A method for establishing a plant line expressing an influenza b virus ha protein in nicotiana benthamiana

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a method for establishing a plant line of an influenza B virus HA protein expressed by Nicotiana benthamiana. According to the method, a nucleotide sequence coding the BV-HA protein is first connected to a plant binary expression vector pHREAC to construct a recombinant plasmid, then the recombinant plasmid is transformed into an E. coli competent cell, an Agrobacterium competent cell in sequence, and the Agrobacterium competent cell is used to infect the Nicotiana benthamiana, so that the BV-HA protein is obtained through transient expression in the Nicotiana benthamiana cell. The BV-HA protein purified through the method has high biological activity, and can be used for further biological function research. The method can realize one-step purification of the protein, is simple and fast, and has high protein purity. The exogenous recombinant protein obtained through the method can be used for biological safety evaluation and biological function research.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a method for establishing a plant line expressing influenza B virus HA protein in tobacco Benzoenta. Background Technology

[0002] Influenza virus (IV) belongs to the family Orthomyxoviridae and can cause seasonal or pandemic influenza in humans and other animals. Based on the antigenicity of its nucleoprotein (NP) and matrix protein (M1), it can be divided into four types: A, B, C, and D.

[0003] Influenza B virus causes pandemics every so often, mainly in densely populated places such as schools, and may also break out in institutions where the elderly live. Influenza B has a rapid onset, and the main symptoms include fever, headache, muscle pain, malaise, chills, shivering, and loss of appetite. In severe cases, it can lead to pneumonia and endanger life.

[0004] Influenza B virus (IBV) is a single-stranded, negative-sense, segmented RNA virus. Its genome is divided into 8 segments, encoding 11 proteins. Hemagglutinin (HA) protein is one of the main components of the influenza virus envelope spikes. HA protein is a transmembrane glycoprotein with a molecular weight of approximately 75 kDa. HA protein is a typical type I glycoprotein (amino terminus outside the envelope, carboxyl terminus inside the envelope) containing four domains: signal peptide, cytoplasmic region, transmembrane region, and extracellular region. Under natural conditions, HA protein exists in a trimer form on the surface of the viral capsid. The HA gene is approximately 1800 bp in length, encoding approximately 560 amino acid residues, mainly comprising three parts: the signal peptide, the hemagglutinin head region (HA1), and the hemagglutinin stem region (HA2).

[0005] Since the 1970s, bacteria, fungi, insect cells, mammalian cells, and plants have been used to produce various recombinant proteins, depending on the type, demand, and production cost. In addition, they have been used to explore the production of biopharmaceuticals. However, due to cost limitations, complicated processes, and the pursuit of high quality, some expression systems are unable to meet the expression requirements of heterologous proteins.

[0006] Nicotiana benthamiana is the most widely used experimental host in plant virology, primarily due to its ability to successfully infect a large number of different plant viruses. Because of its efficient gene transformation and regeneration, and its suitability for virus-induced gene silencing or transient protein expression, Nicotiana benthamiana has rapidly gained popularity in plant biology, particularly in studies requiring protein localization, interactions, or plant-based protein expression and purification systems. Summary of the Invention

[0007] The first objective of this invention is to provide a recombinant carrier for producing immunogenic substances from tobacco benthamiana.

[0008] The second objective of this invention is to provide a method for preparing the BV-HA-pHREAC recombinant plasmid.

[0009] A third objective of this invention is to provide a method for expressing the HA protein of influenza B virus in Tobacco Benedict.

[0010] To achieve the above objectives, the technical solution created by this invention is implemented as follows: The present invention first provides the influenza B virus HA protein (i.e., BV-HA protein).

[0011] The present invention also provides a nucleic acid molecule that encodes the above-mentioned influenza B virus HA protein (i.e., BV-HA protein).

[0012] Specifically, the nucleic acid molecules involved in this invention can be obtained using genetic engineering recombination technology or chemical synthesis methods.

[0013] The present invention further provides a recombinant expression vector comprising the above-mentioned nucleic acid molecules.

[0014] Furthermore, the recombinant expression vector is selected from prokaryotic or eukaryotic expression vectors.

[0015] Furthermore, the recombinant expression vector is selected from bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0016] Specifically, the recombinant expression vector is constructed based on the binary plant expression vector pHREAC.

[0017] The present invention further provides a model organism comprising the above-mentioned recombinant expression vector, or having the above-mentioned nucleic acid molecules integrated into its genome.

