A method for expressing human serum albumin using a tobacco cell suspension culture

Abstract

The present application belongs to the field of biotechnology, and specifically relates to a method for expressing human serum albumin by using Nicotiana sylvestris NS suspension cell line, which comprises the following steps: establishing a stably cultured Nicotiana sylvestris suspension cell system, and producing by amplification, and the obtained cell biomass can reach up to 500 g / L. Then, taking human serum albumin as the research object, transformation screening is carried out in the Nicotiana sylvestris NS suspension cell system to realize high expression of HSA. The present application removes the complex tissue regeneration step, and through plant codon optimization and subcellular targeting strategy design, aims to overcome the problem of low expression of exogenous genes in plants, so as to realize efficient enrichment of target proteins in specific organelles (such as vacuole / endoplasmic reticulum, etc.). The technical scheme of the present application can be popularized and applied to the expression and screening of other types of functional proteins, and provides important technical reference and practical support for the application of plant suspension cell lines in the field of recombinant protein expression.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a method for expressing human serum albumin using a tobacco suspension cell line. Background Technology

[0002] Tobacco, as an important chassis cell in synthetic biology, is an ideal platform for the reconstruction of secondary metabolic pathways, the synthesis of heterologous proteins, and the production of natural products. Compared to other plants, tobacco, as a non-food crop, accumulates a large amount of biomass within a relatively short life cycle and possesses a weak RNA silencing pathway, which reduces the degradation of exogenous RNA. Furthermore, because it contains precursors for the synthesis of certain secondary metabolites (such as geraniol pyrophosphate, a diterpenoid precursor related to paclitaxel synthesis), it is more suitable for constructing pathways for secondary metabolite synthesis. Current research has found that the Agrobacterium-mediated transient expression system of Nicotiana benthamiana has become a relatively mature and widely used method. However, this technology is limited by land area and temperature, making it difficult to scale up cultivation, and the transient transformation products are unstable. Therefore, it is mostly limited to laboratory use and has not been extended to industrial production.

[0003] In 2023, Zhang Fangqing et al. constructed the *Nicotiana benthamiana* suspension cell line (NBS-1), which, according to the paper, achieved a maximum biomass of 476.39 g / L. This invention, through systematic optimization of the nitrogen source ratio and phosphate concentration in the culture medium, for the first time determined the nutritional conditions suitable for the rapid growth of *Nicotiana benthamiana* suspension cells, ultimately achieving a biomass of 502.22 g / L. Therefore, this invention uses *Nicotiana benthamiana* as the research object, utilizing plant tissue culture technology, with the aboveground parts of 7-day-old and 1-month-old *Nicotiana benthamiana* plants as explants, inducing dedifferentiation to produce callus tissue through different hormone inductions, obtaining loose callus tissue through hormone gradient screening, and ultimately transforming it into suspension cells.

[0004] Chinese patent CN200510019084.4 discloses a technical solution for producing recombinant human serum albumin using rice endosperm cells as a bioreactor. However, rice faces problems such as large planting area, susceptibility to environmental influences, and long production cycles. This invention constructs a Nicotiana benthamiana NS suspension cell line and establishes a stable cell line transfer system. Simultaneously, it achieves efficient accumulation of human serum albumin in different subcellular organelles in existing cultivated tobacco BY2 and a de novo Nicotiana benthamiana NS suspension cell line. This allows for large-scale culture of human serum albumin in a short time (7-10 days), avoiding environmental differences and providing an excellent "platform" for the production of other functional proteins. Summary of the Invention

[0005] The purpose of this invention is to provide a method for expressing human serum albumin using the Nicotiana benthamiana NS suspension cell line, so as to enable the heterologous synthesis and production of mammalian-specific functional proteins through stable transformation and screening.

[0006] Therefore, the present invention provides the following technical solution.

[0007] One aspect of the present invention provides a method for expressing human serum albumin using the Nicotiana benthamiana NS suspension cell line, comprising the following steps: Screening and establishing a high-growth NS suspension cell system of Nicotiana benthamiana; Construct a recombinant plasmid containing the gene encoding human serum albumin; Using Agrobacterium-mediated genetic transformation, recombinant plasmids were transformed into the NS suspension cell system of Nicotiana benthamiana to achieve the expression of human serum albumin.

[0008] In a preferred embodiment of the present invention, the establishment of the *Tobacco Benzovia* NS suspension cell system includes: The aboveground parts of sterile seedlings of Nicotiana benthamiana were used as explants for induction culture to obtain callus tissue; The obtained callus tissue was subcultured and screened to obtain loose callus tissue; The obtained loose callus tissue was subjected to single-point suspension culture and screening to obtain a NS suspension cell system of Tobacco Benzoviae var. ...

