A cho cell line of tgev strain s protein and application thereof

Abstract

This invention relates to the field of vaccine technology and provides a CHO cell line for the TGEV strain S protein and its application. The CHO cell line has a TGEV S protein encoding gene with codon optimization carrying the TPA signal peptide integrated into its chromosome. The nucleotide sequence of the TGEV S protein encoding gene is shown in SEQ ID NO.1. This invention not only significantly improves the exocrine expression efficiency and stability of the TGEV S protein but also increases protein quantity and yield, establishing an expression system that can operate stably for a long time and is suitable for large-scale production, demonstrating clear technological advancement and practical application value.

Description

Technical Field

[0001] This invention belongs to the field of vaccine technology, and in particular relates to a CHO cell line for the S protein of TGEV strain and its application. Background Technology

[0002] Transmissible Gastroenteritis Virus (TGEV) is a coronavirus that primarily infects the epithelial cells of the small intestine in pigs, causing severe diarrhea, dehydration, and weight loss. It is particularly harmful to piglets. The virus spreads rapidly and is endemic or periodically prevalent in some farming areas, posing a continuous threat to the safety of livestock production.

[0003] The S protein is a major structural protein on the surface of TGEVs, responsible for viral binding to host cell receptors and membrane fusion. It is a key antigenic determinant for inducing neutralizing antibody production. The native structure of the S protein is a highly glycosylated trimer conformation, and its spatial structural integrity directly determines the correct presentation of the antigenic epitope. Therefore, obtaining a structurally intact and conformationally stable S protein is of great significance in antigen preparation and related product development.

[0004] However, the existing technology has the following problems: First, regarding exocrine expression efficiency, existing expression systems mostly use natural signal peptides or universal heterologous signal peptides to guide protein secretion. However, TGEVS proteins have a large molecular weight, complex structure, many glycosylation modification sites, and contain transmembrane domains, making them highly dependent on the secretion pathway. Conventional signal peptides have limited efficiency in binding to signal recognition particles and transporting in the endoplasmic reticulum, resulting in a high proportion of the target protein remaining in the cell and a low content of effectively secreted protein in the supernatant, which increases the difficulty of purification and reduces the yield per unit volume. Secondly, in terms of protein structure design, some existing technologies have failed to properly truncate the transmembrane region or construct secretory expression structures, making it difficult for proteins to be effectively released into the culture supernatant. Simultaneously, the lack of optimized design for stable trimer structures may affect protein conformational stability, thereby impacting antigen quality. Secondly, regarding expression stability, some technical solutions use transient transfection expression or cell lines that have not undergone rigorous single-clone screening. Such expression systems can achieve a certain yield in the short term, but as the number of passages increases, the expression level is prone to decline or fluctuation, making it difficult to meet the requirements for continuous and stable production.

[0005] In summary, the existing technology has not yet established a CHO cell expression system that can be systematically optimized for the structural characteristics of TGEV S protein, can be stably and efficiently expressed through exocrine expression, and is suitable for large-scale production. Summary of the Invention

[0006] The purpose of this invention is to provide a CHO cell line for the S protein of TGEV strain, aiming to solve the problems mentioned in the background art.

[0007] The present invention is implemented as follows: a CHO cell line for the S protein of TGEV strain, wherein the chromosome of the CHO cell line integrates a TGEV S protein encoding gene carrying a TPA signal peptide after codon optimization, and the nucleotide sequence of the TGEV S protein encoding gene is shown in SEQ ID NO.1.

[0008] Another objective of this invention is to provide a method for constructing a CHO cell line containing the S protein of the TGEV strain, comprising the following steps: (1) A genetically codon-optimized TGEV S protein-coding gene, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) Construct a recombinant vector containing a chimeric signal peptide, a TGE V1 protein-coding gene, and a dual-selection marker; (3) The recombinant vector was transfected into CHO-S cells, and CHO cells expressing the TGEV strain S protein were obtained by antibiotic pressure screening.

[0009] Another objective of this invention is to provide an application of a CHO cell line containing the S protein of the TGEV strain in the preparation of a vaccine against the TGEV strain.

[0010] Compared with the prior art, the embodiments of the present invention have the following advantages: First, regarding exocrine expression efficiency, the embodiments of the present invention improve signal recognition and endoplasmic reticulum transport efficiency by optimizing the codon of the S protein coding sequence and combining it with an efficient signal peptide screening strategy. This allows the target protein to smoothly enter the secretory pathway and be effectively released into the culture supernatant. Compared with expression methods using conventional signal peptides or unoptimized structures, the expression level of the target protein in the supernatant is significantly increased, reducing intracellular retention and degradation, thereby increasing the effective antigen content per unit volume of culture medium. Secondly, regarding protein conformation and quality, the embodiments of the present invention remove the transmembrane region and construct a secretory expression structure, while rationally introducing a trimer stabilizing structure at the C-terminus of the protein. This helps maintain the native trimer conformation of the S protein, improves the correctness of protein folding and structural stability, and the CHO cell system has a complete glycosylation modification capability, making the expression product closer to the native state in terms of spatial conformation and post-translational modification, thereby improving antigen quality. Furthermore, regarding expression stability, the embodiments of this invention establish a stable integrated expression system. Chromosomal integrated CHO cell lines are obtained through resistance screening and monoclonal screening. After multiple generations of continuous passage verification, the cell line can still maintain a stable high level of secretory expression during long-term culture, avoiding the problems of large expression fluctuations or gradual decay in transient expression systems, and has good genetic stability and reproducibility. In summary, the embodiments of the present invention not only significantly improve the exocrine expression efficiency and expression stability of TGEVS protein, but also increase protein quantity and yield, and establish an expression system that can operate stably for a long time and is suitable for large-scale production, which has clear technological progress and practical application value. Attached Figure Description