[0018] Specifically, the present invention also provides host cells transformed, infected, or induced using the recombinant expression vector.

[0019] Specifically, the host cell is a bacterial cell, preferably Agrobacterium tumefaciens.

[0020] Specifically, the present invention also provides plant cells that are infiltrated, transformed, infected, or induced by the host cells.

[0021] Specifically, in some preferred embodiments, the plant is *Nicotiana benthamiana*.

[0022] Furthermore, the present invention provides an expression system capable of transiently expressing influenza B virus HA protein (i.e., BV-HA protein), wherein the expression system is a bacterial, yeast, filamentous fungus, mammalian cell, insect cell, plant cell, or cell-free expression system.

[0023] The present invention further provides a method for preparing influenza B virus HA protein (i.e. BV-HA protein). First, the nucleotide sequence encoding BV-HA protein is ligated into the plant binary expression vector pHREAC to construct a recombinant plasmid. Then, it is transformed into Escherichia coli competent cells and Agrobacterium competent cells in sequence, and infected with Nicotiana benthamiana. Transient expression is performed in Nicotiana benthamiana cells to obtain BV-HA protein.

[0024] Furthermore, based on a general inventive concept, the present invention also provides a method for the transient expression of influenza B virus HA protein (i.e., BV-HA protein) mediated by Agrobacterium tumefaciens in Nicotiana benthamiana, comprising the following steps: (I) Construction of recombinant plasmids 1) Based on the amino acid sequence of the influenza B virus HA protein (i.e., BV-HA protein), the codons corresponding to the amino acid sequence of the BV-HA protein were optimized to Benson's tobacco-loving codons using a codon optimization tool, resulting in the optimized BV-HA protein coding sequence and the optimized BV-HA protein amino acid sequence. The codons corresponding to the PR-1a signal peptide of Nicotiana benthamiana were optimized into Nicotiana benthamiana-biased codons using a codon optimization tool, resulting in the optimized signal peptide PR-1a coding sequence. Then, based on the optimized BV-HA protein coding sequence and the optimized signal peptide PR-1a coding sequence, a BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence was artificially synthesized in vitro. Simultaneously, the BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence and the 6 x H is tag coding sequence was artificially synthesized in vitro; 2) Insert the optimized BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence and the 6 x H is tag coding sequence into the plasmid pHREAC to obtain the recombinant plasmid BV-HA-pHREAC. (ii) Transformation and expression 3) After transforming the recombinant plasmid BV-HA-pHREAC into competent E. coli cells, the cells were cultured on LB solid medium plates containing antibiotics. Single clones were selected for colony PCR or sequencing verification. Colonies with correct sequences were selected to extract the recombinant plasmid, which was then transformed into competent Agrobacterium cells and cultured on LB solid medium containing antibiotics. Single clones were selected for colony PCR or sequencing verification to obtain Agrobacterium engineered bacteria containing the recombinant plasmid, which were then fermented. Meanwhile, the helper plasmid was transformed into Agrobacterium competent cells using the same method described above to obtain Agrobacterium engineered bacteria containing the helper plasmid, and then fermented and cultured. 4) After culturing the seeds of Nicotiana benthamiana in culture soil, the seedlings of Nicotiana benthamiana were obtained. After transplanting them into mixed soil for secondary cultivation, the leaves of Nicotiana benthamiana were obtained for later use. 5) Culture Agrobacterium-derived bacterial culture containing recombinant plasmid and Agrobacterium-derived bacterial culture containing helper plasmid to the same OD. 600 The bacterial culture of Agrobacterium tumefaciens containing recombinant plasmids was then mixed with that of Agrobacterium tumefaciens containing helper plasmids. After centrifugation and removal of the supernatant, the bacterial cells were resuspended in the infection solution to obtain the mixed resuspended bacterial culture. The culture was allowed to stand at room temperature (25±5℃) in the dark for 3-4 hours. Then, the culture was used to infect Tobacco Benedict leaves and treated in the dark for a certain period of time, followed by light / dark treatment for a certain period of time. The infected leaves were taken, weighed, and ground into powder with liquid nitrogen. Protein extraction buffer (lysis buffer) was added and mixed thoroughly. The mixture was incubated at 4℃ for 20-40 minutes and centrifuged to obtain the protein supernatant. (III) Purification 6) The protein supernatant obtained in step 5) is purified using a nickel column to obtain the influenza B virus HA protein (i.e., BV-HA protein).