[0009] In a preferred embodiment of the present invention, the sterile seedlings of Nicotiana benthamiana are obtained by culturing Nicotiana benthamiana seeds on a rooting medium for 7 to 30 days.

[0010] In a preferred embodiment of the present invention, the rooting culture medium comprises the following components: MS powder 4.4 g / L, sucrose 30 g / L, and Phytagel 0.3~0.7%.

[0011] In a preferred embodiment of the present invention, the culture conditions are: a temperature of 25 ℃ and a photoperiod of 16 h light / 8 h darkness.

[0012] In a preferred embodiment of the present invention, the culture medium for induction culture comprises the following components: MS medium, IAA 0~1.5 mg / L, 6-BA 0.5~1.5 mg / L, and 2,4-D 0~1.5 mg / L.

[0013] In a preferred embodiment of the present invention, the conditions for induction culture are: temperature of 25 °C and photoperiod of 24 h in darkness.

[0014] In a preferred embodiment of the present invention, the culture medium for subculture comprises the following components: MS medium, NAA 0.5~1.5 mg / L, and 6-BA 0.5~1.0 mg / L.

[0015] In a preferred embodiment of the present invention, the culture medium for subculture comprises the following components: MS medium, NAA 1.0 mg / L, and 6-BA 1.0 mg / L.

[0016] In a preferred embodiment of the present invention, the conditions for the subculture are: a temperature of 25 °C and a photoperiod of 24 h in darkness.

[0017] In a preferred embodiment of the present invention, the number of domestication and screening cycles in the subculture is 4 to 6.

[0018] In a preferred embodiment of the present invention, the culture medium for the single-point suspension culture is MS liquid culture medium.

[0019] In a preferred embodiment of the present invention, the conditions for single-point suspension culture are: temperature of 25 ℃, shaking speed of 130 r / min, photoperiod of 24 h in darkness, and culture time of 7~10 d.

[0020] In a preferred embodiment of the present invention, the recombinant plasmid uses a plasmid carrying GFP fluorescent protein as an expression vector, and the expression vector includes the pCHL1 plasmid.

[0021] In a preferred embodiment of the present invention, the Agrobacterium is Agrobacterium GV3101.

[0022] In a preferred embodiment of the present invention, the conversion includes: The recombinant plasmid was transformed into Agrobacterium, and the Agrobacterium was successfully transformed into the recombinant plasmid after verification by bacterial PCR. Agrobacterium that has been successfully transformed into recombinant plasmids was cultured in triple-antibiotic medium for 12-16 h to obtain Agrobacterium bacterial suspension. The obtained Agrobacterium tumefaciens bacterial culture was added to the Tobacco Benzovia NS suspension cell line and co-cultured at 25 °C for 2 days to obtain the transformed suspension cell line.

[0023] In a preferred embodiment of the present invention, the triple-antibiotic culture medium is LB medium containing 25 mg / L rifampicin, 50 mg / L spectinomycin, and 50 mg / L gentamicin.

[0024] In a preferred embodiment of the present invention, the transformation process further includes steps of Agrobacterium removal and positive cell screening: The transformed suspension cell lines were washed with water in liquid MS medium containing 100-200 mg / L carbenicillin to remove residual Agrobacterium. The cells that have been washed are placed on MS resistance selection medium for screening to obtain positive callus tissue successfully transformed by Agrobacterium.

[0025] In a preferred embodiment of the present invention, the MS resistance screening medium is an MS solid medium containing 200 mg / L carbenicillin and 30 mg / L kanamycin.

[0026] In a preferred embodiment of the present invention, the number of screening times is 3 to 4 generations.

[0027] In a preferred embodiment of the present invention, the positive cell screening is followed by a step of stepwise identification and yield assessment of human serum albumin: Positive callus tissue was subjected to suspension culture and initial screening for human serum albumin expression was performed using fluorescence microscopy. Expression was validated using Western blot to further determine the expression of human serum albumin; The study also included protein purification of the expression lines, comparative analysis of HSA production in tobacco NS and BY2 cell lines, and evaluation of the regulatory role of subcellular localization on protein accumulation levels.

