CHO-K1 cell strain capable of stably expressing IBV JS-96 S1 protein, domestication method, IBV S1 protein and application

Abstract

The invention relates to the field of biology, and discloses a CHO-K1 cell strain capable of stably expressing IBV JS-96S1 protein, the cell strain can be well domesticated to obtain a suspended CHO-K1 cell strain, and the suspended CHO-K1 cell strain has the advantages of short domestication time and good cell state. Meanwhile, the invention also discloses a method for obtaining the suspended CHO-K1 cell strain by carrying out suspension domestication on the cell strain and application of the cell strain. In addition, the invention further discloses the IBV S1 protein and application.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a CHO-K1 cell line stably expressing an IBV JS-96 S1 protein, an acclimation method, an IBV S1 protein and uses. Background Art

[0002] Infectious bronchitis (IB) is a highly contagious animal disease caused by infectious bronchitis virus (IBV). It has been prevalent in China for a long time and chickens of different ages are susceptible. IBV can invade the urogenital system, respiratory system and digestive system of chickens and has a wide range of tissue tropism. Infection in laying hens is often mainly reproductive tract type, and the egg quality is significantly reduced, with deformed eggs, small eggs and soft eggs; in chicks and broilers, respiratory type, renal type, glandular stomach type, etc. are the main types. When mixed with other diseases or secondary infections occur in severe cases, the incidence rate can be as high as 90%, and the mortality rate can reach more than 25%.

[0003] IBV belongs to the genus Gammacoronavirus of the order Nidovirales and the family Coronaviridae. Since its replication depends on RNA polymerase, which lacks proofreading ability, IBV has a high degree of genetic variability and numerous serotypes. IBV is a single-stranded positive-strand RNA virus with a genome size of approximately 27.6Kb. The structural gene located at the 3' end of the genome accounts for only one-third of the viral genome and encodes four major structural proteins, namely, nucleocapsid protein (N), spike protein (S), membrane protein (M) and small membrane protein (E). S protein is the main surface structural protein of IBV, composed of S1 and S2 subunits. The S1 subunit contains a receptor binding domain, which is responsible for the virus to attach to host cells and can stimulate the host to produce neutralizing antibodies. Most of the antigenic determinants of IBV are located at the N-terminus of the S1 protein, and viral recombination and host selection pressure cause the S1 protein to mutate at a high rate, resulting in the continuous generation of new serotypes and genotypes. Therefore, whether it is the establishment of IB diagnostic methods or the research of IB genetic engineering subunit vaccines, S1 protein is the preferred target protein.

[0004] The applicant previously applied for an invention patent application CN202410389072.3 Construction and application of CHO cell line of infectious bronchitis virus S1 protein, which discloses the construction of a CHO cell line of infectious bronchitis virus S1 protein of chicken. The scheme first deletes the original ampicillin resistance gene in the pXJ40 plasmid and replaces it with the neomycin-kanamycin resistance gene to obtain the eukaryotic expression vector pXJ40-NeoR / KanR, and then codon optimizes the IBV S1 gene to obtain the codon-optimized IBV S1 gene, and then inserts the codon-optimized IBV S1 gene, IRES2, and EGFP into the eukaryotic expression vector pXJ40-NeoR / KanR prepared in step 1 to obtain the recombinant plasmid pXJ40-S1, and finally transfects the recombinant plasmid pXJ40-S1 into CHO cells to obtain a CHO cell line expressing infectious bronchitis virus S1 protein of chicken.

[0005] After verification, the cell line is only suitable for stable adherent culture, and the expression of S1 protein cannot reach the expected level. In the industrial production of biological products, the yield of adherent cells cannot meet the actual production needs, and the use of culture medium containing animal serum will further increase production costs and increase the instability of the cell culture process. With the continuous development of bioengineering technology, serum-free suspension culture technology of mammalian cells has become the key to the industrialization and scale of biological products.

[0006] The problem to be solved in this scheme is: how to provide a CHO cell line that can express the S1 protein of avian infectious bronchitis virus and can be well suspended and domesticated. Summary of the invention

[0007] One of the purposes of the present application is to provide a CHO-K1 cell line that stably expresses IBV JS-96 S1 protein, which can be well domesticated to obtain a suspension CHO-K1 cell line, which has the advantages of short domestication time and good cell state.

[0008] At the same time, the present invention also discloses a method for obtaining a suspended CHO-K1 cell strain by suspension domestication using the cell strain and the use of the cell strain;

[0009] In addition, the invention also discloses IBV S1 protein and its use.

