CN114634948A

CN114634948A - Bioreactor for instantaneously expressing foreign protein by using medlar as tobacco mosaic virus

Info

Publication number: CN114634948A
Application number: CN202011477593.2A
Authority: CN
Inventors: 张玉满; 谢传淼; 方荣祥; 张莉莉; 肖娜; 王莹莹; 黄卫阔
Original assignee: Institute of Microbiology of CAS
Current assignee: Institute of Microbiology of CAS
Priority date: 2020-12-15
Filing date: 2020-12-15
Publication date: 2022-06-17

Abstract

The invention discloses a bioreactor for instantaneously expressing foreign protein by using medlar as tobacco mosaic virus. The invention discloses a recombinant tobacco mosaic virus expression vector containing a coding gene of exogenous protein is introduced into agrobacterium to obtain recombinant agrobacterium; the recombinant agrobacterium obtained is used for infecting the medlar, and the exogenous protein can be expressed in the medlar. The GFP exogenous reporter gene is successfully expressed in the medlar leaves which are rich in nutrition and have medicinal value through a TMV viral vector and an agrobacterium tumefaciens instant expression system. The invention can create new preparation (such as oral preparation) of exogenous nutrient protein, medical polypeptide and protein (such as vaccine), can promote people to improve the immunity of the public and the health of the people by eating the functional food 'daily oral vaccine' containing vaccine components, is an important supplement for the plant production vaccine industry, and has important social significance and application prospect.

Description

Bioreactor for instantaneously expressing foreign protein by using medlar as tobacco mosaic virus

Technical Field

The invention relates to the field of biotechnology, and discloses a bioreactor for instantaneously expressing foreign protein by using medlar as tobacco mosaic virus.

Background

The vaccine is not only used for preventing important diseases (such as BCG vaccine, poliomyelitis vaccine, diphtheria-pertussis-tetanus combined vaccine, measles vaccine, hepatitis B vaccine and the like) and annual epidemic diseases (such as human or livestock flu) which are abused by human beings, but also is an effective measure for dealing with sudden pestilence (such as novel coronary pneumonia COVID-19), and has important significance in human health and improvement of life quality. The use of large quantities of pathogens of animal origin (e.g. chicken embryos) in the production of conventional vaccines (e.g. influenza vaccines) presents a potential risk of contamination and a sudden large demand that is difficult to meet due to the time frame of production. In recent years, scientists have used plant virus vectors in combination with agrobacterium transient expression (differentiation) technology to produce medical protein and subunit vaccines, and the system has the advantages of high foreign protein expression, short time, low cost, safety, large-scale production and the like, so that the system is currently applied to European and American medicine commercial production, such as the company of plant biotechnology, Dow AgroScience, and the company of Protalix, Biolex, SemBosys, and the company of medical, and the company of Dow AgroScience, respectively and successfully develop alpha-galactosidiase A, human interferon alpha, human insulin, H5N1 influenza subunit vaccines. The large-scale application of Magnifection technology promotes the vaccine industry to enter a new era of producing vaccines by plants! At present, China still belongs to the stage of overtaking and filling up the blank in the field.

Plant viral vectors involved in the production of vaccines by plants include various expression vectors such as Tobacco viral viruses (TMV), Potato Viral X (PVX), Alfalfa viral viruses (AMV), Cowpea viral viruses (CPMV), and DNA viruses Bean yellow dewar viruses (BeYDV), and only TMV vectors are currently successfully used in commercial production. Plant materials used for producing vaccines by using TMV viral vectors, namely 'vaccine processing workshops', are Nicotiana benthamiana (Nb). Due to the inedibility of tobacco, foreign protein needs to be purified from tobacco in the process flow of producing vaccine by plants, and the problems of processing cost and protein yield rate caused by expensive price/recycling and the like of the used protein purification chromatographic column are limiting factors in production. Therefore, it is possible to find an edible plant to bring new breakthrough to oral vaccine production, and it has been reported that the application of vegetable plants such as lettuce (Lactuca sativa), eggplant (Solanum melongena) and the like in the agrobacterium transient expression system is attempted, but the plant propagation production process is limited to the laboratory research stage due to low protein expression level and the like.