[0028] By employing the above technical solution, the present invention has at least the following advantages: The *Nicotiana benthamiana* NS suspension cell line of this invention can be cultured at high density in shake flasks without light dependence, making it suitable for large-scale, continuous production of specific exogenous proteins. This *Nicotiana benthamiana* NS suspension cell line can directly achieve heterologous expression of target genes via Agrobacterium-mediated genetic transformation, eliminating the need for complex tissue regeneration steps and significantly shortening the research and development cycle. Compared with conventional BY2 suspension cells, the *Nicotiana benthamiana* NS suspension cell line established in this invention exhibits significant advantages: its endoplasmic reticulum-targeted HSA-positive cell line achieves a protein yield 7.8 times that of BY2 cells, strongly demonstrating the application potential of this system in the efficient production of exogenous proteins. Furthermore, this invention fills the gap in cryopreservation technology for *Nicotiana benthamiana* NS suspension cells, establishing for the first time an efficient cryopreservation and thawing process, laying the foundation for the long-term preservation and subsequent large-scale application of high-quality cell lines.

[0029] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below. Attached Figure Description

[0030] Figure 1A flowchart of the method for expressing human serum albumin using the Nicotiana benthamiana NS suspension cell line of the present invention is shown.

[0031] Figure 2 The present invention illustrates the screening and acquisition of loose callus tissue from *Tobacco Benzoinus*; wherein, a is the induction culture of callus tissue from *Tobacco Benzoinus*; b is browned callus tissue from *Tobacco Benzoinus* and callus tissue with high hardness; c is the subculture culture and screening of loose callus tissue from *Tobacco Benzoinus*.

[0032] Figure 3 The invention demonstrates the establishment of the NS suspension cell line of Tobacco Benzovia in accordance with the present invention.

[0033] Figure 4 This invention illustrates the acquisition of a high-density suspension cell line from *Nicotiana benthamiana*. Specifically, a represents the selection of superior *Nicotiana benthamiana* callus and the constructed suspension cell line; b represents the effect of callus inoculation amount on the fresh weight of the suspension cell line, reflecting the growth curve of the *Nicotiana benthamiana* suspension cell line; and c represents the biomass measurement of *Nicotiana benthamiana* cells in a 2L shake flask, expressing the final biomass of *Nicotiana benthamiana* cells after 1L of expanded culture.

[0034] Figure 5 The cryopreservation and thawing of *Tobacco Benzovia* NS suspension cells of the present invention are illustrated.

[0035] Figure 6 The vector map of the target protein gene of the present invention in the genetic transformation of NS suspension cells of Nicotiana benthamiana is shown.

[0036] Figure 7 A fluorescence micrograph of human serum albumin expression in the *Nicotiana benthamiana* NS suspension cell line of the present invention is shown.

[0037] Figure 8 This demonstrates the detection of the target protein, human serum albumin, using immunofluorescence blotting.

[0038] Figure 9 The SDS-PAGE image of the target protein, human serum albumin, is shown. Detailed Implementation

[0039] To make the technical means, creative features, achieved objectives, and effects of this invention readily understandable, the technical solutions in the embodiments of this invention will be clearly and completely described below in conjunction with the embodiments of this invention. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0040] like Figure 1As shown, this invention first uses the aboveground parts (preferably leaves + part of the stem) of seven-day-old and one-month-old *Nicotiana benthamiana* seedlings as explants. Through induction culture, subculture, suspension culture, and screening, a *Nicotiana benthamiana* NS suspension cell line was established. Subsequently, using the *Nicotiana benthamiana* NS suspension cell line as the chassis cell, stable transformation and expression of the target protein gene were performed. The specific method includes: first, using a plasmid carrying GFP fluorescent protein, such as the pCHL1 plasmid, as an expression vector, it was recombined with the gene encoding the target protein using molecular cloning technology to obtain a recombinant plasmid containing the target protein gene. Then, using Agrobacterium-mediated transformation, the recombinant plasmid containing the target protein gene was transformed into the *Nicotiana benthamiana* NS suspension cell line. After culture and screening, the expression of the target protein in the plant cell line was achieved. For detection, GFP fluorescent protein was used as a marker protein; the detection of GFP fluorescent protein expression further indicates the expression of the target protein. The method of this invention can be used for the expression of other exogenous genes in plant suspension cell lines.

[0041] Specifically, the above method includes the following steps: Screening and establishing a high-growth NS suspension cell system of Nicotiana benthamiana; Construct a recombinant plasmid containing the gene encoding human serum albumin; Using Agrobacterium-mediated genetic transformation, recombinant plasmids were transformed into the NS suspension cell system of Nicotiana benthamiana to achieve the expression of human serum albumin.

[0042] Specifically, establishing the *Nicotiana benthamiana* NS suspension cell system includes: The aboveground parts of sterile seedlings of Nicotiana benthamiana were used as explants for induction culture to obtain callus tissue; The obtained callus tissue was subcultured and screened to obtain loose callus tissue; The obtained loose callus tissue was subjected to single-point suspension culture and screening to obtain a NS suspension cell system of Tobacco Benzoviae var. ...

