阅读说明：本技术 生产含重组人碱性磷酸酶的转化植物细胞的方法以及含重组人碱性磷酸酶的转化植物细胞的用途 (Method for producing transformed plant cells containing recombinant human alkaline phosphatase and use of transformed plant cells containing recombinant human alkaline phosphatase ) 是由 娜塔莉亚·安娜托里耶夫娜·施米科娃 罗马·亚历山德罗维奇·科马金 谢尔盖·阿列克山德罗维奇·斯坦 于 2019-01-29 设计创作，主要内容包括：本发明涉及食品工业和医学,并描述了生产含有重组人碱性磷酸酶的转化植物细胞的方法。本方法包括构建具有人碱性磷酸酶基因的植物表达载体,将具有人碱性磷酸酶基因的植物表达载体导入农杆菌菌株,生产植物愈伤组织细胞,使用农杆菌菌株农杆菌转化愈伤组织细胞；从转化的愈伤组织细胞产生体细胞胚,并在悬浮培养中培养含有人碱性磷酸酶基因的体细胞胚。本发明的目的是调节胃肠道微生物群落以及保护和恢复人体免疫系统。(The present invention relates to the food industry and medicine and describes a method for producing transformed plant cells containing recombinant human alkaline phosphatase. The method comprises constructing a plant expression vector containing human alkaline phosphatase gene, introducing the plant expression vector containing human alkaline phosphatase gene into Agrobacterium strain to produce plant callus cells, and transforming the callus cells with Agrobacterium strain Agrobacterium; somatic embryos were generated from transformed callus cells and somatic embryos containing the human alkaline phosphatase gene were cultured in suspension culture. The aim of the invention is to regulate the gastrointestinal microflora and to protect and restore the human immune system.)

1. A method for producing a transformed plant cell containing recombinant human alkaline phosphatase, which comprises constructing a plant expression vector having a human alkaline phosphatase gene, introducing said plant expression vector having a human alkaline phosphatase gene into an Agrobacterium strain, producing a plant callus cell, and subjecting said callus cell to Agrobacterium transformation with the Agrobacterium strain; producing somatic embryos from the transformed callus cells, and culturing the somatic embryos containing the human alkaline phosphatase gene in a suspension medium.

2. The method of claim 1, wherein after the agrobacterium transforms the callus cells, the transformed callus cells having the human alkaline phosphatase gene are cultured in suspension culture.

3. The method of claim 1, wherein said cultured transformed plant cells containing the human alkaline phosphatase gene are dried.

4. The method of claim 1, wherein the recombinant human alkaline phosphatase is a tissue-specific alkaline phosphatase, such as intestinal alkaline phosphatase and placental alkaline phosphatase, or a non-tissue-specific alkaline phosphatase.

5. The method of claim 1, wherein the development of the embryos is synchronized.

6. The method of claim 1, wherein the somatic embryos are cultured in a liquid nutrient medium supplemented with an osmolality-increasing compound, such as polyethylene glycol, mannitol, and other osmotically-increasing compounds.

7. The method of claim 1, wherein the transformed plant cells containing the human alkaline phosphatase gene are used as ready-to-feed capsules, tablets, sachets and other forms.

8. Use of transformed plant cells produced using the method of claim 1 for the prevention of gastrointestinal microflora diseases and their restoration under toxic and stress conditions.

9. Use of transformed plant cells produced using the method of claim 1 for the prevention of immune system diseases associated with gut microflora disruption and gut barrier function, and as immunostimulants to restore the disrupted immune system.

Technical Field

The present invention relates to the food industry and medicine and describes a method for producing transformed plant cells containing recombinant human alkaline phosphatase and their use in maintaining the homeostasis of the gastrointestinal tract (GI tract).

