CN103614340B - The clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application - Google Patents

Abstract

The invention discloses the clone of stably express chicken interleukin-2 10 albumen and construction process thereof and application.The present invention will go the chicken interleukin-2 10 encoding gene directed cloning of signal peptide in containing in the carrier for expression of eukaryon of neomycin resistance gene, obtain recombinant plasmid by its transfected Chinese hamster ovary cell line CHO-K1 cell, pressurizeing through G418, screening, purifying, acquisition are stablized, the clone CHO-chIL-10M of high expression chIL-10 albumen, and its microbial preservation number is CGMCC NO.7659.Clone CHO-chIL-10M constructed by the present invention has wide practical use preparing in recombination chicken IL-10 albumen and chicken interleukin-2 10 monoclonal antibody etc.

Description

Cell line stably expressing chicken interleukin-10 protein and its construction method and application

technical field

The present invention relates to a cell line expressing recombinant chicken interleukin, in particular to a cell line stably expressing chicken interleukin-10 fusion protein. The present invention further relates to the construction method and application of the cell line, belonging to the preparation of recombinant chicken interleukin-10 protein field.

Background technique

Interleukin, referred to as interleukin, is a cytokine produced by white blood cells that mediates intercellular interactions and plays a regulatory role in cell activation, proliferation and differentiation. With the development of molecular biology, research on chicken interleukin genes and functions has gradually become a hot spot. So far, a variety of interleukins have been isolated from chickens, such as: IL-1, IL-2, IL-6, IL- 10. IL-12, IL-16, IL-18, etc. These interleukins can be used in immunotherapeutic agents, immune enhancers, and construction of new genetic engineering vaccines, etc., using interleukins and other cytokines to enhance or regulate the body's non-specific immunity, It will not only effectively prevent and control livestock and poultry diseases, but also generate huge economic benefits in animal husbandry production.

The existing genetic engineering methods for preparing recombinant chicken interleukin-10 protein have defects such as low expression efficiency and unstable expression to varying degrees, which need to be improved.

Contents of the invention

One of the purposes of the present invention is to provide a cell line stably expressing chicken interleukin-10 protein;

The second object of the present invention is to provide a method for constructing the cell line stably expressing chicken interleukin-10 protein;

Above-mentioned purpose of the present invention is achieved through the following technical solutions:

The present invention respectively amplifies the entire coding gene of chicken chIL-10 (euchIL-10F) and the chicken de-signal peptide coding gene (euchIL-10M), and directionally clones them respectively in the true gene containing reporter gene eGFP and neomycin resistance gene (neo). In the nuclear expression vector pEGFP-N1, recombinant plasmids N1-euchIL-10F and N1-euchIL-10M were obtained after enzyme digestion and sequencing identification. In the present invention, the obtained recombinant plasmids N1-euchIL-10F and N1-euchIL-10M were respectively transfected into the Chinese hamster ovary cell line CHO-K1 cells, and were screened and purified under G418 pressure to obtain the cell line CHO-K1 expressing chIL-10 protein. chIL-10M and CHO-chIL-10F. The CHO-chIL-10M and CHO-chIL-10F cell lines were purified twice in a row and subcultured to passage 26. Under the FITC filter channel, CHO-chIL-10M could still observe the activity of the eGFP protein stimulated by the reporter gene. Strong green fluorescence, while CHO-chIL-10F fluorescence is weak; the experimental results show that the two recombinant eukaryotic expression plasmids can express recombinant chicken interleukin-10 protein in CHO-K1 cells, but the cell line CHO-chIL-10M expresses recombinant chicken The expression efficiency of interleukin-10 protein is much higher than that of CHO-chIL-10F expressing recombinant chicken interleukin-10 protein, and the stability of cell line CHO-chIL-10M expressing recombinant chicken interleukin-10 protein is far better than that of CHO-chIL-10M Stability of chIL-10F.

The results of RT-PCR identification showed that the entire coding gene of chIL-10 (euchIL-10F) and chicken signal peptide coding gene (euchIL-10M) were stably expressed in CHO-chIL-10F and CHO-chIL-10M cell lines, respectively. However, the expression intensity of chicken signal peptide coding gene in CHO-chIL-10M cell line is about 4-6 times that of all chIL-10 coding genes in CHO-chIL-10F cell line.