[0043] On one hand, sterile seedlings of *Nicotiana benthamiana* are obtained by culturing *Nicotiana benthamiana* seeds on a rooting medium for 7–30 days. Any suitable rooting medium in the art can be used for the rooting of sterile *Nicotiana benthamiana* seedlings. For example, a suitable rooting medium may consist of the following components: 4.4 g / L MS powder, 30 g / L sucrose, and 0.3–0.7% Phytagel; suitable culture conditions are: a temperature of 25 °C and a photoperiod of 16 h light / 8 h dark.

[0044] The aforementioned induction culture medium for inducing callus tissue comprises the following components: MS medium, IAA 0–1.5 mg / L, 6-BA 0.5–1.5 mg / L, and 2,4-D 0–1.5 mg / L. Suitable IAA concentrations in the aforementioned induction medium can be, for example, 0 mg / L, 0.5 mg / L, 1.0 mg / L, or 1.5 mg / L; suitable 6-BA concentrations can be, for example, 0.5 mg / L, 1.0 mg / L, or 1.5 mg / L; and suitable 2,4-D concentrations can be, for example, 0 mg / L, 0.5 mg / L, 1.0 mg / L, or 1.5 mg / L.

[0045] The conditions for callus induction culture were: temperature 25℃, photoperiod 24 h, and darkness.

[0046] After obtaining callus tissue, subculture screening is required to obtain loose callus tissue. A suitable subculture medium includes the following components: MS medium, NAA 0.5–1.5 mg / L, and 6-BA 0.5–1.0 mg / L. Suitable NAA concentrations in this subculture medium can be, for example, 0.5 mg / L, 1.0 mg / L, or 1.5 mg / L; suitable 6-BA concentrations can be, for example, 0.5 mg / L or 1.0 mg / L. After screening different ratios to determine the optimal hormone concentrations, the final optimal medium composition is determined to be: MS medium supplemented with 1.0 mg / L NAA and 1.0 mg / L 6-BA.

[0047] The conditions for subculture screening were: temperature 25 ℃, photoperiod 24 h dark. The number of subculture acclimatization screenings was 4 to 6 times.

[0048] After obtaining loose callus tissue, further single-point suspension culture and screening are needed to obtain a *Nicotiana benthamiana* NS suspension cell system with uniform cell morphology and relatively rapid growth. MS liquid medium was used for single-point suspension culture. The conditions for single-point suspension culture were: temperature 25 ℃, shaker speed 130 r / min, photoperiod 24 h dark, and culture time 7–10 days.

[0049] On the other hand, it is also necessary to construct a recombinant plasmid containing the human serum albumin gene encoding the target protein. This recombinant plasmid uses a plasmid carrying GFP fluorescent protein as an expression vector, and the gene encoding the human serum albumin protein is inserted into the expression vector. The preferred expression vector is the pCHL1 plasmid.

[0050] Finally, the recombinant plasmid was transformed into a Nicotiana benthamiana NS suspension cell system using Agrobacterium-mediated genetic transformation to achieve the expression of the target protein, human serum albumin. The Agrobacterium used was Agrobacterium GV3101.

[0051] Specifically, the transformation includes: First, the recombinant plasmid was transformed into Agrobacterium, and the successfully transformed Agrobacterium was confirmed by colony PCR. Then, the successfully transformed Agrobacterium was cultured in triple-antibiotic medium for 12–16 h to obtain Agrobacterium colony. Finally, the obtained Agrobacterium colony was added to Nicotiana benthamiana NS suspension cell line and co-cultured at 25 °C for 2 days to obtain the transformed suspension cell line.

[0052] The three-antibiotic culture medium used above was LB medium containing 25 mg / L rifampin, 50 mg / L spectinomycin, and 50 mg / L gentamicin.

[0053] Following transformation, the process also includes steps such as removing Agrobacterium from the transformed suspension cell line and screening for positive cells, specifically including: First, the transformed suspension cell lines were washed with water in liquid MS medium containing 100-200 mg / L carbenicillin to remove residual Agrobacterium. Then, the washed cells were placed on MS resistance selection medium for 3-4 passages to obtain positive callus tissue successfully transformed with Agrobacterium.

[0054] The MS resistance screening medium used for resistance screening was MS solid medium containing 200 mg / L carbenicillin and 30 mg / L kanamycin.

