 cycles per annealing temperature, finally 55 ℃ for 1min,30 cycles, 72 ℃ for 3min.

And (4) recovering the PCR amplification product by agarose gel electrophoresis, and purifying the PCR amplification product to be sent to a biological company for sequencing verification. The result shows that the amplified DNA fragment is completely consistent with the EGFP sequence published by GenBank.

2) Construction of expression vector containing EGFP sequence

PCR amplification of EGFP was double digested with EcoRV, nheI (the correct sequence was verified by sequencing), while the pIRES-Neo plasmid (from Clontech, USA) was double digested with E coRV, nheI. Identifying the enzyme digestion result by agarose gel electrophoresis, and recovering the EGFP sequence fragment and pIRES-Neo linear plasmid DNA after enzyme digestion by gel;

the enzyme digestion system of the PCR amplification product of the EGFP is as follows: mu.L of 10 XNEB Buffer 2.1, 10U/. Mu.L of EcoRV, nheI enzyme each 0.5. Mu.L, 1.3. Mu.g/. Mu.L of EGFP amplification product 0.78. Mu.L, and water to 20. Mu.L. After fully and evenly mixing, incubating for 6h at 37 ℃;

the restriction enzyme system of pIRES-Neo plasmid is as follows: mu.L of 10 XNEB Buffer 2.1, 10U/. Mu.L of EcoRV, nheI enzyme each 0.5. Mu.L, 1.0. Mu.L of plasmid pIRES-Neo (1.0. Mu.g/. Mu.L), and water to 20. Mu.L. After fully and evenly mixing, incubating for 3h at 37 ℃;

the EGFP sequence fragment and pIRES-Neo linear plasmid DNA after enzyme digestion are taken and connected by T4 ligase (the connector system is 10 mu L of 2 Xquick Ligation Buffer, 200ng of pIRES-Neo linear plasmid DNA, 87.2ng of the EGFP sequence fragment after enzyme digestion, 1 mu L of 350U/. Mu.L of T4 ligase, water is supplemented to 20 mu L), and the EGFP sequence fragment and the pIRES-Neo linear plasmid DNA are connected at 16 ℃ overnight. Adding the ligation product into E.coli JM109 competent bacterial suspension for transformation, inoculating 100 μ L of transformed bacterial liquid on an LB solid culture plate containing ampicillin, culturing overnight at 37 ℃, and picking out a single colony for shake culture. Extracting bacterial plasmids, carrying out enzyme digestion verification on the recombinant plasmids, selecting the plasmids with correct enzyme digestion verification, carrying out sequencing verification, and naming the vector with the completely correct target gene sequence as pIRES-EGFP, namely the EGFP recombinant expression vector to be constructed.

EXAMPLE 3 construction of expression vectors containing different promoters

This example provides a method for constructing recombinant expression vectors comprising different promoters, comprising the steps of:

seven promoter fragments of PGK (GenBank: KJ 175229.1), CAG enhancer (GenBank: AJ 575208.1), mCMV (GenBank: KT 343252.1), CHEF-1 alpha (GenBank: KY 447299.1), CMV mutant, HEF-1 alpha (GenBank: AY 393 188. 1) and CAG (GenBank: GU 299216.1) were artificially synthesized. The original promoters on pIRES-EGFP are respectively replaced by adopting a seamless cloning technology, as shown in figure 1. The correctly constructed plasmids are named pIRES-PGK, pIRES-CAGen, pIRES-mCMV, pIRES-CHEF, pIRES-CMVmut, pIRES-HEF and pIRES-CAG respectively.

Example 4 construction of recombinant expression vector containing HBsAg Gene

Artificially synthesizing an HBsAg gene sequence shown as SEQ ID NO.1, and respectively introducing EcoRV enzyme cutting sites and NheI enzyme cutting sites into the 5' end of the gene for convenient cloning.

The HBsAg gene fragment synthesized by double digestion with EcoRV and NheI, and the pIRES-PGK, pIRES-CAGen, pIRES-mCMV, pIRES-CHEF, pIRES-CMVmut, pIRES-HEF and pIRES-CAG are simultaneously digested with EcoRV and NheI. And (3) identifying the enzyme digestion result by agarose gel electrophoresis, and recovering the HBsAg gene sequence fragment and the linear plasmid DNA after enzyme digestion by gel.

The enzyme cutting system of the HBsAg gene fragment is as follows: mu.L of 10 XNEB Buffer 2.1, 10U/. Mu.L of EcoRV, nheI enzyme each 0.5. Mu.L, 1.3. Mu.g/. Mu.L of HBsAg gene fragment 0.78. Mu.L, and make up water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 6h.