Background

It is well known that the intestine is an important barrier organ, consisting of the normal (ecologically human-associated) microflora, the mucosal layer, the epithelium and the subepithelial immune system. The main function of the intestinal barrier is to protect the organism from bacterial transfer into the systemic circulation. The normal microflora can remain in the intestinal lumen without stimulating the host's immune response, whereas the same bacteria can induce immune responses and inflammation if transported across the intestinal barrier into the blood and to other organs. Disruption of the intestinal wall barrier can lead to excessive immune responses to homeotropic bacterial components, which in turn leads to the development of chronic Inflammatory Bowel Disease (IBD), such as crohn's disease and nonspecific ulcerative colitis (Podolsky, 2010). Stress, infection, aging and toxic effects of xenobiotics, including drugs, also play an important role in the development of these diseases (Cadwell et al, 2010).

Currently, the only widely available means of maintaining gastrointestinal homeostasis includes the modulation of the gastrointestinal microflora and the immune system, which are drugs and products based on probiotics, live microbial cultures consisting of species normally present in the intestinal tract. However, there is no convincing evidence that such products have clinical efficacy. In addition, externally introduced microbial communities are generally not viable in humans and are rejected by naturally occurring microbial communities. Moreover, probiotics are generally two bacteria: lactobacillus and bifidobacterium, while the human microflora includes over 300 bacteria. Thus, the initial instability of gastrointestinal homeostasis often reoccurs after discontinuation of probiotic use.

However, new approaches based on the use of Alkaline Phosphatase (AP) enzymes to regulate the gastrointestinal homeostasis are considered promising, as the development of microbial communities, disturbances of the immune system and intestinal barrier function, as well as IBD, is largely triggered by inflammation inducers, some of which are physiological substrates of the AP enzymes. The most effective causes of inflammation are: 1) lipopolysaccharide (LPS) -bacterial endotoxin of escherichia coli and other gram-negative bacteria; 2) extracellular ATP and other nucleotides and nucleosides. The intestinal microflora constantly releases LPS, which enters the blood when the intestinal barrier is disrupted, causing inflammation even at the lowest concentration (ng/kg). In this process, LPS binds to the membrane protein CD14 on the surface of macrophages, leading to its activation, which in turn leads to the expression of tens of biologically active compounds: prostaglandins, nitric oxide and cytokines, including interleukins and TNF α (enson, 1987). LPS and extracellular ATP are important factors for the development of systemic immune and inflammatory responses in response to "foreign" and "dangerous" signals (Matzinger, 2002).

Intestinal alkaline phosphatase produced by intestinal mucosal cells plays an important role in intestinal homeostasis by inactivating (detoxifying) LPS and preventing LPS translocation, regulating intestinal microflora and intestinal surface pH (Eskandari et al, 1999; weemes et al, 2002; Riggle et al, 2013; tube et al, 2009). The dephosphorylation of LPS catalyzed by AP abolishes its pro-inflammatory activity, thereby protecting the body from the development of various inflammatory and autoimmune diseases (Park, Lee, 2013).

Disruption of normal levels (activity) of intestinal AP due to decreased expression of the gene encoding AP was observed in inflammatory bowel disease, celiac disease, and obesity. Oral administration of AP to mice with colitis can reduce mRNK levels of the pro-inflammatory cytokines TNF α and nitric oxide (Muginova et al, 2007). Molnar et al (2012) have demonstrated that AP levels in inflamed intestinal mucosa of infants with Crohn's disease and nonspecific ulcerative colitis are significantly lower than in the control group. Exogenous AP administration to patients with active intestinal inflammation prevents disease progression. In another study it has been demonstrated that exogenous AP administration can reduce intestinal inflammation in newborn animals, so AP has been proposed for the prevention of inflammatory diseases in newborn infants (Biesterveld et al, 2015).

There are several prior art patents and patent applications describing the use of AP in the regulation of intestinal microflora, immune system activity, prevention and treatment of various diseases: US20130280232 on 24/10/2013 for detoxification of LPS toxins on mucosal barriers with alkaline phosphatase, US20110206654 on 25/8/2011 for methods of modulating gastrointestinal microbiota drowning with alkaline phosphatase (US 20130022591 on 24/1/2013 for reducing or inhibiting toxic effects associated with bacterial infection with alkaline phosphatase (US 8574863 on 5/11/2013 for treating gastrointestinal inflammatory diseases).