[0055] Further steps, following positive cell screening, include the step-by-step identification and yield assessment of human serum albumin: Positive callus tissue was subjected to suspension culture and initial screening for human serum albumin expression was performed using fluorescence microscopy. Expression was validated using Western blot to further determine the expression of human serum albumin; The study also included protein purification of the expression lines, comparative analysis of HSA production in tobacco NS and BY2 cell lines, and evaluation of the regulatory role of subcellular localization on protein accumulation levels.

[0056] The above methods can be used to obtain a high-biomass, uniformly morphologically grown, and relatively fast-growing NS suspension cell system of *Nicotiana benthamiana*. In addition, the *Nicotiana benthamiana* NS suspension cell system established using this invention can achieve high expression of human serum albumin.

[0057] The following is a detailed description of specific embodiments.

[0058] Example 1: Establishment of the NS suspension cell system of *Nicotiana benthamiana* 1. Obtaining sterile vaccines Prepare the rooting medium according to the formula in Table 1: After preparing the rooting medium according to the formula in Table 1, sterilize it at 121℃ under high temperature and high pressure for 30 min. After it has cooled slightly to room temperature, pour it evenly into the tissue culture bottle, adjust the air volume of the ultra-clean workbench, and use it after the surface of the medium is dry and solidified. The poured medium can be stored in a refrigerator at 4℃ for about 7 days.

[0059] Table 1. Rooting medium for Tobacco Benedict.

[0060] Tobacco Benedict seeds ( Nicotiana benthamiana) The seeds were placed in 1.5 mL EP tubes containing an appropriate amount of ultrapure water and vernalized at 4 ℃ for 1 day. On the second day, the ultrapure water used for soaking the seeds was removed in a clean bench, and an appropriate amount of 5% NaClO solution was added for disinfection for 10 min. During disinfection, the EP tubes were inverted approximately every 2 minutes to ensure thorough disinfection of the seeds. After disinfection, the 5% NaClO solution was discarded, and the seeds were rinsed 5-6 times with sterile water to remove any residual 5% NaClO from the seed surface (NaClO is hazardous waste and was recovered using 1.5 mL EP tubes and placed in a hazardous waste container). The cleaned seeds (approximately 6 seeds) were evenly placed on rooting medium, sealed, and cultured in an incubator at 25 ℃ under 16 h light and 8 h darkness conditions. Germination occurred in 2-3 days, yielding *Nicotiana benthamiana* seedlings.

[0061] 2. Induction of callus from *Tobacco Bunsenii* and screening of loose callus subculture Inverted induction medium: First, allow the MS solid medium (Coolaber, PM10111) that has been autoclaved for 30 min to cool slightly at room temperature. Then, use a 25 mL pipette to transfer 30 mL of MS culture medium into 100 mL Erlenmeyer flasks. After the medium in the 100 mL Erlenmeyer flasks has cooled to room temperature, add appropriate concentrations of 0.5–1.5 mg / L IAA (indoleacetic acid) and 0.5–1.5 mg / L 6-BA (6-benzylaminopurine), as well as 0.5–1.5 mg / L 2,4-D (2,4-dichlorophenoxyacetic acid) and 0.5–1.5 mg / L 6-BA (6-benzylaminopurine) according to the formula shown in Table 2. Use the two growth hormones (IAA, 2,4-D) and cytokinin (6-BA) in combination, then shake to mix well, pour into plates, and mark the concentrations on the plates. Adjust the airflow of the clean bench to the maximum, and it is ready for use after the surface of the culture medium is dry and solidified.

[0062] Table 2. Induction medium for *Tobacco Benzovia*

[0063] The above-ground parts of sterile *Nicotiana benthamiana* seedlings cultured for 7 or 30 days were used for callus induction: The above-ground parts of the seedlings were removed using a sterile scalpel and used as explants for callus induction (explants: leaves + part of the stem; seedlings grown on different plates were selected). The cut explants (1cm*1cm) were dispersed on the induction medium shown in Table 2 (explants from different parts of the seedlings grown on different plates were placed in the same concentration of induction medium), sealed, and incubated at 25℃ in the dark for 24 hours to form callus tissue. No obvious callus tissue was observed after 10 days when IAA and 6-BA were used together at different hormone concentrations. However, around 10 days, white callus tissue could be initially observed growing on the induction medium with the hormone combination of 2,4-D and 6-BA. Callus tissue could be induced at all concentrations of this combination (2,4-D + 6-BA).