[0025] Specifically, the optimized coding sequence of the influenza B virus HA protein (i.e., BV-HA protein) in step 1) is shown in SEQ ID No. 1.

[0026] Specifically, the amino acid sequence of the optimized influenza B virus HA protein (i.e., BV-HA protein) in step 1) is shown in SEQ ID No. 2.

[0027] Specifically, the nucleotide coding sequence of the optimized PR-1a signal peptide in step 1) is shown below: ATGGGATTTGTTCTTTTTTCCAACTTCCATCTTTTCTTCTTGTTTCTACTCTTCTTCTTTTTCTTGTTATTTCTCATTCTTGTCGTGCT.

[0028] Specifically, the base sequence encoding the 6 x H is tag (HHHHHH) in step 1) is: GTGGTGATGGTGATGAT.

[0029] Specifically, the helper plasmid is pSoup-p19.

[0030] Specifically, in step 3), the resistance is rifampicin and kanamycin.

[0031] Specifically, in step 4), the mixed soil is prepared from vermiculite and black soil in a mass ratio of 1:1.

[0032] Specifically, in step 4), the secondary culture conditions are 25℃; 16 hours of light, 8 hours of darkness, for 2-3 weeks.

[0033] Specifically, in step 4), the time for cultivating tobacco plants is 24 to 72 hours, preferably 60 hours.

[0034] Specifically, in step 4), the leaves of the cultivated tobacco plant are new leaves; preferably, the new leaves are leaves that have grown for 28-42 days, and more preferably leaves that have grown for 30 days.

[0035] Specifically, in step 5), when preparing the mixed resuspension, the volume of Agrobacterium-mediated bacterial culture containing the helper plasmid pSoup-p19 required for each 5 mL of infection solution is calculated as 0.2 / (2×OD). 600 () × 5 mL, the volume of Agrobacterium-mediated bacterial culture containing recombinant plasmid required per 5 mL of infection solution = 0.5 / (2 × OD) 60 Mix 5 mL of BV-HA-pHREAC / GV3101 bacterial solution and pSoup-p19 / GV3101 bacterial solution.

[0036] Specifically, in step 5), the formulation of the inoculum is: 10mM MgCl2; 10mM 2-(N-morpholino)ethanesulfonic acid (MES)-KOH, pH 5.6; 100μM acetylsuccinone (As).

[0037] Specifically, in step 5), the dark processing time is 4 to 10 hours, preferably 8 hours.

[0038] Specifically, in step 5), the light / dark interval processing is to perform light processing for 10 to 18 hours, preferably 16 hours, and dark processing for 4 to 10 hours, preferably 8 hours.

[0039] Specifically, in step 5), the dosage ratio for infecting tobacco leaves is 2-5 mL of protein extraction buffer per gram of leaf.

[0040] Specifically, in step 5), the Lysis buffer consists of: 50mM Tris, 150mM NaCl, 5mM MgCl2, 10% (volume percentage) glycerol, 0.1% (volume percentage) NP-40, 0.5mM DTT, 1mM PMSF, 16% (volume percentage) Western lysate (IP cell lysis buffer), 1mM EDTA, and a proteinase inhibitor cocktail tablet (1 tablet / 50ml).

[0041] Specifically, the protein purification steps in step 6) are as follows: a. Configure buffer A and buffer B; b. Equilibrate the Ni affinity medium by washing it three times with buffer A (each time using 5 times the volume of the Ni affinity medium); add the crude protein extract to the Ni affinity medium and incubate at 4°C for 10 hours by rotation. c. After filtering the crude protein extract, add it to the affinity medium of step (1) and mix by rotating at 4°C for 12 hours. d. Pack the crude protein extract and nickel affinity medium together into a column, release the flow-through after the protein has bound to the nickel affinity medium, and wash the nickel affinity medium with buffer A to obtain the eluent; e. Use different concentrations of buffer B buffer (concentration gradients of 20 mM imidazole, 500 mM imidazole, 100 mM imidazole, and 20 mM imidazole) to perform gradient elution of the Ni affinity medium bound to the protein to obtain a solution containing purified BV-HA protein. f. Western blotting was performed on the protein solutions obtained by elution of crude protein extract, flow-through buffer, elution buffer, and buffer B buffer at various concentration gradients using a mouse primary antibody against his tag (Solepro, K200060).

[0042] Specifically, buffer A consists of 20 mM Tris-HCl (pH=8) buffer and solute; the solute and its concentration are as follows: 150 mM NaCl; Buffer B: Composed of buffer A (pH=8) and solute; the solute and its concentration are as follows: 20-200mM imidazole.