[0011] Figure 1 This is a schematic diagram illustrating the construction of the pcDNA3.1-S eukaryotic expression vector provided in an embodiment of the present invention; Figure 2 A schematic diagram of the recombinant vector pcDNA3.1-S containing the TPA signal peptide provided in an embodiment of the present invention; Figure 3 The enzyme digestion identification results of the pcDNA3.1-S recombinant vector provided in the embodiments of the present invention are as follows: M: 1Kb plus, 1: HindIII digestion of pcDNA3.1-S, 2: double digestion of pcDNA3.1-S with HindIII and EcoRI. Figure 4 The protein expression identification results after pcDNA3.1-S transfection of cells provided in the embodiments of the present invention are shown below. M: 26619 marker, 1: supernatant of cells transfected with blank vector, 2: supernatant of cells transfected with pcDNA3.1-S. Figure 5 This refers to the exocrine expression efficiency of cells transfected with pcDNA3.1-S provided in this embodiment of the invention. Figure 6 The cell supernatant purification and identification results of pcDNA3.1-S provided in the embodiments of the present invention are as follows: M: marker26619, 1: CHO-TGEV / S cell supernatant, 2: flow-through, 3: 25mM, 4: 50mM, 5: 250mM, 6: 500-1mM, 7: 500-2mM, 8: 500-3mM, 9: 500-4mM; Figure 7 The stability test results of the CHO-TGEV / S cell line provided in the embodiments of the present invention. Detailed Implementation

[0012] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0013] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0014] Example 1: Construction of the eukaryotic recombinant vector for TGEV S protein and establishment of a stable cell line, specifically including the following steps: 1. Identification of the S protein and construction of a eukaryotic recombinant expression vector for the exocrine signal peptide: The study analyzed the S protein in existing vaccine strains and compared it with current circulating strains using multiple sequence comparison. It found that the sequence was highly conserved. The sequence was then subjected to identity analysis and phylogenetic analysis, and the amino acid sequence of the S protein of the WH1 strain (ADY39740) with the highest homology (over 97.7%) was selected. Tissue plasminogen activator (TPA, amino acid sequence: MDAMKRGLCCVLLLCGAVFVSA, as shown in SEQ ID NO. 2) was selected for the construction of a recombinant eukaryotic expression vector. A kozak sequence (GCCGCCACC) was added before the start codon, and four signal peptide sequences were added after the start codon. The transmembrane and intracellular regions of the S protein were removed. A trimer motif (GYIPEAPRDGQAYVRKDGEWVLL STFL, as shown in SEQ ID NO. 3) and a 6×His tag were added to the C-terminus of the S protein and linked via a GGSGG short peptide. Finally, a [missing information - likely a typo, should be inserted here] was added to the 5' end of the entire sequence. Hin dIII, 3' end added Eco The RI restriction site was cloned into the pcDNA3.1 empty vector and sent to General Biotechnology Co., Ltd. for optimization of codon bias, GC content, mRNA secondary structure, repetitive sequence regions, and gene synthesis using CHO cells as the host. After plasmid extraction using Tiangen Biotech's endotoxin-free small-volume extraction kit, double enzyme digestion and sequencing verification were performed (the nucleotide sequence of the TGAVS protein encoding gene carrying the TPA signal peptide is shown in SEQ ID NO.1). 2. Expression and identification of TGEV S protein: CHO-S cells in good growth condition with a cell viability greater than 97% were centrifuged, and the cell volume was 1 mL, with a cell count of 1 × 10⁶ cells. 7After mixing, centrifuge at 650 rpm for 5 min, discard the supernatant, and resuspend the contents in 800 μL of Eden B100S basal medium. Add 40 μg of plasmid, and set up a control group with no transfection wells. Add the contents to a pre-chilled electrode cup and mix slowly. Perform electroporation using a 4 mm Bourne electroporator at 300 V, 950 uF, and 4 mm. After electroporation, transfer the liquid from the electroporator to 15 mL of Eden B100S basal medium. Incubate the culture flasks in a cell culture incubator at 130 rpm (37℃, 8% CO2) for two days, then change the medium. Add 800 μL of plasmid to all groups. Cells were subjected to pressure selection with g / mL G418 for 3 weeks until the cells in the blank control group died completely. The cells that survived at this time were the successfully transfected cell pool. The cell pool was seeded into 96-well plates using the limiting dilution method for screening of single-clonal cell lines. At this time, the culture medium did not contain G418. After screening, the single cells were cultured for one week and then seeded into 24-well plates for further culture. At this time, the culture medium contained G418. After the cells were stable, the supernatant was collected and centrifuged at 8000 rpm for 5 min. Western blotting was used to verify whether the S protein was successfully expressed. Cell lines with specific target protein bands were further expanded and frozen. The cell line with the best expression level was selected for subsequent experiments and named CHO-TGEV / S. 3. Identification of stable cell lines: When CHO-TGEV / S cells were verified to express the target protein, they were recorded as the first generation. After the cells were cultured to the 25th generation, samples of cells from the 5th, 15th and 25th generations were taken and Western blot was performed to detect the expression of the target protein. 4. Purification of TGEV S protein: CHO-TGEV / S cells were cultured in shake flasks at a volume of 30 mL. On day 5, the cell supernatant was harvested and centrifuged at 6000 rpm for 5 min at 4°C. The supernatant was then filtered through a 0.22 μm filter. The protein supernatant was mixed 1:1 with equilibration buffer (20 mM Tris, 200 mM NaCl, 25 mM imidazole, pH 7.4) and added to equilibrated nickel ion packing material. After standing for 1 h, four different concentration gradients of imidazole (25 mM, 50 mM, 250 mM, and 500 mM) were used for elution. Small amounts of each solution were used to prepare samples. The purification status of the target protein was detected by SDS-PAGE electrophoresis, and its concentration was determined using a BCA protein concentration kit. 5. Results and Analysis: 5.1 Construction of the eukaryotic recombinant expression vector pcDNA3.1-S containing the TPA signal peptide: Bioinformatics prediction of the WH1 strain S protein (1477AA, 4434nt) revealed a signal peptide at amino acid positions 1-16, a transmembrane domain at amino acid positions 1389-1408AA, and an intracellular domain at amino acid positions 1409-1447AA. After removing the transmembrane and intracellular regions of the S protein, a recombinant expression vector was constructed according to the established strategy (e.g., ...). Figure 1 As shown), and the recombinant eukaryotic expression vector plasmid map was constructed using SnapGene software (as shown). Figure 2 (As shown), using restriction endonucleases Eco RI and Hin Enzyme digestion of the recombinant expression plasmid with dIII showed that after double digestion, two bands of the expected size were obtained (e.g., ...). Figure 3 As shown in the figure, the sequencing results were also completely correct, indicating that the recombinant expression vector pcDNA3.1-S was successfully constructed.