[0064] Callus induced by different concentrations of combinations (2,4-D + 6-BA) was then dispersed on the subculture media shown in Table 3 for subculture screening of different hormones (25 ℃, 24 h dark conditions). The optimal hormone concentration was determined through screening at different ratios. Finally, it was determined that adding 1.0 mg / L NAA and 1.0 mg / L 6-BA to MS medium, after 4-6 subcultures, yielded loose callus tissue from *Nicotiana benthamiana*. Callus tissues with other hormone concentration combinations could only maintain cell growth or cell browning. The callus tissue particles were large and hard, and not suitable for shaking in a shake flask, therefore, they were not suitable for subsequent suspension culture.

[0065] Table 3. Subculture medium for *Tobacco Benzovia*

[0066] Callus tissues obtained from the above-mentioned different induction and subculture media are shown below. Figure 2 .

[0067] like Figure 2 As shown in Figure a, callus formation was induced in the aerial parts of *Nicotiana benthamiana* (from left to right) in MS medium containing different hormone ratios (2,4-D + 6-BA). The figure shows that the wound sites on the aerial parts swelled after induction and formed callus through dedifferentiation. The induced callus tissues were then placed on subculture media (Table 3) for further selection. Finally, under MS medium + 1.0 mg / L NAA + 1.0 mg / L 6-BA conditions, after 5-6 generations of selection, the following results were obtained: Figure 2 c shows loose callus tissue from *Nicotiana benthamiana*, while callus tissue cultured under other combinations of 2,4-D and 6-BA concentrations is partially browned, hard, and grows slowly. Figure 2 b).

[0068] 3. Obtaining the NS suspension cell system of *Nicotiana benthamiana* Loose callus tissue from *Nicotiana benthamiana* that grew rapidly per unit area within the specified time period was collected and inoculated into MS liquid medium. The culture was then incubated at 25°C, with a shaking speed of 130 r / min, in complete darkness for 10 days to establish a preliminary suspension culture. Large cell clusters were then filtered through a 50 μm filter to remove them, and the filtrate was collected. This filtrate was inoculated into MS liquid medium, and multiple generations of suspension culture screening were performed using the same method (25°C, shaking speed of 130 r / min, in complete darkness) to obtain a stable *Nicotiana benthamiana* NS suspension cell line (NS suspension cells, NS).

[0069] The process of establishing suspension cell lines can be found in [link to documentation]. Figure 3 The resulting suspension cell lines are shown in Figure 4 .

[0070] like Figure 3 As shown, after suspension culture of callus tissue from different single points (corresponding to NS xx in suspension cells), there are differences in growth density and cell state. The NS1-1 / NS4-1 / NS4-2 suspension cell line has a higher growth rate and cell density compared to other suspension cell lines. It was used as the original cell line for subsequent suspension acclimatization culture.

[0071] like Figure 4 As shown in Figure a, after domestication, the NS4-2 cell line became a high-density suspension cell line, specifically characterized by dispersed particles and rapid cell proliferation per unit time. According to... Figure 4 In Figure b, the cell growth curves show that different initial callus inoculum amounts have different effects on the growth of suspension cells. When the initial callus inoculum amounts are approximately 0.6 g and 0.9 g, the callus reaches a rapid growth phase in 6–8 days. When the initial callus inoculum amounts are approximately 1.2 g and 1.6 g, the callus reaches a rapid growth phase in 4–6 days, after which cell growth enters a plateau phase. Figure 4 The cell mass shown in b represents a final wet cell weight of 457–513 g / L. Since the volume of the shake-flask culture affects biomass, expanding the cell culture from 100 mL to 2 L shake flasks resulted in a maximum biomass of 502.22 g / L. Figure 4 c), which is also related to the biological state of the cells and the initial inoculum size. Thus, a high-density, high-biomass, and highly dispersed *Nicotiana benthamiana* NS suspension cell line was screened.

[0072] 4. Establishment of methods for cryopreservation and resuscitation of Nicotiana benthamiana NS suspension cell lines 4.1 Cryopreservation of Nicotiana benthamiana NS suspension cell line Take 10 mL of the above-mentioned suspension cells grown for 5-7 days (rapid growth phase), centrifuge to remove the supernatant, and collect the cell pellet. Add LSP cryopreservation medium (composition shown in Table 4 below) to the obtained cell pellet to obtain suspension cells. Add the suspension cells to cell cryopreservation tubes, then place them in a programmed cryopreservation box and slowly freeze at -40℃ for 5 h. Remove the frozen cells and immediately place them in liquid nitrogen. They can be stored for about one year.