[0010] To achieve the above objectives, the present application discloses a CHO-K1 cell line stably expressing IBV JS-96 S1 protein, which is prepared by the following steps:

[0011] Step 1: Connect the S1 gene and the vector to obtain a plasmid;

[0012] Step 2: Transfect the plasmid into CHO-K1 cells. After successful transfection, a CHO-K1 cell line can be obtained;

[0013] The nucleotide sequence of the S1 gene is shown in SEQ ID NO.1.

[0014] At the same time, the present invention discloses a method for acclimating a suspended CHO-K1 cell line, wherein the CHO-K1 cell line prepared in step 1 is subjected to multiple stages of suspension culture, and the amount of FBS in the suspension culture medium used in each stage is gradually reduced from the beginning to the end, until the FBS in the suspension culture medium is reduced to 0 and is successfully subcultured for 3 consecutive times, which means that the acclimation is successful, and a suspended CHO-K1 cell line is obtained.

[0015] In the above-mentioned acclimation method, the cycle is divided into at least 6 stages. The suspension culture medium used in the first stage contains 8-12wt% FBS; the suspension culture medium used in the second stage contains 4-6wt% FBS; the suspension culture medium used in the third stage contains 2-3wt% FBS; the suspension culture medium used in the fourth stage contains 0.8-1.2wt% FBS; the suspension culture medium used in the fifth stage contains 0.05-0.2wt% FBS; the suspension culture medium used in the fifth stage does not contain FBS.

[0016] In the above-mentioned acclimation method, the suspension culture medium is CD CHO 031 culture medium.

[0017] In addition, the present invention also discloses a suspended CHO-K1 cell line, which is domesticated by any of the domestication methods described above.

[0018] And the use of the above-mentioned suspension CHO-K1 cell line to harvest IBV S1 protein.

[0019] Finally, the present invention also discloses an IBV S1 protein, which is obtained by culturing the above-mentioned suspension CHO-K1 cell line.

[0020] And use of the IBV S1 protein as described above in preparing polyclonal antibodies.

[0021] The beneficial effects of this application are:

[0022] The present invention uses the vector pcDNA3.1(+) to construct a CHO-K1 cell line that stably expresses the IBV S1 protein. After the cell line is suspended and acclimated, the S1 protein expression level of the cell line is compared with that of the adherent stable cell line that has not been suspended and the CHO cell line constructed by the commercial expression vector pXJ40-S1, and it is found that the cell line has the following advantages:

[0023] 1. Can be domesticated, and the cell activity is good after domestication;

[0024] 2. The expression of S1 protein is high;

[0025] 3. S1 protein has few impurities and high purity;

[0026] 4. Antibodies prepared using S1 protein have high titer and strong specificity. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the plasmid map of the recombinant plasmid;

[0028] Figure 2 This is the PCR amplification result of the target fragment, and the band size is 3073 bp;

[0029] Figure 3 The result diagram of double enzyme digestion verification of the recombinant plasmid;

[0030] Figure 4 The pictures are of cells transfected at different times;

[0031] Figure 5 The figure shows the Western blot protein detection results of the cell pellet and cell supernatant after transfection;

[0032] Figure 6 The results of the optimal concentration screening of G418 for screening monoclonal cells, where A: 9th day (G418 concentration of 300ug / mL group); B: 9th day (G418 concentration of 400ug / mL group); C: 9th day (G418 concentration of 500ug / mL group); D: 9th day (G418 concentration of 600ug / mL group); E: 9th day (G418 concentration of 700ug / mL group); F: control cells (without G418);

[0033] Figure 7 The figure below shows the protein expression results of the supernatant of the monoclonal cell line; the left figure shows the Western blot results of the monoclonal cell line, and the right figure shows the bar chart comparison results based on the grayscale value of the protein bands. Among the eight monoclonal cell lines (C2, C3, C21, C27, C29, C39, C51, and C55), C55 has the best expression, so C55 was selected for downstream experiments;

[0034] Figure 8 A comparison chart of the S1 protein expression levels of CHO cell lines constructed by the cell strain of the present invention and the vector pXJ40-S1;

[0035] Fig. 9 This is a diagram showing the detection results of cell supernatants of the cell lines of the present invention after different passages;

[0036] Fig.10The results of cell density testing at different culture times;

[0037] Fig.11 The results of cell activity detection at different culture times;

[0038] Fig.12 The Western-blot results after purification, where E1-E5 on the left are the first to fifth purification elutions, FT is the flow-through, and W1-W4 are the first to fourth purification washes; in the right figure, 1 is the S1 protein before concentration and replacement, and 2 is the S1 protein after concentration and replacement;