Lycium barbarum (Lycium barbarum) is a multi-branched shrub plant of the genus Lycium (Lycium) of the family Solanaceae (Solanaceae). The whole body of medlar is treasure, and the Ming Li Shizhen's book of compendium of materia Medica: spring collected folium Lycii, named Tianjing grass; summer flower, named Changsheng grass; named as fructus Lycii; collected root in winter, named cortex lycii radicis. Lycium chinense seeds are well known as health-preserving food materials and high-immunity medicinal materials, tender leaves of Lycium chinense can be used as vegetables and vegetables, the nutritional value of Lycium chinense is increasingly valued by people, and related varieties of Lycium chinense are cultivated and applied.

Disclosure of Invention

The technical problem to be solved by the invention is how to express the medical or edible exogenous protein in edible plants in a transient and high-efficiency manner by a non-transgenic technology.

In order to solve the above technical problems, the present invention provides a method for expressing a foreign protein in a plant, wherein the plant is lycium barbarum, the method comprising: introducing a recombinant tobacco mosaic virus expression vector containing a coding gene of exogenous protein into agrobacterium to obtain recombinant agrobacterium; and infecting the medlar by using the recombinant agrobacterium to realize the expression of the foreign protein in the plant.

In the above method, the recombinant tobacco mosaic virus expression vector may be obtained by inserting a gene encoding the foreign protein into a tobacco mosaic virus vector.

In the above method, the tobacco mosaic virus vector may be p 35S-30B.

In the above method, the agrobacterium may be agrobacterium strain GV 3101.

In the above method, the Lycium barbarum can be a Lycium plant.

The Lycium plant may be Lycium barbarum L, Lycium barbarum Mill, Lycium ruthenicum Murr, Lycium barbarum Pojark, Lycium barbarum Y.C.Wang, Lycium cylindricum Kuang, or Lycium yunnanense Kuang.

In one embodiment of the invention, the Lycium barbarum (Lycium barbarum L) is Lycium barbarum No. 9.

The invention also provides a kit comprising the tobacco mosaic virus vector and the agrobacterium.

The kit may further comprise said wolfberries.

The kit can be composed of the tobacco mosaic virus vector and the agrobacterium, and can also be composed of the tobacco mosaic virus vector, the agrobacterium and the medlar.

The kit can be used for expressing a foreign protein in a plant, or preparing a product for expressing a foreign protein in a plant, or producing a medical protein, vaccine or nutritional protein, or preparing a product for producing a medical protein, vaccine or nutritional protein.

Any of the following uses of the kit also fall within the scope of the invention:

x1, expressing a foreign protein in a plant;

x2, preparation of a product for expressing a foreign protein in a plant;

x3, production of a protein for medical use, vaccine or nutritional protein;

x4, preparation of products for the production of proteins for medical use, vaccines or nutritional proteins.

The vaccine may be an oral vaccine.

In the above application, the plant may be fructus Lycii. The Lycium barbarum can be a Lycium plant.

In one embodiment of the present invention, the exogenous protein is green fluorescent protein.

The GFP exogenous reporter gene is successfully expressed in the medlar leaves which are rich in nutrition and have medicinal value through a TMV viral vector and an agrobacterium tumefaciens instant expression system. Because the plants of the lycium genus and the tobacco which is widely applied to the plants for producing the vaccines belong to the solanaceae family, the lycium macrocarpum has nutritional and medicinal values, is suitable for asexual propagation such as cuttage, pile cutting and the like, keeps the stable and consistent genotype, has low cost, can be produced in a large scale and the like. The present invention provides safe and edible oral preparation of medical polypeptide and protein, such as vaccine, without high purity and difficult preparation steps. The invention can create new preparation (such as oral preparation) of exogenous nutrient protein, medical polypeptide and protein (such as vaccine), can promote people to improve the immunity of the public and the health of the people by eating the functional food 'daily oral vaccine' containing vaccine components, is an important supplement for the plant production vaccine industry, and has important social significance and application prospect.