[0073] Table 4 LSP Frozen Culture Medium

[0074] 4.2 Resuscitation of Nicotiana benthamiana NS suspension cell line Remove the frozen *N. benthamiana* NS suspension cells from the liquid nitrogen tank and agitate them continuously for 2 minutes in a 35°C water bath. In a clean bench, thoroughly mix the *N. benthamiana* cells by aspiration and then evenly spread them onto a subculture medium (composition shown in Table 3) containing two layers of filter paper. Seal the container and incubate at 25°C in the dark for 1 day. On the second day, place a new filter paper in a fresh subculture medium and transfer the cells that have recovered from the 1-day thawing incubator onto the new filter paper. Seal the container and incubate at 25°C in the dark until cell growth occurs, indicating successful cell thawing. See below for details on the cryopreserved and thawed cell culture. Figure 5 .

[0075] like Figure 5 As shown, after being cryopreserved in liquid nitrogen, the NS suspension cells of Nicotiana benthamiana were revived. After about 7 days, granular callus tissue growth could be observed on filter paper. Microscopic examination showed that the cells were in normal condition, proving that the cell revival was successful.

[0076] Example 2: Construction of a recombinant plasmid containing the target protein (human serum albumin) Using the pCHL1 plasmid carrying GFP fluorescent protein as an expression vector, it was recombined with the gene encoding human serum albumin (HSA) using molecular cloning technology to obtain a recombinant plasmid containing the mature HSA gene. A map of the obtained recombinant plasmid is shown below. Figure 6 .

[0077] The amino acid sequence of the above-mentioned human serum albumin is shown in SEQ ID NO. 1, and the nucleotide sequence is shown in SEQ ID NO. 2: SEQ ID NO. 1: 。

[0078] SEQ ID NO. 2:

[0079] Example 3: Genetic Transformation Using Agrobacterium GV3101 (catalog number B528430-0010, purchased from Sangon Biotech Co., Ltd.) mediated genetic transformation technology, the recombinant plasmid encoding the target protein gene human serum albumin prepared in Example 2 was used to infect and co-culture the Nicotiana benthamiana NS suspension cell line prepared in Example 1. The specific steps included: (1) The recombinant plasmid containing the mature HSA gene constructed in Example 2 was transformed into Agrobacterium GV3101, and Agrobacterium was successfully transformed into the recombinant plasmid after verification by bacterial PCR.

[0080] (2) Agrobacterium that has been successfully transformed into recombinant plasmids was cultured in LB medium containing 25 mg / L rifampicin, 50 mg / L spectinomycin and 50 mg / L gentamicin for 12-16 h to obtain Agrobacterium bacterial culture.

[0081] (3) Take 10 μL of Agrobacterium tumefaciens bacterial solution (OD) 600 =0.6~1.0) were added to 10 mL of Tobacco Benzovia NS suspension cell line and cultivated Tobacco BY2 suspension cell line respectively for infection and co-culture. They were co-cultured in MS medium at 22~28℃ for 2 days to obtain the transformed suspension cell line.

[0082] (4) The transformed suspension cell line was washed twice with water in liquid MS medium containing 200 mg / L carbenicillin to remove residual Agrobacterium.

[0083] (5) Add 200 mg / L carbenicillin and 30 mg / L kanamycin to MS solid medium as selection medium. Take 0.5-1 mL of the washed cells and place them on the selection medium for resistance selection (25℃, 24 h in the dark, initial selection takes about three weeks). After 3-4 generations of resistance selection (25℃, 24 h in the dark, 12-14 days), positive callus tissue successfully transformed by Agrobacterium is obtained.

[0084] To further verify the reliability of the above method, using human serum albumin (designated RL32 in this application) as the target protein, a recombinant plasmid containing the human serum albumin gene was constructed by simulating the mature form of human serum albumin in the human body using the method in Example 2. Subsequently, the plasmid was transformed into the *Tobacco Bengal* NS and cultivated tobacco BY2 suspension cell lines respectively according to the above genetic transformation method.

[0085] Callus tissue successfully transformed with Agrobacterium was examined under a fluorescence microscope to observe the fluorescence of GFP in order to characterize the expression of the target protein. Results are shown below. Figure 7 .

[0086] like Figure 7 As shown, under the GFP fluorescence channel, NS suspension cells of *Nicotiana benthamiana* transformed with the target protein gene exhibited GFP fluorescence, preliminarily indicating that the recombinant protein containing the target protein gene was expressed in *Nicotiana benthamiana* NS suspension cells.

[0087] Callus tissue from *Nicotiana benthamiana*, which successfully exhibited GFP fluorescence, was cultured in suspension on MS liquid medium (composition as shown in Table 3, but without agar) (25 °C, 24 h in the dark). The expression of the target protein was then detected by Western blotting. Results are shown below. Figure 8 .