The results of Western blot detection showed that the entire coding gene of chIL-10 (euchIL-10F) and chicken de-signal peptide coding gene (euchIL-10M) were correctly expressed in the cells, but the chicken de-signal peptide coding gene in CHO-chIL-10M cell line The expression level of the gene encoding interleukin-10 in chicken is much higher than the expression level of all genes encoding chicken interleukin-10 in CHO-chIL-10F.

The results of IFA detection showed that mouse anti-chIL-10 serum showed specific red fluorescence with cell lines CHO-chIL-10F and CHO-chIL-10M, while rabbit anti-chIL-10 serum did not react with any cell lines.

The effect of whether there is a signal peptide on the expression of IL family members in vitro is difficult to predict in advance. Li Xing (Li Xing, Establishment of Cell Line Stably Expressing Chicken IL-18 Protein and Preparation of Chicken IL-18 Monoclonal Antibody, 2012, Master Thesis) found that under the same conditions, compared with no signal peptide The signal peptide of chicken IL-18 will obviously promote the efficiency or expression amount of its in vitro expression; the experiment of the present invention is very surprising to find that after the signal peptide of chicken IL-10 is removed, its expression efficiency or expression amount in cells It should be far higher than the expression efficiency or expression amount of chicken interleukin-10 full-length gene.

In view of the expression amount or expression efficiency of chicken interleukin 10 encoding gene without signal peptide in CHO-chIL-10M cell line is much higher than the expression amount or expression efficiency of all chicken interleukin 10 encoding genes in CHO-chIL-10F, the present invention will The cell line CHO-chIL-10M is submitted to a patent-approved institution for preservation, and its microbial deposit number is: CGMCC NO.7659; the classification is named: Chinese hamster ovary cell line. Deposit unit: General Microbiology Center of China Microorganism Preservation Management Committee; preservation time is May 29, 2013: preservation address: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences;

The cell line CHO-chIL-10M constructed by the invention can efficiently and stably express the recombinant chicken interleukin-10 protein, and has broad application prospects in the preparation of the recombinant chicken interleukin-10 protein and the chicken interleukin-10 monoclonal antibody.

The invention involves terms

The term "polynucleotide" or "gene" means deoxyribonucleotides, deoxyribonucleosides, ribonucleosides or ribonucleotides and polymers thereof in single- or double-stranded form. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.) . Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., MolCell.Probes 8:91 -98(1994)).

The term "reporter gene" is a gene encoding a detectable protein or enzyme, and a gene whose expression product can be easily identified. Fusion its coding sequence and gene expression regulatory sequence to form a chimeric gene, or fuse it with other target genes, and express it under the control of the regulatory sequence, so that its expression product can be used to mark the expression regulation of the target gene, and the transformation can be obtained by screening body.

The term "host cell" means a cell comprising a polynucleotide of the invention, regardless of the method used for insertion to produce a recombinant host cell, such as direct uptake, transduction, pairing, or other methods known in the art. Exogenous polynucleotides may remain as non-integrating vectors such as plasmids or may integrate into the host genome.

The term "transformation": A method of introducing a heterologous DNA sequence into a host cell or organism.

The term "expression": the transcription and/or translation of an endogenous or transgene in a cell.

The term "coding sequence": a nucleic acid sequence transcribed into RNA.

The term "eukaryotic expression vector": one or more DNA vectors used to achieve eukaryotic transformation, containing regulatory elements required for eukaryotic cell transcription and translation, such as promoters, poly A tailing signals, etc.; in addition , there can also be a selection marker gene, a gene of interest, and a replication site that can be recognized by eukaryotic cells.

Description of drawings

Figure 1 The formation of G418 pressure-selected 12d resistant colonies (F1, 100×).

Figure 2 Confocal microscope scans 24 hours after transfection.

Fig. 3 The result of stable expression of chIL-10 gene in CHO-K1 cells (100×).

Figure 4 RT-PCR identification of CHO cell lines stably expressing chIL-10F; 1: CHO-chIL-10F; 2: CHO-N1; 3: CHO; 4: water; 5: CHO-chIL-10M; M: DNA molecular mass standard.