[0043] Furthermore, the influenza B virus HA protein (i.e., BV-HA protein) obtained by this invention can be used to prepare vaccines, induce animals to produce antibodies against influenza viruses, and prevent viruses from infecting animals.

[0044] Furthermore, based on a general inventive concept, the present invention also provides the application of the prepared influenza B virus HA protein (i.e., BV-HA protein) in the preparation of influenza B immunizing drugs or influenza B virus vaccines.

[0045] Compared with the prior art, the advantages of the present invention are: 1. This invention clones a foreign protein gene into a pHREAC vector, and then uses tobacco leaves as a reactor to transiently and efficiently express the foreign protein in tobacco leaves using Agrobacterium infection and transformation. Then, based on the MYC affinity tag carried by the recombinant protein, the expressed protein is efficiently purified using an affinity medium with corresponding tag antibodies. Finally, the affinity medium is competitively eluted to obtain the purified target protein. The purified protein exhibits high biological activity and can be used for further biological function studies. This invention achieves one-step protein purification, which is simple, rapid, and yields high protein purity. The recombinant protein obtained using this method can be used for biosafety assessment and biological function studies.

[0046] 2. This invention provides a method for efficient expression of exogenous proteins. By cloning the exogenous BV-HA protein gene with a 6×his tag into the pHREAC vector, and then using Agrobacterium infection and transformation, the exogenous protein can be expressed instantaneously and efficiently in tobacco leaves, which has good application prospects. Attached Figure Description

[0047] Figure 1 This is a nucleic acid electrophoresis image of the transformation results after the recombinant plasmid was transformed into Agrobacterium and identified by PCR in the bacterial culture; in the image, 1: 2000 marker; 2-5: bands after Agrobacterium bacterial culture PCR; Figure 2 This is a graph showing the optimal time for protein expression after transformation. In the graph, 1: protein expressed in leaves 24 hours after transfection with recombinant Agrobacterium; 2: protein expressed in leaves 36 hours after transfection with recombinant Agrobacterium; 3: protein expressed in leaves 48 hours after transfection with recombinant Agrobacterium; 4: protein expressed in leaves 60 hours after transfection with recombinant Agrobacterium; 5: protein expressed in leaves 72 hours after transfection with recombinant Agrobacterium. Figure 3The image shows the results of protein purification. In the image, 1: crude protein extract; 2: flow-through protein; 3: washing buffer; 4: protein extracted with 20mM buffer B; 5: protein extracted with 50mM buffer B; 6: protein extracted with 100mM buffer B; 7: protein extracted with 200mM buffer B. Detailed Implementation

[0048] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in this technical field. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as known to those skilled in the art. Furthermore, any methods and materials similar to or equivalent to those described herein can be applied to the methods of the present invention.

[0049] The terms “comprising,” “including,” “having,” “containing,” etc., as used herein are open-ended, meaning they include but are not limited to. Terminology: As used herein, the terms “transformation,” “induction,” and “infiltration” refer to any process that introduces or delivers exogenous DNA into or into a host cell using methods well-known in the art.

[0050] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA / RNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA / RNA.

[0051] In the following examples, room temperature or normal temperature refers to 25±5℃.

[0052] Biomaterials, experimental reagents, culture media 1. Infection solution (pH 5.6): 10mM MgCl2; 10mM 2-(N-morpholino)ethanesulfonic acid (MES)-KOH, pH 5.6; 100μM acetylsuccinone (As); 2. Lysis buffer: 50mM Tris, 150mM NaCl, 5mM MgCl2, 10% (volume percentage) glycerol, 0.1% (volume percentage) NP-40, 0.5mM DTT, 1mM PMSF, 16% (volume percentage) Western lysate (IP cell lysis buffer), 1mM EDTA, Proteinase inhibitor cocktail tablet (1 tablet / 50ml); 3. Buffer A: Composed of 20 mM Tris-HCl (pH=8) buffer and solute; solute and its concentration are as follows: 150 mM NaCl; 4. Buffer B: Composed of buffer A (pH=8) and solute; the solute and its concentration are as follows: 20-200mM imidazole.

[0053] 5. Culture medium: LB solid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, 18.0 g agar powder, add deionized water to 1.0 L; pH 7.0~7.2, sterilize at 121℃ for 20 min.