Drawings

FIG. 1 fluorescent detection of GFP reporter gene expression on Lycii folium by Agrobacterium transient expression system.

30, 35 and 80d seedlings of the medlar seeds are respectively injected with agrobacterium GV3101 containing TMV viral vector (pAT-His-eGFP) of exogenous reporter gene and plant expression vector (130-GFP), wherein the injection of MMA buffer solution is Mock negative control. Fluorescence observation was performed when the culture was continued for 3,5,7 and 10dpa (days post agroinfiltration), respectively, after the Agrobacterium injection. In the figure, p130-GFP represents 130-GFP.

FIG. 2 detection of His-eGFP expressing proteins in Lycii folium.

And extracting total soluble protein of the medlar leaf blade injected with pAT-His-eGFP agrobacterium, and carrying out His-eGFP protein expression detection. (A) Coomassie blue staining for His-eGFP expression, M: a protein Marker; MMA: a Mock negative control; #1, #2 and #3 represent different lycium barbarum plants; the sample amounts in the lanes were 4.5mg total soluble protein from fresh weight leaves. The red arrow indicates His-eGFP.

(B) Western blot analysis was performed using His-tag antibody. The loading of lanes was 2.7mg fresh weight of total soluble protein.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st 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.

Examples 1,

1. Materials and methods

1.1 materials:

lycium barbarum seed (Lycium barbarum) (ningxia Lycium barbarum No. 9); agrobacterium strain GV 3101.

1.2 method:

(1) constructing a vector and a recombinant bacterium:

Agrobacterium-TMV virus expression vector p35S-30B (ref. Hongge Jia, Yongqi Page, Rongxiang Fang, agriculture infection as a simple way to a background a. tobaco biological virus-based expression vector, Acta Botanica Sinica 2003,45: 770-773; preserved in the institute of microbiology of the national institute of plant genomics, publicly available from the institute of microbiology of the national institute of sciences), was digested with PacI and KpnI and inserted into a His-eGFP reporter gene fragment with a tag in combination with a recombination system, and the resulting recombinant plasmid was named pAT-His-eGFP. pAT-His-eGFP is a recombinant vector obtained by replacing the DNA fragment between the PacI and KpnI recognition sequences of p35S-30B with the DNA fragment shown in sequence 1 in the sequence table.

Wherein, the 76 th to 807 th sites of the sequence 1 are eGFP gene sequences.

In addition, the reference plant expression vector 130-GFP [ i.e., P ] was also used in the experiments_35SeGFP, reference Wang H, Zhang Y, Xiao N, Zhang G, Wang F, Chen X, and Fang R (2020) Rice Transformer-LIKE PROTEIN 2-1Functions in selected university under the Control of Abscisic Acid and Gibberellic Acid signalling pathways plant Physiology 183:1157 1170, publicly available from the national academy of sciences microbiology]。

Introducing the pAT-His-eGFP into an agrobacterium strain GV3101 to obtain a recombinant strain GV 3101/pAT-His-eGFP; the reference plant expression vector 130-GFP is introduced into the agrobacterium strain GV3101 to obtain the recombinant strain GV 3101/130-GFP.

(2) Culturing medlar seedlings:

soaking the medlar seeds in water for one day, and then directly sowing the seeds in vermiculite: in a culture medium of nutrient soil (1:1), transplanting seedlings after culturing for 10 days at 22 ℃ in a plant greenhouse (illumination for 16 h/darkness for 8h), and respectively carrying out agrobacterium tumefaciens transient expression experiments when the seedlings are continuously cultured in the plant greenhouse to 30d, 35d and 80 d.