[0039] Fig.13 The results of SDS-PAGE Coomassie blue staining of the purification, where CS is the cell supernatant before purification, FT is the flow-through, W1-W4 are the first to fourth purification washes, and E1-E5 are the first to fifth purification elutions;

[0040] Fig.14 This is the result of SDS-PAGE Coomassie blue staining after purification and concentration;

[0041] Fig.15 The protein standard curve result obtained by BCA protein concentration determination of purified S1 protein;

[0042] Fig.16 This is a graph showing the potency of S protein at different dilutions after being applied to mice;

[0043] Fig.17 Figure 2. Antibody specificity detection results after mouse immunization. DETAILED DESCRIPTION

[0044] The present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. In the description of the present invention, it should be noted that if no specific conditions are specified in the embodiments, the process is carried out according to conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used without specifying the manufacturer are all conventional products that can be purchased from the market.

[0045] In order to better illustrate the contents of the present invention, the materials listed below are some instruments and equipment and main experimental reagents.

[0046] Materials used in this application:

[0047] Table 1 Instruments and equipment

[0048]

[0049]

[0050] Table 2 Main reagents

[0051]

[0052] Example 1 Preparation of CHO-K1 cell line

[0053] 1.1 Construction of recombinant plasmid

[0054] Using the pIRSE2-S1 stored in the laboratory as a template, primers S1-F and S1-R were used to amplify the S1 gene and the downstream EGFP gene (a total of 3073 bp, the sequence is shown in the S1 gene sequence below), and the vector pcDNA3.1(+) was linearized by double digestion with restriction endonucleases BamH I and Not I, and then recovered from the gel. Figure 2 As shown in FIG. 1 ) and the linearized vector were homologously recombined to obtain a recombinant plasmid; the plasmid map is shown in FIG. Figure 1 After transformation to DH5a, plate the cells and pick single clones for expansion culture.

[0055] The primer sequences are:

[0056] S1-F:cttggtaccgagctcggatccGCCACCATGGAGACAGACACA (SEQ ID NO. 2);

[0057] S1-R: gccctctagactcgagcggccgcTTACTTGTACAGTCGTCCATGCC (SEQ ID NO.3);

[0058] The sequence of S1 gene (SEQ ID NO.1) is:

[0059]

[0060] 1.2 Plasmid extraction, identification and sequencing

[0061] The above-mentioned expanded culture solution was mixed with ampicillin resistance medium at a ratio of 1:200 and incubated overnight in a shaker at 37° and 220 r / min. The next day, the recombinant plasmid was extracted and double enzyme digestion was performed on the plasmid. The results are referenced to Figure 3 The plasmids with correct enzyme digestion were sent for sequencing identification. After the sequencing results were correct, the plasmids were extracted after endotoxin removal. Figure 3 This is the result of double enzyme digestion verification of the recombinant plasmid.

[0062] 1.3 Identification of IBV S1 protein expression

[0063] When the density of CHO-K1 cells in the six-well plate reached 80%-100%, transfection was started. 2ug of recombinant plasmid was transfected into each well of the experimental group, and 2ug of empty plasmid was transfected into each well of the control group. The procedure was carried out according to the instructions. After 6-8 hours, the cell status was observed under a microscope, the original culture medium was discarded, and replaced with 2% FBS and complete culture medium without dual antibodies. Cells were observed and photographed using a fluorescence microscope 24h and 48h after transfection ( Figure 4 At 48 h, the cell pellet and cell supernatant were collected for Western blot protein detection ( Figure 5 ).

[0064] 1.4 Pressure screening of single clones

[0065] The optimal G418 concentration was screened out as follows: CHO-K1 cells were inoculated in a 24-well plate, and the culture medium was replaced with 300ug / ml, 400ug / ml, 500ug / ml, 600ug / ml, 700mg / ml, 800ug / ml, and 900ug / ml of antibiotic G418. Three replicates were performed for each concentration, and a control group was set up. The optimal pressurization concentration was the one that caused all cells to die within 9 days ( Figure 6 ). Transfect pcDNA3.1-S1 according to the above operation and set up a negative control group. Start pressurization after 48 hours. Observe the cell status every 3D and take photos and record. Replace the culture medium every 3-5D. After the cells in the control group die completely, single clone cell sorting can be performed using flow cytometry.