The closest approach to the food production method disclosed in the present invention is the method disclosed in patent application US20110206654, method for regulating gastrointestinal microbiota level with alkaline phosphatase, dated 2011 8/25, which describes the regulation of GI microbiota with AP, including the protection and restoration of normal microbiota when antibiotics are used. In this prototype and other published sources, the authors used AP as a pure protein and purified it from in vitro proteins and other impurities. The AP is administered to animals or humans (in clinical trials) in the form of a pure protein or pharmaceutical preparation (comprising purified AP and different excipients). Purified AP obtained by various methods, including: isolating AP from bovine intestinal mucosa; isolation of AP from mammalian placenta: recombinant human AP is produced in mammalian cell culture, followed by isolation and purification. A common feature of the known processes is the production of AP as a pure protein, which leads to several disadvantages listed below and virtually eliminates the possibility of using it as a food or food ingredient.

A very important disadvantage of AP isolated from the mammalian intestinal tract or other organs is that it is a foreign protein to humans (due to interspecies genetic differences) and thus the use of AP in humans may cause allergic reactions and develop dangerous allergic reactions after repeated use. In addition, animal tissues have low AP content, requiring expensive protein extraction and purification procedures. There is also a risk of contamination by prions and pathogenic microorganisms. Thorough purification can add significant cost to the final product and severely limit its practical use.

The in vitro production of recombinant human AP in mammalian cell systems has its own drawbacks, including: limited scalability of the process, high cost and risk of contamination by human pathogens.

Due to difficulties in isolation, purification and quality control of recombinant proteins, a common drawback of the AP process for producing what is described as a pure protein is the high cost of the final product (up to $ 1000-2000 per package). This virtually eliminates the possibility of using it as a food for the prevention and treatment of diseases.

Another common disadvantage of the described methods for AP production as a pure protein is the high sensitivity of AP to external conditions such as temperature and pH (acidity) of the culture medium. For example, AP is completely inactivated in the acidic medium of the stomach. This eliminates the possibility of using such AP in food products or the possibility of complex dosage forms that are required to produce gastric resistant effects.

The above-mentioned disadvantages of AP production preclude the use of AP as a widely available product for maintaining the normal microflora and immune system of the gut and for preventing inflammatory and autoimmune diseases associated with disturbances of the gastrointestinal homeostasis. Thus, the possibility of using AP as pure protein or mixed with standard pharmaceutical excipients to modulate the gastrointestinal microflora described in the prototype (US20110206654, date 25/8/2011) is severely limited by the above-mentioned drawbacks and cannot be used in the manipulation of food products or food ingredients.

Disclosure of Invention

The object of the present invention is to develop a new, effective, safe and affordable AP-containing food product for maintaining the normal intestinal microflora and immune system and preventing the development of inflammation and autoimmune diseases associated with disturbances of the homeostatic balance of the gastrointestinal tract.

This object is achieved by the development of a food product wherein the recombinant human alkaline phosphatase is located in a plant cell. Plant cells containing AP (unlike prototype cells using purified AP) provide natural protection from AP inactivation in acidic gastric fluid and delivery to the gut where AP performs its function-dephosphorylation (inactivation) of bacterial endotoxins produced by the gastrointestinal pathogenic microbial flora. Thus, the AP-containing food product maintains, prevents the dysregulation of, restores under toxic and stressful conditions, and also prevents the development of inflammation and autoimmune diseases. The AP-containing plant cells contained in the food product are safe for human consumption, do not cause allergic and dangerous allergic reactions upon repeated use, and do not infect humans with prions and animal pathogenic microorganisms (unlike AP derived from mammalian organs). Finally, foods based on plant cells containing recombinant human AP are not only safer than drugs based on purified recombinant human AP produced in mammalian cells, but are also 2-3 orders of magnitude cheaper. This makes it useful as a food product for large-scale prevention of gastrointestinal microflora disorders and immune system diseases and prevention of inflammatory and autoimmune intestinal diseases in the general population.

A method for producing a food product based on plant cells containing recombinant human alkaline phosphatase comprising: constructing a plant expression vector with human AP genes, introducing the plant expression vector with the human AP genes into agrobacterium to generate plant callus cells, and performing agrobacterium transformation on the callus cells by using agrobacterium strains; the food product is produced by culturing transformed plant callus cells containing the human AP gene in suspension culture.