The enzyme cutting system of the plasmid is as follows: mu.L of 10 XNEB Buffer 2.1, 10U/. Mu.L each of EcoRV, nheI enzyme 0.5. Mu.L, 1.0. Mu.L of plasmid (1.0. Mu.g/. Mu.L), and make up water to 20. Mu.L. After mixing well, incubate at 37 ℃ for 3h.

The digested HBsAg gene fragment sequence fragment and the linear plasmid DNA are taken and connected by T4 ligase (the connector system is 10 mu L of 2 Xquick Ligation Buffer and 200ng of linear plasmid DNA, the digested HBsAg gene fragment sequence fragment is 87.2ng, 350U/. Mu.L of T4 ligase is 1 mu L, water is added to 20 mu L), and the mixture is connected overnight at 16 ℃. Adding the ligation product into E.coli JM109 competent bacterial suspension for transformation, inoculating 100 μ L of transformed bacterial liquid on an LB solid culture plate containing ampicillin, culturing overnight at 37 ℃, and picking out a single colony for shake culture. Extracting bacterial plasmids, carrying out restriction enzyme digestion verification on the recombinant plasmids, selecting the plasmids with correct restriction enzyme digestion identification, carrying out sequencing verification, and naming the vectors with completely correct target gene sequences as pIRES-PGK-HBsAg, pIRES-CAGen-HBsAg, pIRES-mCMV-HBsAg, pIRES-CHEF-HBsAg, pIRES-CMVmut-HBsAg, pIRES-HEF-HBsAg and pIRES-CAG-HBsAg.

Example 5 cell transfection

The HEK293 cells were cultured in DMEM high-glucose complete medium containing 10% fetal calf serum, and the degree of cell fusion in the next day reached 80-90%. 1 freshly sterilized 1.5ml EP tube was added diluted in DMEM high-sugar medium (serum-free)

3000, the mixture was thoroughly aspirated and mixed, and each plasmid was transfected with 1. Mu.L of Lipo3000 dissolved in 25. Mu.L of DMEM high-sugar medium. 48h after transfection, the wells were washed free of dead and non-adherent cells with PBS, 500 microliters of fresh complete DMEM high-glucose medium was added again, and the transient expression levels were analyzed by inverted fluorescence microscopy and flow cytometry.

Example 6HBsAg Gene expression assay

The HBsAg gene transfected HEK293 cells were transferred 48 hours later to a 125ml Canning shake flask supplemented with 30ml HEK293 suspension medium (purchased from Thermo Fisher Scientific) to inoculate the cells at a concentration of 70 ten thousand/ml. Placing the culture on a shaking table with the set rotating speed of 120rpm/min, incubating for 7 days under the set temperature condition, adding culture medium additives according to the experimental design scheme during the incubation culture process, observing the growth condition of the cells on time every day, and staining and counting by using 0.8% trypan blue. When the cell density reaches 7X 10 ⁶ At/ml, the culture broth was collected and centrifuged to obtain a supernatant to examine the expression level of HBsAg. And analyzing the expression condition of the HBsAg gene by Western blot and ELISA.

Test example 1 comparison of promoter results

After HEK293 cells are transfected by the seven promoter constructs in the experiment, the EGFP transgenes driven by the CAG, HEF-1 alpha, CHEF-1 alpha and CMV mutant promoters have stronger fluorescence intensity under a fluorescence microscope compared with the CMV promoter, and the CAG promoter shows the strongest fluorescence (figure 2). Flow-through results showed the highest transfection efficiency of the vector containing the CAG promoter (81.3%), followed by HEF1- α (75.1%), CMV mutant (67.7%), CHEF1- α (63.4%) and CMV (59.1%; FIG. 3). The flow cytometry detected the average fluorescence intensity, and the results showed that the fluorescence enhancement of the CAG promoter was the highest compared to the CMV promoter (fig. 4), followed by HEF1- α, CMV mutant, CHEF-1 α and CAG enhancer.

Test example 2 HBsAg Low temperature expression results

HBsAg transfected cells were transferred to 125mL suspension culture flasks, added with 30ml HEK293 serum free medium (purchased from Thermo Fisher Scientific Co., ltd.), suspension cultured at 120rpm, analyzed for cell density by a serum cell counter every day, and analyzed for cell activity by trypan blue staining.

The experimental setup was divided into 3 groups, one group being cultured at room temperature 37 ℃; two groups are cultured at the low temperature of 33 ℃ at 37 ℃ after suspension culture for 24 hours; the third group is cultured at low temperature of 33 ℃, cell supernatants are collected after the seventh day of culture, the expression quantity of HBsAg of each group is detected by ELISA, the result shows that the volume expression quantity of HBsAg of the three groups is respectively 19.15mg/L, 34.46mg/L and 23.55mg/L, the expression quantity of the second group is the highest, and the next group is the next group.