[0015] 5.2 S protein expression and identification: Cell supernatant was collected, and the target protein in the cells was identified by Western blotting. The result of a single band of 230 kDa indicates that the S protein was successfully expressed in the supernatant of CHO-S cells transfected with the recombinant expression vector of TPA signal peptide (e.g., Figure 4 As shown), subsequently, when all cells expressed the protein on day 5, samples were collected and processed uniformly, and the expression level of the target protein in the supernatant was preliminarily assessed using a cocoagulase staining assay (e.g., ...). Figure 5 (As shown), the cell supernatant was purified by nickel column affinity chromatography and the BCA concentration was determined, as follows: Figure 6 As shown, a single target band of 230 kDa was successfully purified, and the supernatant secretion expression level was measured to be 0.48 mg / mL.

[0016] 5.3 Screening of cell lines stably expressing S protein: CHO-TGEV / S cells were selected using a pressure screening method. G418 expression was used to remove non-resistant CHO cells; therefore, this concentration was chosen as the drug concentration for cell line selection. Cells were seeded into 96-well plates using a limiting dilution method for monoclonal selection. After one week, the cells were passaged for expansion culture until they reached full growth, indicating successful monoclonal cell selection. Western blot results are shown below. Figure 7 As shown, the secretory target protein was stably and efficiently expressed in the 5th, 15th and 25th generation CHO- / S cell lines, indicating that the CHO cell lines have good stability.

[0017] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A CHO cell line containing the S protein of a TGEV strain, characterized in that, The chromosome of the CHO cell line integrates a TGAVS protein-coding gene carrying the TPA signal peptide after codon optimization, and the nucleotide sequence of the TGAVS protein-coding gene is shown in SEQ ID NO.

1.

2. A method for constructing a CHO cell line of the TGEV strain S protein as described in claim 1, characterized in that, Includes the following steps: (1) A genetically codon-optimized TGEV S protein-coding gene, the nucleotide sequence of which is shown in SEQ ID NO.1; (2) Construct a recombinant vector containing a chimeric signal peptide, a TGE V1 protein-coding gene, and a dual-selection marker; (3) The recombinant vector was transfected into CHO-S cells, and CHO cells expressing the TGEV strain S protein were obtained by antibiotic pressure screening.

3. The method for constructing a CHO cell line for the TGEV strain S protein according to claim 2, characterized in that, In step (2), the signal peptide is the TPA signal peptide.

4. The use of a CHO cell line containing the S protein of the TGEV strain as described in claim 1 in the preparation of a vaccine against the TGEV strain.

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