Figure 5 Western blot analysis of CHO cell lines stably expressing chIL10 fusion protein; 1: CHO-N1; 2: CHO; 3: CHO-chIL-10F; 4: CHO-chIL-10M; 5: pET-chIL-10; M : DNA molecular mass standard; A: rabbit anti-chIL-10 serum; B: mouse anti-GFP tag monoclonal antibody; C: mouse anti-chIL-10 serum.

Figure 6 IFA identification of CHO cell lines stably expressing chIL-10 (100×); A: CHO-chIL-10F; B: CHO-chIL-10M; C: CHO-N1; D: CHO.

detailed description

The present invention will be further described below in conjunction with specific embodiments, and the advantages and characteristics of the present invention will become clearer along with the description. However, these embodiments are only exemplary and do not constitute any limitation to the scope of the present invention. Those skilled in the art should understand that the details and forms of the technical solutions of the present invention can be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the protection scope of the present invention.

1 Experimental materials

1.1 Experimental materials

The eukaryotic expression plasmid vectors pEGFP-N1 and CHO-K1 cells were preserved by the Experimental Animal Group of the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences; specific pathogen-free (SPF) chickens, clean-grade male New Zealand rabbits, and BALB/c mice were maintained by the Harbin Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. Provided by the Experimental Animal Center of the Veterinary Research Institute, the experimental animal production license number is SCXK (black) 2011-007.

1.2 Main Reagents

Ex Taq, EcoR I, Xho I, Pst I and competent Escherichia coli DH5α were purchased from Bao Bioengineering Dalian Co., Ltd.; Lipofectamine ^TM 2000 transfection reagent was purchased from Invitrogen; Shenzhen) Co., Ltd.; fetal bovine serum, RPMI 1640, and DMEM/F12 medium were purchased from HyClone; plasmid mini-extraction kits and DNA gel recovery kits were purchased from Omega; G418 sulfate was purchased from Amresco; DyLight 594 Labeled goat anti-rabbit IgG was purchased from EarthOx Company, DyLight 594-labeled goat anti-mouse IgG was purchased from Jackson Company, IRDye ^TM 700DX-labeled goat anti-rabbit IgG and IRDye ^TM 700DX-labeled goat anti-mouse IgG (1:5000) were purchased from Rockland Company, anti-GFP tag monoclonal antibody was purchased from Rockland Company; chicken lymphocyte separation solution was purchased from BD Company; lipopolysaccharide (LPS) and concanavalin A (ConA) were purchased from Invitrogen Company, and other reagents were domestic analytical grade.

Example 1 Construction of a cell line stably expressing chicken interleukin-10 protein

1. Experimental method

1.1 Isolation, culture and total RNA extraction of chicken peripheral blood lymphocytes

Collect 10 mL of sodium citrate anticoagulant blood from an adult SPF chicken through sterile wing vein, and separate peripheral blood lymphocytes with chicken lymphocyte separation medium according to the product instructions. Adjust the cell density to 1×10 ⁶ /mL with complete RPMI1640 medium containing 10% fetal bovine serum, add it to a 6-well cell plate for culture, and add LPS and final concentrations of 10ug/mL and 20ug/mL to it, respectively. ConA with concentrations of 10ug/mL, 25ug/mL, and 30ug/mL was used as a stimulant, cultured in a 5% CO ₂ incubator at 37°C, shaken every 4 hours, and cells were collected at different times. Total cellular RNA was extracted with Trizol Reagents total RNA extraction kit, and the specific method was carried out according to the instructions.

1.2 Construction and expression of chIL-10 prokaryotic expression vector and preparation of antiserum

According to the chIL-10 gene registered in GenBank (NM_001004414.2), prokaryotic expression primers were designed to amplify the gene encoding chIL-10 mature protein;

The upstream primer sequence is:

proChIL10-F: 5'-CG GAATTC TTGGAGCCCACCTGCCTG-3' (EcoR I) (SEQ ID NO. 1),

The downstream primer sequences are:

proChIL10-R: 5'-CC CTCGAG TCACTTCTCTCCTCCTCATCAGC-3' (Xho I) (SEQ ID NO. 2).