Another variation of the method for producing a food product based on plant cells containing recombinant human alkaline phosphatase comprises constructing a plant expression vector having a human AP gene, introducing the plant expression vector having the human AP gene into an Agrobacterium strain to produce plant callus cells, subjecting the callus cells to Agrobacterium transformation using the Agrobacterium strain, and producing somatic embryos from the transformed callus cells; the food product is produced by culturing somatic embryos containing the human AP gene in suspension culture.

During the growth of somatic embryos, embryos are synchronously developed through filtering by sieves with different sizes, centrifuging, automatic separation or other synchronization methods. Somatic embryos are cultured in liquid nutrient medium supplemented with an osmolality-increasing compound. The optimum concentration of such compounds does not exceed 10%. Polyethylene glycol, mannitol and other compounds may be used as the compound for increasing osmotic pressure.

Tissue-specific alkaline phosphatases, such as intestinal alkaline phosphatase and placental alkaline phosphatase or tissue-nonspecific alkaline phosphatase, as well as other isoforms of alkaline phosphatase, may be used as recombinant human alkaline phosphatase of the invention.

Transformed callus cells or somatic embryos cultured in suspension culture are dried and used as food products, e.g., as capsules, tablets, sachets and other ready-to-eat forms, or added to other food products.

The food product is produced for regulating the gastrointestinal microflora, for preventing immune system diseases and as an immunomodulator for restoring an impaired immune system.

Plant producers are known to be the most promising system for producing high quality, safe and relatively inexpensive proteins (Conley et al, 2011; Sharma, Sharma, 2009; Sabalza et al, 2014). We have taken advantage of plant producers to create a fundamentally novel product comprising plants containing recombinant human alkaline phosphatase instead of pure AP. Any edible plant that can be used to produce recombinant proteins can be used for this purpose, including, but not limited to, carrot (Daucus carota), lettuce (Latuca sativa), cabbage (Brassica oleracea), Chinese cabbage (Brassica pekinensis), dill (Anethum graveolens), celery (Apium graveolens), cucumber (Cucumis sativus), pumpkin (Cucurbitappepo), Stevia (Stevia rebaudiana), tobacco (Nicotiana tabacum), rice (Oryza sativa), alfalfa (Medicago sativa), tomato (Solanum lycopersicum), and other plants. Preferably, plants are used which are capable of somatic embryogenesis in at least 60% of the cells. All or a portion of these plants, including but not limited to cells, embryos, leaves, stems, and other parts, can be used. Recombinant human AP-containing plants or parts thereof can be consumed in a dried form, both in their original as intact or ground (even to individual cells) and with or without different drying methods. The food product may comprise tissue-specific AP (e.g. gut, placenta) or tissue-non-specific AP or other human alkaline phosphatase.

The method of the present disclosure (variations thereof) includes the following.

A first variation of the method comprises the steps of: constructing a plant expression vector with human AP genes, introducing the plant expression vector with the human AP genes into an agrobacterium strain to generate plant callus cells, and performing agrobacterium transformation on the callus cells by using the agrobacterium strain. Food products are produced by culturing transformed plant callus cells containing the human AP gene in suspension culture.

A second variation of the method comprises the steps of: constructing a plant expression vector with human AP genes, introducing the plant expression vector with the human AP genes into an agrobacterium strain to generate plant callus cells, performing agrobacterium transformation on the callus cells by using the agrobacterium strain, and generating somatic embryos from the transformed callus cells. Food products are produced by culturing somatic embryos containing the human AP gene in suspension culture.

Plant cells containing recombinant human alkaline phosphatase may also be used for the production and growth of plants. The resulting plant biomass can be used as a feedstock for the production of food products based on plant cells containing recombinant human alkaline phosphatase.