[0054] LB liquid medium: 10.0 g tryptone, 5.0 g yeast extract, 10.0 g NaCl, add deionized water to 1.0 L; pH 7.0~7.2, sterilize at 121℃ for 20 min.

[0055] Example 1 Construction of recombinant plasmid 1. Based on the latest influenza strain information recommended by the World Health Organization for 2023-2024, and the whole genome information of influenza B virus (B / Austria / 1359417 / 2021 (B / Victoria lineage)), the amino acid sequence and coding sequence of influenza B virus HA protein (i.e. BV-HA protein) were obtained. 2. Use GenSmart TM The Codon Optimization tool optimizes the codons corresponding to the amino acid sequence of the influenza B virus HA protein (i.e., BV-HA protein) to Benson's tobacco-biased codons, resulting in the optimized nucleotide sequence and amino acid sequence of the BV-HA protein. The optimized nucleotide sequence encoding the BV-HA protein is shown in SEQ ID NO.1; The optimized amino acid sequence of the BV-HA protein is shown in SEQ ID NO.2.

[0056] 3. Use GenSmart TM The Codon Optimization tool optimized the codons corresponding to the PR-1a signal peptide of Nicotiana benthamiana (MGFVLFSQLPSFLLVSTLLLFLVISHSCRA) into Nicotiana benthamiana-preferred codons. The optimized codon sequence of the Nicotiana benthamiana PR-1a signal peptide (Genbank: ABV21361.1) is as follows: ATGGGATTTGTTCTTTTTTCCAACTTCCATCTTTTCTTCTTGTTTCTACTCTTCTTCTTTTTCTTGTTATTTCTCATTCTTGTCGTGCT.

[0057] 4. Based on the optimized coding sequence of BV-HA protein and the optimized coding sequence of tobacco PR-1a signal peptide, Qingke Biotechnology Co., Ltd. was commissioned to artificially synthesize the BV-HA protein coding sequence containing the coding sequence of signal peptide PR-1a in vitro. The optimized coding sequence of tobacco PR-1a signal peptide was added to the 5' end of the BV-HA protein coding sequence. Meanwhile, Qingke Biotechnology Co., Ltd. was commissioned to synthesize in vitro a BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence and the 6 x H is tag (HHHHHH) coding sequence. The 6 x H is tag (HHHHHH) was added to the 3' end of the target nucleic acid sequence (i.e. the optimized nucleotide sequence encoding the BV-HA protein). The base sequence encoding the 6 x H is tag (HHHHHH) is: GTGGTGATGGTGATGAT.

[0058] 5. Using conventional methods in the existing technology, the recombinant plasmid BV-HA-pHREAC was artificially synthesized in vitro by Qingke Biotechnology Co., Ltd. An optimized BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence and the 6 x H is tag coding sequence was inserted into the plasmid pHREAC. The specific construction of this plasmid followed conventional methods in the existing technology and is not the inventive point of this invention; therefore, it will not be described in detail here. Please refer to the following reference: Wen-Hao Yan, Peng Wang, Hua-Xia Chen, Hong-JuZhou, Qiu-Ping Li, Chong-Rong Wang, Ze-Hong Ding, Yu-Shan Zhang, Si-Bin Yu, Yong-Zhong Xing, Qi-Fa Zhang, A Major QTL, Ghd8, Plays Pleiotropic Roles in Regulating Grain Productivity, Plant Height, and Heading Date in Rice, Molecular Plant, Volume 4, Issue 2, 2011, Pages 319-330, ISSN 1674-2052.

[0059] The pHREAC plasmid nucleotide sequence used in this invention is shown below:

[0060] Example 2 Construction of recombinant Agrobacterium The recombinant plasmid BV-HA-pHREAC was transformed into Agrobacterium tumefaciens GV3101 (purchased from Biomed Biotechnology, catalog number: BC304-01) using a chemical transformation method. The specific steps are as follows: (1) Take 100 µL of GV3101 Agrobacterium competent cells stored at -80℃ and thaw them on ice or at room temperature; (2) Under sterile conditions, add the BV-HA-pHREAC plasmid to be transformed (about 500 ng) to the freshly thawed competent cell suspension, mix gently, and let stand in an ice bath for 10 min. (3) Place the EP tube containing the recombinant plasmid and competent cells in liquid nitrogen and freeze for 1 min to complete the transformation; (4) Let stand at room temperature until Agrobacterium thaws; (5) Under aseptic conditions, add 800 μl of antibiotic-free LB liquid medium to the EP tube containing the transformed Agrobacterium and incubate at 28°C with shaking for 2-3 hours; (6) Take about 150 μl of LB medium supernatant to resuspend the bacterial cells, add them to an LB solid medium plate containing 50 μg / ml rifampicin and 50 μg / ml kanamycin, and spread the cells evenly with a sterile bacterial spreader; after the liquid in the plate is completely absorbed, invert the plate and incubate at 28°C for 1-2 days; (7) Six single clones were randomly selected and cultured in liquid for 8 hours. Then, 50 μl of bacterial culture was taken, flash-frozen in liquid nitrogen for 1 min, and incubated in a water bath at 65°C for 1 min. This process was repeated three times. The bacterial culture was then collected by centrifugation at 6000 rpm for 5 min. Amplification was performed using the F1 / R1 primer pair and rTaq enzyme (Takara, RR901A). The agarose gel electrophoresis was then performed (e.g., ...). Figure 1 As shown in the figure, the recombinant plasmid has been successfully transformed into GV3101, that is, the Agrobacterium engineered strain GV3101-HA containing BV-HA-pHREAC was obtained.

[0061] The primer pairs are as follows: F1: 5'-cccttatcgggaaac-3'; R1: 5'-actatccttcgcaagacc-3'.

[0062] The results of the running glue are as follows Figure 1 As shown in the figure, the presence of the target band of the corresponding size indicates that the recombinant plasmid has been successfully transformed into GV3101.

[0063] The helper plasmid pSoup-p19 (reference: Zhang, Y., Qiu, L., Zhang, Y. et al. A high-efficiency transient expression system mediated by Agrobacterium tumefaciens in Spinacia oleracea leaves. Plant Methods 20, 100 (2024).) was transformed into GV3101 using the same method described above to obtain the Agrobacterium engineered strain GV3101-p19 containing pSoup-p19.

[0064] Example 3: Preparation of recombinant BV-HA protein I. Cultivation of Tobacco Benedict before Conversion 1. Seeds of Nicotiana benthamiana (obtained by the method described in the reference Barzigar R, Haraprasad N, Kumar BYS, Mehran MJ, Fakrudin B. Transient recombinant expression of highly immunogenic CagA, VacA and NapA in Nicotiana benthamiana. Biotechnol Rep (Amst). 2021 Dec 29;33:e00699. doi: 10.1016 / j.btre.2021.e00699. PMID: 35028298; PMCID:PMC8739878.) were placed in culture pots filled with 0-10mm general-purpose Nicotiana benthamiana peat moss. The pots were covered with a sealing film and placed in a plant room for humidification (25℃; 16 hours light, 8 hours darkness, for 4-5 days). 2. Transplant the cultivated seedlings into a mixed soil (vermiculite: black soil in a 1:1 mass ratio), water thoroughly, and continue cultivation (25℃; 16 hours of light, 8 hours of darkness) for 3 weeks (2-3 weeks is also acceptable). Obtain the leaves of the cultivated tobacco (the leaves are new leaves; the new leaves are leaves that have grown for 28-42 days, preferably leaves that have grown for 30 days) for later use.

[0065] II. Instantaneous Transformation of Tobacco Agrobacterium GV3101 containing BV-HA-pHREAC and pSoup-p19 respectively was mixed and injected into tobacco to transiently express BV-HA protein.

[0066] The specific operating method is as follows: (1) Transfer 0.15 ml of engineered bacterial culture containing BV-HA-pHREAC and pSoup-p19 culture, which were cultured overnight at 28°C, to 15 ml of LB liquid medium containing 25 ug / ml rifampicin and 50 ug / ml kanamycin, respectively, and culture at 28°C for 24 h. (2) Detection of OD of overnight cultured bacterial culture 600 The value; (3) The volume of pSoup-p19 Agrobacterium required per 5 mL of infection solution = 0.2 / (2 × OD) 600 ) × 5 mL, based on the required volume of BV-HA-pHREAC Agrobacterium per 5 mL of infection solution = 0.5 / (2 × OD) 60 Mix BV-HA-pHREAC / GV3101 bacterial suspension and pSoup-p19 / GV3101 bacterial suspension in 5 mL of centrifuge. Centrifuge at 5000g for 5 min, remove the supernatant, and resuspend the bacterial cells in infection solution (pH 5.6, composition: 10mM MgCl2; 10mM 2-(N-morpholino)ethanesulfonic acid (MES)-KOH, pH 5.6; 100μM acetylsyleugenone (As)) to obtain the mixed resuspended bacterial suspension. Let it stand in the dark for 3-4 h. (4) After scratching the lower epidermis of the leaf with a syringe needle, hold the front of the leaf with your thumb and inject 1 ml of mixed resuspended bacterial solution into the leaf of 4-week-old tobacco from the wound on the lower epidermis of each leaf. After successful injection, the tobacco leaf will appear moist. (5) The infected leaves were treated in the dark for 8 hours. After the dark treatment, they were cultured at 25°C for 16 hours of light and 8 hours of darkness. The timer was started, and 0.5 g of infected tobacco leaves were taken at 24 hours, 36 hours, 48 ​​hours, 60 hours and 72 hours respectively.