PCR reaction program: 95°C for 5min; 32 cycles of 94°C for 30s, 68.3°C for 30s, 72°C for 30s; 72°C for 10min. The amplified and purified target gene and pET-30a(+) were double-digested with restriction endonucleases EcoR I and Xho I, and the digested products were purified by a gel recovery kit, ligated with T ₄ DNA Ligase, and transformed into Competent DH5α cells. The prokaryotic recombinant expression plasmid obtained after transformation was identified by PCR method, restriction endonuclease analysis and nucleic acid sequence determination, and named pET-chIL-10. Transform pET-chIL-10 into E.coli BL21(DE3), and induce expression with 0.1mmol/L IPTG. The bacteria were collected, ultrasonically disrupted in an ice bath, and the precipitate and supernatant were collected respectively, and the expression form of the target protein was analyzed by SDS-PAGE. A large number of recombinant bacteria were induced to express, and the gel strip where the target protein was located was cut off, ground, resuspended with an appropriate amount of PBS, and immunized with New Zealand rabbits and BALB/c mice respectively to prepare rabbit antiserum and mouse antiserum.

1.3 Construction of chIL-10 gene eukaryotic expression plasmid

According to the gene sequence of NM_001004414.2, 2 pairs of eukaryotic expression primers were designed to amplify the full length of chIL-10 (called euchIL-10F) and the sequence without signal peptide (called euchIL-10M) respectively.

The primer sequence of euchIL-10F is:

euChIL10F-U: 5'- CTCGAG ACCATGCAGACCTGCTGCCAAG-3' (Xho I) (wherein ACC is a Kozak sequence) (SEQ ID NO.3);

euChIL10F-L: 5'-AA CTGCAG CTTCTCCTCCTCATCA-3' (Pst I) (stop codon removed) (SEQ ID NO. 4).

The primer sequence of euchIL-10M is:

euchIL-10M-U: 5'- CTCGAG GCCATGTTGGAGCCCACCT-3'(Xho I) (SEQ ID NO.5);

euchIL-10M-L: 5'-AA CTGCAG CTTCCTCCTCCTCATC-3' (Pst I) (SEQ ID NO. 6).

The amplified and purified target gene and the eukaryotic expression vector pEGFP-N1 containing the reporter gene eGFP were double digested with Xho I and Pst I, and the digested products were purified by the gel recovery kit and ligated with T ₄ DNA Ligase. Transform DH5α competent cells. The positive clones obtained after transformation were identified by PCR method, restriction endonuclease analysis and nucleic acid sequence determination. Use the endotoxin-free mini-plasmid extraction kit to extract plasmids from the identified clones. The resulting eukaryotic recombinant expression plasmids were named N1-chIL-10F and N1-chIL-10M, respectively.

1.4 Establishment of CHO-K1 cell line stably expressing chIL-10 gene

Passage the CHO-K1 cells in a 96-well cell plate. When the cells reach 80%, add DMEM/F12 culture medium containing different concentrations (100-1200mg/L) of G418 and 10% fetal bovine serum, and culture continuously for about 2 weeks , to determine the minimum lethal dose of G418 cells. Referring to the kit instructions, Lipofectamine ^TM 2000 was used to transfect CHO-K1 cells, and the vector pEGFP-N1 was also transfected as a negative control. After 24 hours, resistance selection was carried out with DMEM/F12 complete culture medium containing G418 at a final concentration of 800 mg/L. The transfection-positive cells were serially monocloned by limiting dilution method; at the same time, a control cell line (CHO-N1) expressing the empty vector pEGFP-N1 was established. Routine trypsinization and serial subculture of cells.

2 Experimental results

Figure 1 is the result of G418 pressurized screening for 12 days of resistant colony formation (F1, 100×); Figure 2 is the confocal microscope scanning image after 24 hours of transfection. From the results in Figure 3, it can be seen that CHO-chIL-10M and CHO-chIL-10F cells have undergone two consecutive monoclonal purifications and subcultured to the 26th generation. Under the FITC filter channel, CHO-chIL-10M can still be observed The strong green fluorescence excited by the reporter gene eGFP protein, while the fluorescence of CHO-chIL-10F was weak (Figure 3), indicated that the two recombinant eukaryotic expression plasmids could be expressed in CHO-K1 cells, but the expression intensity of the reporter gene or There are very significant differences in efficiency, and the expression efficiency and expression stability of CHO-chIL-10M are significantly better than those of CHO-chIL-10F. The experimental results show that after the chicken IL-10 signal peptide is removed, its expression efficiency or expression level in cells is much higher than that of the chicken IL-10 full-length gene.