[0088] like Figure 8 As shown, using untransformed Agrobacterium wild-type cells as a negative control (wildtype, WT), the expression of the target protein in RL32-1 (selected from the first callus of RL32 for suspension culture) and RL32-2 (selected from the second callus of RL32 for suspension culture) in the BY2 and NS suspension cell lines was successfully detected by immunofluorescence. After amplification of RL32-1 culture, SDS-PAGE detection by affinity chromatography clearly showed the expression of HSA (marked with red *), and the expression of HSA in the Nicotiana benthamiana NS suspension cell line was 7.8 times that in the cultivated tobacco BY2 suspension cell line. Figure 9 ).

[0089] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the methods and techniques disclosed above without departing from the scope of the present invention to create equivalent embodiments. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for expressing human serum albumin using the Nicotiana benthamiana NS suspension cell line, characterized in that, Includes the following steps: Screening and establishing a high-growth NS suspension cell system of Nicotiana benthamiana; Construct a recombinant plasmid containing the gene encoding human serum albumin; Using Agrobacterium-mediated genetic transformation, recombinant plasmids were transformed into the NS suspension cell system of Nicotiana benthamiana to achieve the expression of human serum albumin.

2. The method according to claim 1, characterized in that, The establishment of the *Tobacco Benzovia* NS suspension cell system includes: The aboveground parts of sterile seedlings of Nicotiana benthamiana were used as explants for induction culture to obtain callus tissue; The obtained callus tissue was subcultured and screened to obtain loose callus tissue; The obtained loose callus tissue was subjected to single-point suspension culture and screening to obtain a NS suspension cell system of Tobacco Benzoviae var. ...

3. The method according to claim 2, characterized in that, The sterile seedlings of Nicotiana benthamiana were obtained by culturing Nicotiana benthamiana seeds on a rooting medium for 7 to 30 days. The rooting medium comprises the following components: MS powder 4.4 g / L, sucrose 30 g / L, Phytagel 0.3~0.7%; The culture conditions were: a temperature of 25 ℃ and a photoperiod of 16 h light / 8 h darkness.

4. The method according to claim 2, characterized in that, The culture medium for induction culture comprises the following components: MS medium, IAA 0~1.5 mg / L, 6-BA 0.5~1.5 mg / L, and 2,4-D 0~1.5 mg / L; The conditions for induction culture were: temperature 25 ℃, photoperiod 24 h, and darkness.

5. The method according to claim 2, characterized in that, The culture medium for the subculture includes the following components: MS medium, NAA 0.5~1.5 mg / L, and 6-BA 0.5~1.0 mg / L; The conditions for the subculture were: temperature 25 ℃, photoperiod 24 h dark; The number of domestication and screening cycles for the subculture is 4 to 6.

6. The method according to claim 2, characterized in that, The culture medium for the single-point suspension culture is MS liquid medium; The conditions for single-point suspension culture were: temperature 25 ℃, shaker speed 130 r / min, photoperiod 24 h dark, and culture time 7~10 d.

7. The method according to claim 1, characterized in that, The recombinant plasmid uses a plasmid carrying GFP fluorescent protein as an expression vector, and the expression vector includes the pCHL1 plasmid.

8. The method according to claim 1, characterized in that, The Agrobacterium is Agrobacterium GV3101.

9. The method according to claim 1, characterized in that, The transformation includes: The recombinant plasmid was transformed into Agrobacterium, and the Agrobacterium was successfully transformed into the recombinant plasmid after verification by bacterial PCR. Agrobacterium that has been successfully transformed into recombinant plasmids was cultured in triple-antibiotic medium for 12-16 h to obtain Agrobacterium bacterial suspension. The obtained Agrobacterium tumefaciens bacterial solution was added to the Nicotiana benthamiana NS suspension cell line and co-cultured at 25 °C for 2 days to obtain the transformed suspension cell line; The triple-antibiotic culture medium is LB medium containing 25 mg / L rifampin, 50 mg / L spectinomycin, and 50 mg / L gentamicin.

10. The method according to claim 1, characterized in that, The transformation process also includes steps for Agrobacterium removal and positive cell screening: The transformed suspension cell lines were washed with water in liquid MS medium containing 100-200 mg / L carbenicillin to remove residual Agrobacterium. The cells that have been washed with water were placed on MS resistance selection medium for screening to obtain positive callus tissue that has been successfully transformed by Agrobacterium. The MS resistance screening medium was an MS solid medium containing 200 mg / L carbenicillin and 30 mg / L kanamycin; The number of screening cycles is 3 to 4.

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