Test example 3 Effect of Small molecule supplements (Medium supplements) on HBsAg expression

HKE293 cells were cultured and analyzed according to the culture method of the second group of Experimental example 2, and sodium butyrate and hydrocinnamic acid were added to the cells on the second day of suspension culture at the concentrations shown in the following Table. Cell density was analyzed daily using a serum cytometer and trypan blue staining for cell viability. Cell supernatants were collected by culture to day seven and the expression of HBsAg was measured by ELISA for each group as shown in table 2 below:

TABLE 2

The ELISA results showed that the HBsAg expression levels in the nine groups were 36.05mg/L, 39.46mg/L, 46.34mg/L, 51.49mg/L, 42.06mg/L, 43.14mg/L, 41.12mg/L, 41.21mg/L, 40.32mg/L and 33.14mg/L, respectively, and the highest expression level in the fourth group and the third group were obtained.

The sodium butyrate and the hydrocinnamic acid can play a role in synergism to promote the stable high-level expression of the HBsAg in the HEK293 cell, and the expression quantity of the HBsAg is obviously improved. The invention verifies the effect of low temperature and sodium butyrate and hydrocinnamic acid on the expression of recombinant HBsAg of HEK293 cells through experiments.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Sequence listing

<110> Xinxiang medical college of biologicals institute of Henan Punuo Yi

Culture medium additive for expression of recombinant protein of humanized HEK293 cell line, recombinant hepatitis B vaccine and expression method

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ctgctgacca ggatcctgac catcccccag agcctggaca gctggtggac cagcctgaac 120

ttcctgggcg gcgcccccac ctgccccggc cagaacagcc agagccccac cagcaaccac 180

agccccacca gctgcccccc catctgcccc ggctacaggt ggatgtgcct gaggaggttc 240

atcatcttcc tgttcatcct gctgctgtgc ctgatcttcc tgctggtgct gctggactac 300

cacggcatgc tgcccgtgtg ccccctgctg cccggcacca gcaccaccag caccggcccc 360

tgcaagacct gcaccatccc cgcccagggc accagcatgt tccccagctg ctgctgcacc 420

aagcccagcg acggcaactg cacctgcatc cccatcccca gcagctgggc cttcgccagg 480

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cagtggttcg tgggcctgag ccccaccgtg tggctgagcg tgatctggat gatgtggtac 600

tggggcccca gcctgtacaa catcctgagc cccttcctgc ccctgctgcc catcttcttc 660

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Claims (8)

1. The culture medium additive for the humanized HEK293 cell line to express the recombinant protein is characterized by comprising sodium butyrate and hydrocinnamic acid; transfecting HEK293 cells by the recombinant protein expression vector, and adding the culture medium additive in the suspension culture process; wherein the addition amount of the sodium butyrate is 2.0-2.5 mol/L, and the addition amount of the hydrocinnamic acid is 0.6-0.8 mol/L.

2. The humanized HEK293 cell line expressing recombinant protein media additive of claim 1 wherein the HEK293 cells are transfected with the recombinant protein vector and the media additive is added after 24 hours of suspension culture.

3. The application of the culture medium additive in the aspect of expression of the recombinant hepatitis B vaccine according to any one of claims 1 to 2, characterized by comprising the steps of transfecting HEK293 cells with an expression vector containing hepatitis B surface antigen sequences, and adding the culture medium additive in the suspension culture process; the medium additives comprise sodium butyrate and hydrocinnamic acid; the addition amount of sodium butyrate is 2.0-2.5 mol/L, and the addition amount of hydrocinnamic acid is 0.6-0.8 mol/L.

4. The use according to claim 3, wherein the expression vector comprising the hepatitis B surface antigen sequence is transfected into HEK293 cells and the medium supplement is added after 24h of suspension culture.

5. The use according to claim 3, wherein the expression vector containing the HBsAg sequence is transfected into HEK293 cells in DMEM high-glucose medium containing transfection reagent for 48 hours, then transferred for suspension culture, and the medium additive is added after 24 hours of suspension culture.

6. The use as claimed in claim 3, wherein the expression vector comprising the hepatitis B surface antigen sequence is transfected into HEK293 cells, and after suspension culture at 37 ℃ for 24 hours, the temperature is reduced to 33 ℃ for culture.

7. The use of claim 3, wherein the promoter of the expression vector comprising the hepatitis B surface antigen sequence is a CAG promoter.

8. The use of claim 7, wherein the hepatitis B surface antigen sequence is as set forth in SEQ ID NO:1 is shown.

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