Example 2 Identification of cell lines stably expressing chicken interleukin-10 protein

1. Experimental method

1.1 RT-PCR detection

Trizol Reagents extraction kit was used to extract the total RNA of CHO-chIL-10F and CHO-chIL-10M cells constructed in Example 1, and the specific method was carried out according to the instructions of the kit. The reverse transcription product cDNA was taken, and the chIL-10 gene was identified by PCR amplification. The reaction program was: 95°C for 5 minutes; 30 cycles of 94°C for 30s, 64°C for 30s, and 72°C for 40s; 72°C for 10 minutes.

1.2 IFA detection

The cloned and purified CHO-chIL-10F and CHO-chIL-10M cell lines were seeded in 96-well plates at ¹ ×104/well, with CHO-N1 cells as negative control and CHO-K1 cells as blank control, respectively. The prepared chIL-10 rabbit antiserum and mouse antiserum were used as primary antibodies (1:50), and DyLight 594-labeled goat anti-rabbit IgG and DyLight 594-labeled goat anti-mouse IgG were used as secondary antibodies (1:200) for IFA detection .

1.3 Western blot detection

The CHO-chIL-10F and CHO-chIL-10M cells were collected after being washed with PBS, and the total cell protein was extracted with RIPA cell lysate. After separation by SDS-PAGE, transfer to PVDF membrane, the prepared rabbit antiserum (1:50), mouse antiserum (1:50), anti-GFP tag monoclonal antibody (1:1000) were used as primary antibodies respectively, and IRDye ^TM 700DX-labeled goat anti-rabbit IgG and IRDye ^TM 700DX-labeled goat anti-mouse IgG (1:5000) were used as secondary antibodies for Western blot detection. Scanned with an Odyssey near-infrared two-color laser imaging system. At the same time, CHO-N1 cells transfected with empty vector were used as negative control, and CHO-K1 cells were used as blank control.

2 Experimental results

2.1 RT-PCR identification results

Total RNA was extracted from CHO-chIL-10F, CHO-chIL-10M, CHO-N1 and CHO cells, respectively. Two pairs of primers euChIL10F-U/euChIL10F-L and euchIL-10M-U/euchIL-10M-L were used for RT-PCR amplification respectively. As a result, 533bp and The 473bp band is consistent with the theoretical size of the full-length and signal peptide-free chIL-10 gene, while CHO-N1 and CHO have no corresponding amplification products, indicating that the target gene has been stably expressed in the cell (Figure 4) The expression results showed that the expression level of chIL-10 without the signal peptide (called euchIL-10M) in vitro was about 4-6 times higher than that of the full-length chIL-10 (called euchIL-10F).

2.2 Western blot detection results

The results of Western blot detection (Figure 5) showed that using GFP-specific monoclonal antibody and mouse polyantibody serum, bands of 46ku and 44ku could be detected in the protein cleavage products of CHO-chIL-10F and CHO-chIL-10M respectively , in line with the theoretical value of the fusion protein of eGFP gene and chIL-10 gene, but no band was detected by using rabbit antiserum. It indicated that both the full-length and the chIL-10 gene without signal peptide were correctly expressed in the cells, but the immunogenicity of the recombinant protein was different between rabbits and mice.

2.3 IFA test results

The results of IFA detection showed that the mouse anti-chIL-10 serum showed specific red fluorescence with CHO-chIL-10F and CHO-chIL-10M, while the rabbit anti-chIL-10 serum did not react with any cell line (Figure 6).

Claims (3)

1. A cell line CHO-chIL-10M stably expressing chicken interleukin-10 protein, characterized in that its microorganism preservation number is: CGMCC NO.7659. 2. The use of the cell line CHO-chIL-10M according to claim 1 in the preparation of recombinant chicken interleukin-10 protein. 3. The use of the cell line CHO-chIL-10M according to claim 1 in the preparation of chicken interleukin-10 monoclonal antibody.