[0067] III. Preparation of BV-HA protein 1) Grind the obtained leaves thoroughly in liquid nitrogen. Add 30 ml of Lysisbuffer buffer to every 6 g (fresh weight) of tobacco leaves and mix by rotation at 4°C for 40 min. Then centrifuge at 4°C and 12000 rpm for 15 min, and take the supernatant to obtain crude protein extract. 2) The crude protein extract was identified by Western blotting using a mouse primary antibody against the His tag (Solepro, K200060). (Western blotting reference: Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels tonitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350–4354.) The results are as follows Figure 2 As shown, lanes 1-5 represent crude protein extracts prepared from leaves infected with Agrobacterium and exposed to light for 24, 36, 48, 60, and 72 hours, respectively. Figure 2 As can be seen, the protein band in lane 4 is significantly thicker than the other bands, and the protein expression level is optimal at 60 hours.

[0068] Example 4 Purification of recombinant BV-HA protein The BV-HA protein expressed in *Nicotiana benthamiana* leaves was purified using a nickel column in Tris-HCl buffer. The specific steps were as follows: 1) Equilibrate the Ni affinity medium (Lambolid, N30210) by washing it three times with buffer A (each time the volume of the Ni affinity medium is 5 times); add the crude protein extract to the Ni affinity medium and incubate at 4°C for 10 h by rotation.

[0069] 2) After filtering the crude protein extract through a 0.22 μM filter, add it to the affinity medium in step (1) and mix by rotating at 4°C for 12 h.

[0070] 3) Pack the crude protein extract and nickel affinity medium together onto a column, release the flow-through after the protein has bound to the nickel affinity medium, and wash the nickel affinity medium with buffer A to obtain the eluent.

[0071] 4) The Ni affinity medium containing the protein was eluted with different concentrations of buffer B buffer (concentration gradients of 20 mM imidazole, 500 mM imidazole, 100 mM imidazole, and 20 mM imidazole) to obtain a solution containing purified BV-HA protein.

[0072] 5) The protein solutions eluted from crude protein extract, flow-through buffer, elution buffer, and buffer B buffers of various concentration gradients were identified by Western blotting using a mouse primary antibody against the His tag (Solepro, K200060). (Reference: Towbin, H., Staehelin, T., & Gordon, J. (1979). Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proceedings of the National Academy of Sciences of the United States of America, 76(9), 4350–4354.) The results are as follows Figure 3 As shown, from Figure 3 As can be seen, there are protein bands in lane 5, and the BV HA protein with the his tag can be eluted and purified by 50mM imidazole-containing buffer B buffer.

Claims

1. A method for preparing influenza B virus HA protein, characterized in that, Includes the following steps: First, the nucleotide sequence encoding the BV-HA protein was ligated into the plant binary expression vector pHREAC to construct a recombinant plasmid. Then, it was sequentially transformed into Escherichia coli competent cells and Agrobacterium competent cells, and infected with Nicotiana benthamiana. Transient expression was performed in Nicotiana benthamiana cells to obtain the BV-HA protein.