Construction of a plant expression vector with the human AP Gene was performed as follows:

a nucleotide sequence of a human AP recombinant gene having a size of 1587 base pairs, which completely coincided with the nucleotide sequence of the natural mRNA of a human alkaline phosphatase gene (homo sapiens alkaline phosphatase, intestinal tract (ALPI), sequence ID: NM-001631.4), was synthesized. To facilitate cloning, the sequence of restriction site Bgl II (agact) was added to the sequence of the recombinant AP gene at the 5 'end before the translation initiation site (atg), and the sequence of restriction site Xba I (atctagaat) was added to the 3' -end after the stop codon (tga). The nucleotide sequence of the human AP recombinant gene having a size of 1602 base pairs with the sequences of the restriction sites Bgl II and Xba I was cloned into a plasmid pAL-T vector and named pAL-T-AP. The nucleotide sequence of the AP gene, 1600 base pairs in size, was removed from the restriction sites Bgl II and Xba I of the pAL-T-AP plasmid and ligated into the earlier constructed plasmid p35S-NLS-recA-licBM3[1], which was previously hydrolyzed at the restriction sites BamH I and Xba I, to produce the plasmid p35S-AP, in which the recombinant AP gene was under the control of the cauliflower mosaic virus 35S promoter [2 ]. The accuracy of the gene construction of the p35S-AP splice was verified by sequencing.

Introduction of a plant expression vector having a human AP gene into an Agrobacterium strain was performed as follows:

using the "triparental hybridization" method, the plant expression vector with human AP gene was introduced into Agrobacterium strains, using E.coli cells with p35S-AP plasmid as donor, E.coli cells of HB101 pRK2013 strain as promoter for conjugative transfer, and Agrobacterium tumefaciens of GV3101 strain or AGL0 strain as acceptor. This resulted in Agrobacterium strains GV3101 p35S-AP and AGL0p35S-AP containing the human AP gene.

The generation of plant callus cells and the transformation of callus cells by agrobacterium.

Plant seeds were sterilized under sterile conditions (laminar flow cabinet) in an aqueous solution of a chlorine-containing commercial disinfectant to which Tween-20 (1 drop per 100ml of solution) was added, and then washed 3 times for 10 minutes each in sterile distilled water. Embryos were then extracted from the sterilized seeds and transferred to vials containing modified Murashige and Skoog Medium (MSM) enriched with growth regulators 2, 4-dichlorophenoxyacetic acid (2,4-D) and kinetin. The vial was placed in a thermostat and incubated at 23 ℃ in the dark until callus formation occurred.

TABLE 1-composition of modified Murashige and Skoog media (Mutsuda et al, 1981)

Bacterial colonies isolated from fresh cultures of Agrobacterium strain GV3101 p35S-AP or AGL0p35S-AP carrying the human AP gene were placed in vials (using sterile inoculating loops) containing 3 sterile liquid medium LB carrying antibiotics corresponding to the bacterial strains in a laminar flow cabinet. The Agrobacterium is cultured for 20-48 hours at 28 ℃ on a circular rotary vibrator (amplitude 5-10 cm, speed 150-200 RPM) equipped with a thermostat.

Agrobacterium is cultured using standard media, e.g. containing per liter: 10 g of tryptone, 5 g of yeast extract, 5 g of sodium chloride and 15 g of bacto agar. The medium is autoclaved for 15-20 minutes under standard conditions. After cooling to 65 ℃, antibiotics were added: for strain AGL 0-each final concentration of 100mg/L kanamycin and rifampicin; for the GV3101 strain-kanamycin and rifampicin each at a final concentration of 100mg/L and gentamicin at a final concentration of 25 mg/L.

Plant callus cells were placed on sterile filter paper in petri dishes in a laminar flow cabinet and 10-25 mcl of overnight cultured AGL0 agrobacterium and GV3101 with alkaline phosphatase gene were applied to each graft. After Agrobacterium culture, the calli were dried slightly and transferred to a vial of MSM nutrient medium supplemented with 0.2mg/L2, 4-D. After 3 days, the calli were transferred to an agar medium of the same composition but supplemented with 500mg/L cefotaxime and 100mg/L kanamycin. Culturing is carried out in the dark at 22-24 ℃ for 10 days. Then, 1-2 transfers were performed on the medium with the same composition until new callus cell colonies appeared. To propagate them, callus cells were transferred to MSM nutrient medium containing 0.2mg/L2,4-D and 200mg/L cefotaxime. The callus may be stored indefinitely in culture, periodically broken into pieces and planted on fresh medium. Selection of transgenic carrot cells was performed by the ability of alkaline phosphatase to dephosphorylate p-nitrophenyl phosphate to yield yellow p-nitrophenol. To produce the desired amount of food product, callus cells with the human AP gene were cultured in suspension cultures in agar-free liquid nutrient medium of the same composition.