(3) And (3) agrobacterium injection:

reference is made to the published articles and slightly modified [ Wang H, Zhang Y, Xiao N, Zhang G, Wang F, Chen X, and Fan R (2020) Rice GERMIN-LIKE PROTEIN 2-1Functions in selected university under the Control of Abscisic Acid and Gibberella Acid signalling pathway plant Physiology 183:1157 Ash 1170 ]. The method comprises the following specific steps:

inoculating the recombinant strain GV3101/pAT-His-eGFP or the recombinant strain GV3101/130-GFP to the strain containingAfter shaking culture at 220rpm/28 ℃ overnight in LB broth containing 50. mu.g/ml Kanamycin (Kanamycin, Kan) and 50. mu.g/ml Rifampicin (Rifamicin, Rif) antibiotics, the cells were diluted at 1/100 and transferred to LB broth (containing 50. mu.g/ml Kan,10mM MES, 20. mu.M AS), and the culture was continued until OD600 became 1.0, and then centrifuged at 12,000rpm for 30sec, the supernatant was discarded, and the pellet was collected. Using MMA buffer (solvent is water, solute and its concentration are respectively 10mM MgCl₂10mM MES and 100 μ M AS) and centrifuged again, and the cells were resuspended with MMA buffer to OD600 ═ 0.8. Standing at room temperature for 2-3 h. Finally, recombinant agrobacterium containing pAT-His-eGFP or 130-GFP is injected into the back of each Chinese wolfberry leaf by a 1ml syringe without a needle, 3 Chinese wolfberry plants are injected into each bacterium, and 9 leaves are counted. And (5) continuously culturing for 3-10 days under illumination, and then analyzing and detecting the exogenous gene. The Mock negative control was the MMA buffer injected to suspend the bacteria.

(4) Detection and analysis of exogenous reporter gene:

1) eGFP fluorescence detection: the whole plant after injection is put under dark condition and ultraviolet lamp (c)

UVP Upland CA91786, u.s.a.) and observing eGFP protein expression. Normal non-expressing control leaves showed red color, while eGFP highly expressed injection leaves showed green fluorescence.

2) Western blot protein detection of eGFP: the leaves of Lycium barbarum injected with recombinant Agrobacterium containing pAT-His-eGFP or 130-GFP were harvested with liquid nitrogen and frozen and stored at-80 ℃ until use. The sample was ground well to a powder in a pre-cooled mortar and 2ml of protein extraction buffer [ solvent ddH ] was added according to a fresh weight of 1g₂O, solute and its concentration were 50mM Tris (pH 7.5),150mM NaCl, 10% glycerol, 0.5% Triton X-100 and complete protease inhibitor cocktail, respectively. Wherein the working concentration of the Protease Inhibitor complete Protease Inhibitor Cocktail (Roche, Mini Protease Inhibitor Cocktail, Cat: 11836153001) is 1 pill/10 ml protein extraction buffer]After rotating at 4 ℃ for 30min, centrifuging at 4 ℃ for 15min at 12,000rpm twice, taking the supernatant and respectively performing protein SDS-PAGE and WeAnd (5) analyzing the sternblot.

The primary antibody used for Western blot was His-tag antibody (Sigma-Aldrich, cat # SAB2702220), and the secondary antibody was Anti-Mouse IgG (H + L), HRP Conjugate (Promega, cat #: w4021).

2. Results of the experiment

2.1 fluorescence detection of transient expression of GFP reporter genes in Lycium barbarum

Agrobacterium containing pAT-His-eGFP and p130-GFP was injected into 30, 35, and 80d extended leaves of Lycium barbarum seedlings at different growth stages by Agrobacterium injection transient expression system, and ultraviolet fluorescence detection analysis revealed that 3-10d of inoculated leaves of Lycium barbarum after injection all had strong blue-green fluorescence, whereas no fluorescence signal was observed in the negative control leaves injected with MMA buffer (FIG. 1), and this experiment result showed successful expression of active exogenous GFP protein in Lycium barbarum leaves by Agrobacterium transient expression system.