2. A method for transient expression of influenza B virus HA protein mediated by Agrobacterium tumefaciens in Nicotiana benthamiana, characterized in that, Includes the following steps: (I) Construction of recombinant plasmids 1) Based on the amino acid sequence of the influenza B virus HA protein (i.e., BV-HA protein), the codons corresponding to the amino acid sequence of the BV-HA protein were optimized to Benson's tobacco-loving codons using a codon optimization tool, resulting in the optimized BV-HA protein coding sequence and the optimized BV-HA protein amino acid sequence. The codons corresponding to the PR-1a signal peptide of Nicotiana benthamiana were optimized into Nicotiana benthamiana-biased codons using a codon optimization tool, resulting in the optimized signal peptide PR-1a coding sequence. Then, based on the optimized BV-HA protein coding sequence and the optimized signal peptide PR-1a coding sequence, a BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence was artificially synthesized in vitro. Simultaneously, the BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence and the 6 x H is tag coding sequence was artificially synthesized in vitro; 2) Insert the optimized BV-HA protein coding sequence containing the signal peptide PR-1a coding sequence and the 6 x H is tag coding sequence into the plasmid pHREAC to obtain the recombinant plasmid BV-HA-pHREAC. (ii) Transformation and expression 3) After transforming the recombinant plasmid BV-HA-pHREAC into competent E. coli cells, the cells were cultured on LB solid medium plates containing antibiotics. Single clones were selected for colony PCR or sequencing verification. Colonies with correct sequences were selected to extract the recombinant plasmid, which was then transformed into competent Agrobacterium cells and cultured on LB solid medium containing antibiotics. Single clones were selected for colony PCR or sequencing verification to obtain Agrobacterium engineered bacteria containing the recombinant plasmid, which were then fermented. Meanwhile, the helper plasmid was transformed into Agrobacterium competent cells using the same method described above to obtain Agrobacterium engineered bacteria containing the helper plasmid, and then fermented and cultured. 4) After culturing the seeds of Nicotiana benthamiana in culture soil, the seedlings of Nicotiana benthamiana were obtained. After transplanting them into mixed soil for secondary cultivation, the leaves of Nicotiana benthamiana were obtained for later use. 5) Culture Agrobacterium-derived bacterial culture containing recombinant plasmid and Agrobacterium-derived bacterial culture containing helper plasmid to the same OD. 600 The bacterial culture of Agrobacterium tumefaciens containing recombinant plasmids was then mixed with that of Agrobacterium tumefaciens containing helper plasmids. After centrifugation and removal of the supernatant, the bacterial cells were resuspended in the infection solution to obtain the mixed resuspended bacterial solution. The solution was allowed to stand at room temperature in the dark for 3-4 hours, and then used to infect Tobacco Benedict's leaves. The leaves were treated in the dark for a certain period of time, followed by light / dark treatment for a certain period of time. The infected leaves were taken, weighed, and ground into powder with liquid nitrogen. The protein extraction buffer (lysis buffer) was added and mixed thoroughly. The mixture was incubated at 4°C for 20-40 minutes and then centrifuged to obtain the protein supernatant. (III) Purification 6) The protein supernatant obtained in step 5) is purified using a nickel column to obtain the influenza B virus HA protein.

3. The method as described in claim 2, characterized in that, The optimized coding sequence of the influenza B virus HA protein (i.e., BV-HA protein) in step 1) is shown in SEQ ID No. 1; The amino acid sequence of the optimized influenza B virus HA protein (i.e., BV-HA protein) in step 1) is shown in SEQ ID No.

2.

4. The method as described in claim 2, characterized in that, The nucleotide coding sequence of the optimized PR-1a signal peptide in step 1) is shown below: ATGGGATTTGTTCTTTTTTCCAACTTCCATCTTTTCTTCTTGTTTCTACTCTTCTTCTTTTTCTTGTTATTTCTCATTCTTGTCGTGCT; In step 1), the base sequence encoding the 6 x H is tag is: GTGGTGATGGTGATGAT.

5. The method as described in claim 2, characterized in that, The helper plasmid is pSoup-p19.

6. The method as described in claim 2, characterized in that, In step 4), the resistance is rifampicin and kanamycin.

7. The method as described in claim 2, characterized in that, When preparing the mixed resuspension, the volume of Agrobacterium-mediated bacterial culture containing the helper plasmid pSoup-p19 required per 5 mL of infection solution is calculated as 0.2 / (2×OD). 600 () × 5 mL, the volume of Agrobacterium-mediated bacterial culture containing recombinant plasmid required per 5 mL of infection solution = 0.5 / (2 × OD) 60 Mix 5 mL of BV-HA-pHREAC / GV3101 bacterial solution and pSoup-p19 / GV3101 bacterial solution.

8. The method as described in claim 2, characterized in that, In step 6), the dark processing time is 4~10 hours; The light / dark interval processing involves sequentially performing light processing for 10~18 hours and dark processing for 4~10 hours.

9. The influenza B virus HA protein prepared by any one of the methods described in claims 2 to 8.

10. The use of the influenza B virus HA protein according to claim 9 in the preparation of influenza B immunotherapy drugs or influenza B virus vaccines.

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