[0066] 1.5 Screening of stable monoclonal cell lines

[0067] The steps are as follows: digest the cells after G418 screening with trypsin, resuspend the cells after centrifugation, and sort the single cells into a 96-well plate pre-added with complete culture medium (15% FBS, 2% double antibody) after setting the parameters of the flow cytometer. After about 6 days, single cell clusters can be observed under the microscope. After the cells are full, they are transferred to a 24-well plate for expansion and culture, and the complete culture medium with 10% FBS and 1% double antibody is replaced. After the cells are full, the supernatant is collected for Western blot detection of protein expression of each cell line ( Figure 7 ), the cell line with the best expression of IBV S1 protein was selected and named ACHO-K1-IBV / S1 adherent cells, and the S1 protein expression of the CHO cell line constructed with the commercial expression vector pXJ40-S1 (CN202410389072.3) was compared ( Figure 8 ).

[0068] 1.6 Identification of stable cell lines

[0069] CHO-K1-IBV / S1 adherent cells were transferred to T25 cell flasks for continuous passage. Each generation should be marked when frozen and the passage was stopped at the 15th generation. The cell supernatant was collected every 5 generations, and the supernatant of the 5th and 15th generations was used for Western blot to identify the stability of the cell line ( Fig. 9 ).

[0070] Example 2 Cell suspension culture of CHO-K1 cell line

[0071] The acclimatization culture was carried out using the gradient serum reduction method, which was divided into 6 stages, as follows:

[0072] Phase 1: suspension medium + 10wt% FBS;

[0073] Phase II: suspension medium + 5wt% FBS;

[0074] Stage 3: suspension medium + 2.5wt% FBS;

[0075] Stage 4: suspension medium + 1wt% FBS;

[0076] Stage 5: suspension medium + 0.1wt% FBS;

[0077] Stage 6: serum-free suspension medium 100wt%;

[0078] The steps for each stage are as follows:

[0079] First, inoculate CHO-K1-IBV / S1 adherent cells in the logarithmic growth phase into a 125 ml shake flask and add CD CHO 031 medium to 30 ml to a final concentration of 6.0-1.2×10 5 pc.ml -1 Place in a 37°C, 5% CO 2 , 120r·min-1 on a shaker, and the cell density and viability were tested and recorded every 24h (the results are shown in Fig.10 Fig.11 The cell density must increase exponentially within 3 days, and the viability must reach over 90% before entering the next gradient. The culture was considered successful if the viability was maintained after three generations of continuous transmission, and the suspension cell line SCHO-K1-IBV / S1 was obtained.

[0080] Example 3 Purification of IBV S1 protein

[0081] The supernatant of about 120 ml of the suspension cell line SCHO-K1-IBV / S1 after culturing for 3-6 days was collected and centrifuged at 14000 rpm for 10 min. Then, the supernatant was concentrated to 20-30 ml using a 50Kda 15 ml ultrafiltration tube at 4000 rpm for 20 min for immediate purification.

[0082] The purification steps are as follows:

[0083] (1) Column equilibration and incubation: Use Ni-NTA Resin with full gold content for nickel column affinity chromatography purification. Assemble a 30 ml chromatography column according to the instructions and evenly add 2 ml of chromatography gel. Slowly add denatured lysis buffer (50 mmol / L Tris, 500 mmol / L NaCl, 8 mol / L urea, pH 7.5) to equilibrate the column. After the lysis buffer has drained away, pour in the concentrated supernatant and add protease inhibitors. Incubate at 4° for 6 h on a rotary mixer.

[0084] (2) Flow-through: After assembling the pipeline and column, collect the supernatant after 6 hours of incubation that flows out of the pipeline, pour the recovered supernatant again along the side of the chromatography column, repeat 5 times, and take the sample of the last flow-through for detection;

[0085] (3) Washing: Slowly and gently add 2 ml of denaturing washing solution (50 mmol / L Tris, 500 mmol / L NaCl, 8 mol / L urea, 15 mmol / L imidazole, pH 7.5) along the inner periphery of the chromatography column to prevent the chromatography gel at the bottom of the column from dispersing. Pay attention to slowing down the flow rate of the pipeline. Repeat the operation 5 times and collect the samples after 5 washes for detection;

[0086] (4) Elution: Slowly and gently add 1 ml of denaturing eluent (50 mmol / L Tris, 500 mmol / L NaCl, 8 mol / L urea, 250 mmol / L imidazole, pH 7.5) along the inner periphery of the column. Pay attention to adjust the pipeline flow rate to the slowest. Repeat the operation 5 times and collect the samples after elution 5 times for detection.

[0087] The purified eluate was concentrated to 500-700ul using a 50Kda 15ml ultrafiltration tube at 4000rpm for 30min, and then PBS replacement buffer was added. The protein obtained after concentration and replacement was detected using a BCA protein concentration determination kit. The results are as follows: Figure 12-15 .