2.2 protein analysis of transient expression of GFP reporter Gene in Lycium barbarum

In order to confirm that the fluorescent signal on the injected leaves of lycium barbarum was a GFP foreign protein, Western blot assay was further performed with His-tagged antibody fused to eGFP (fig. 2), and the results showed that there was signal production with the expected size of His-GFP fusion protein (about 27kD) in the injected samples of lycium barbarum leaves, while the negative control leaves had no protein expression signal.

The invention proves that the Chinese wolfberry leaves are suitable for expressing active exogenous target protein through an agrobacterium transient expression system by combining fluorescence detection and Western blot protein analysis, establishes a practical novel method for producing exogenous polypeptide and protein by using Chinese wolfberry as a virus expression host, can be applied to the production of medical protein, vaccine, nutritional protein and the like, is particularly suitable for the development of oral vaccine, and has important application potential.

Comparative examples 1,

According to the method of example 1, lycium barbarum was replaced with mulberry, chenopodium album, dandelion, lettuce, rehmannia glutinosa, cedrela sinensis, peach tree, capsicum, sweet potato, aralia elata and lycium barbarum, respectively, and the injection of agrobacterium and the expression of eGFP are shown in table 1.

TABLE 1 plant screening for transient expression of GFP by Agrobacterium injection suitable for TMV viral vectors

Note: in table 1, in the column "easy to inject", X indicates that agrobacterium cannot be injected, i.e. the liquid in the injector is completely blocked and cannot be injected), V indicates that agrobacterium can be partially injected, i.e. a small amount of liquid is injected into the local tissue of the blade, VV indicates that agrobacterium is successfully injected, i.e. injection is easy, and liquid can be injected into the whole blade; in the "GFP fluorescence" column, X indicates that no green fluorescence signal is present and GFP cannot be expressed, VV indicates that an obvious fluorescence signal is present and eGFP is successfully expressed; NT means no test (not test).

The results show that, because the cell structures of different plant leaves are different, the affinity difference between the cells and the virus vector is large, and not all plants are suitable for a certain virus vector to express the foreign protein instantly, the medlar of the invention is used as a bioreactor for the tobacco mosaic virus to express the foreign protein instantly, and is a new material successfully obtained by screening various plants, such as various plant materials in the table 1, and only the medlar leaf has successful agrobacterium injection and high-efficiency expression.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.

<110> institute of microbiology of Chinese academy of sciences

<120> bioreactor for transient expression of foreign protein by using medlar as tobacco mosaic virus

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<170> PatentIn version 3.5

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Claims

1. A method of expressing an exogenous protein in a plant, the plant being lycium barbarum, the method comprising: introducing a recombinant tobacco mosaic virus expression vector containing a coding gene of an exogenous protein into agrobacterium to obtain recombinant agrobacterium; and infecting the medlar by using the recombinant agrobacterium to realize the expression of the foreign protein in the plant.

2. The method of claim 1, wherein: the recombinant tobacco mosaic virus expression vector is obtained by inserting the coding gene of the exogenous protein into a tobacco mosaic virus vector.

3. The method of claim 2, wherein: the tobacco mosaic virus vector is p 35S-30B.

4. A method according to any one of claims 1 to 3, wherein: the agrobacterium is agrobacterium strain GV 3101.

5. The method according to any one of claims 1 to 4, wherein: the fructus Lycii is a Lycium plant.

6. The method of claim 5, wherein: the Lycium plant is Lycium barbarum L, Lycium barbarum Mill, Lycium ruthenicum Murr, Lycium barbarum Pojark, Lycium truncatum Y.C.Wang, Lycium cylindracea Kuang, or Lycium yunnanense Kuang.

7. A kit of parts comprising the tobacco mosaic virus vector of any one of claims 2-4 and the Agrobacterium.

8. The kit of claim 7, wherein: the kit of parts further comprising lycium barbarum of any one of claims 1-6.

9. Use of a kit according to claim 7 or 8 for any one of the following applications:

x1, expressing a foreign protein in a plant;

x2, preparation of a product for expressing a foreign protein in a plant;

x3, production of a protein for medical use, vaccine or nutritional protein;

10. Use according to claim 9, characterized in that: the plant is medlar.