[0088] Example 4 Mouse immunization

[0089] The first immunization operation is as follows: take 50ug of purified ultrafiltered IBV S1 protein, dilute it with PBS, and mix it with Freund's complete adjuvant at a volume of 1:1 to 500ul. After emulsification on ice for 30min, inject it into the hands and feet of mice. At the same time, set up a control group of mice and inject 500ul of PBS each. Immunize again every two weeks, the second and third immunizations are subcutaneous injections and intramuscular injections respectively, and the adjuvant is replaced with Freund's incomplete adjuvant. About one week after the third immunization, the mice are bled from the cheeks, and the blood is centrifuged at 5000xg for 10min after standing at 37°. The supernatant is marked and stored at -20°.

[0090] Titer detection after mouse immunization

[0091] The ELISA test steps are as follows:

[0092] (1) Coating: Prepare coating solution at 0.5 ug / ul of IBV S1 protein, coat each well of the ELISA reaction plate with 100 ul of the solution and incubate at 4°C overnight. Wash the plate three times the next day, each time for 6 min.

[0093] (2) Blocking: 2% BSA in PBST, 200 μl per well, overnight at 4°C, wash the next day, 6 min each time;

[0094] (3) Primary antibody: Mouse serum was diluted at 1:1000, 1:2000, 1:4000, 1:8000, 1:16000, 1:32000, 1:64000, and 1:128000. Negative serum control and blank control were also performed. Incubate at 37° for 1 h and wash the plates.

[0095] (4) Secondary antibody: Goat Anti-Mouse IgG / HRP diluted 1:5000 with PBST and added to the sample, incubated at 37° for 1 h, and then washed;

[0096] (5) Color development: 50ul of color development solution per well, 37°C, wash the plate after 5 min;

[0097] (6) Termination and reading: 50ul of the stop solution is placed in each well. After the reaction is terminated, place the plate in an ELISA reader and read the result at a wavelength of 450nm and keep the record (e.g. Fig.16 shown).

[0098] Antibody specificity detection after mouse immunization

[0099] To detect the specificity of antibodies after mouse immunization, the IBV-S1 antibody obtained after mouse immunization was diluted 1:4000 in TBST as the primary antibody for Western-blot detection of IBV S1 protein, and a negative control was set up. The results are shown in Fig.17 shown.

[0100] Through the above experiments, we can draw the following conclusions:

[0101] 1. Compared with the applicant's prior application, the protein expression amount increased by about 20%;

[0102] 2. This cell line has few S1 protein bands and high purity;

[0103] 3. The antibodies prepared using the S1 protein produced by this cell line have high titer and strong specificity;

[0104] 4. This cell line can be domesticated, and the cell viability is good after domestication.

Claims

1. A CHO-K1 cell line stably expressing IBV JS-96S1 protein, characterized in that: Prepared by the following steps: Step 1: Connect the S1 gene and the vector to obtain a plasmid; Step 2: Transfect the plasmid into CHO-K1 cells. After successful transfection, a CHO-K1 cell line can be obtained; The nucleotide sequence of the S1 gene is shown in SEQ ID NO.

1.

2. A method for acclimating a suspension CHO-K1 cell line, characterized in that: The CHO-K1 cell line prepared in step 1 is subjected to multiple stages of suspension culture. From the beginning to the end, the amount of FBS in the suspension culture medium used in each stage is gradually reduced until the FBS in the suspension culture medium is reduced to 0 and is successfully passaged for 3 consecutive times, which means that the cell line is successfully domesticated and a suspension CHO-K1 cell line is obtained.

3. The acclimation method according to claim 2, characterized in that: The cycle is divided into at least 6 stages. The suspension culture medium used in the first stage contains 8-12 wt% FBS; the suspension culture medium used in the second stage contains 4-6 wt% FBS; the suspension culture medium used in the third stage contains 2-3 wt% FBS; the suspension culture medium used in the fourth stage contains 0.8-1.2 wt% FBS; The suspension medium used in the fifth stage contains 0.05-0.2 wt% FBS; The suspension medium used in stage 5 does not contain FBS.

4. The acclimation method according to claim 3, characterized in that: The suspension culture medium is CD CHO 031 culture medium.

5. A suspension CHO-K1 cell line, characterized in that The method is domesticated using any one of claims 2 to 4.

6. Use of the suspension CHO-K1 cell line according to claim 5 to harvest IBV S1 protein.

7. An IBV S1 protein, characterized in that The IBV S1 protein is obtained by culturing the suspension CHO-K1 cell line as claimed in claim 5.

8. Use of the IBV S1 protein according to claim 7 for preparing polyclonal antibodies.

Images