If the second variation of the food production process is used, somatic embryos are produced from transformed plant callus cells and then cultured in suspension culture.

Production of somatic embryos from transformed callus cells was performed as follows:

to produce somatic embryos, callus cell suspensions were cultured in liquid MSM nutrient medium containing 0.2mg/L IAA (indole-3-acetic acid) and kinetin. The resulting embryogenic suspensions comprise different pre-embryogenic structures as well as isolated non-embryogenic cells and cell populations. To obtain a uniform population of somatic embryos (to synchronize them), the suspension cultures were filtered through a nylon screen with a mesh size of 120mcm and then through a 50mcm nylon screen. The cell mass remaining on the second sieve is transferred to fresh medium to form an embryo. On average, up to 7 ten thousand embryos can be produced from 1L of medium.

Dry food based on plant callus cells or somatic embryos carrying the human AP gene was produced.

Washing the callus cells containing human intestinal AP gene from the rest nutrient medium with distilled water, freeze-drying at-55 deg.C, and drying at +30 deg.C to make protein not denaturalized. The activity of alkaline phosphatase in the dried cell pellet was determined by the ability of alkaline phosphatase to dephosphorylate p-nitrophenyl phosphate. The prepared plant cell mass containing the AP gene is packaged into capsules, tablets, sachets or other forms for use as a food ingredient.

In order to better illustrate the above embodiments, the development and optimization of food production technology will be described below.

Plant cells containing human AP gene, such as carrot cells, are freeze-dried at-55 deg.C and dried at 20 deg.C, 30 deg.C, 40 deg.C, 50 deg.C and 60 deg.C. The dried biomass (about 1g) was triturated with 10ml of a buffer solution containing 5mM Tris HCl, 0.1mM magnesium chloride, 0.1mM zinc chloride and centrifuged at 100g for 30 minutes. The dephosphorylation capacity of AP in the supernatant was examined by adding 20mM pNPP (p-nitrophenyl phosphate) to the solution. The reaction mixture was incubated at 37 ℃ for 30 minutes to color the mixture. The reaction was stopped with 2ml of cooled 0.5M NaOH. The amount of enzyme required to produce 1mcM pNP was taken as the unit of activity. Specific activity was calculated in units of per gram of carrot cells.

TABLE 2 Activity of AP in carrot cell Biomass in different drying regimes

Item	Temperature mode	Activity, U/g
			1	Drying at-55 deg.C and 20 deg.C	216
2	Drying at-55 deg.C and 30 deg.C	225
			3	Drying at-55 deg.C and 40 deg.C	200
4	Drying at-55 deg.C and 50 deg.C	150
			5	Drying at-55 deg.C and 60 deg.C	120

Table 2 demonstrates that the temperature profile of the final drying of plant cells affects the AP activity, with optimal final drying temperatures ranging from 20 ℃ to 40 ℃.

The effect of nutrient medium composition on callus formation was studied in carrot cultivars Nantskaya 4 and Moscow Winter A-555, which belong to two different cultivars. Zygotic embryos isolated from mature carrot seeds were used as explants. Embryos are cultured in three of the most widely used media: MS (Murashige, Skoog, 1964), MSM (Masuda et al, 1981) and B-5(Gamborg et al, 1976). To induce callus formation, 2,4-D was added to the medium and the results are listed in Table 3.

TABLE 3 callus formation in carrot zygotic embryo culture in different nutrient media

The table shows that carrots formed callus in all media studied, whereas the best medium for both carrots was MSM.

The effect of growth regulators on the ability of plant callus (e.g., carrot Nantskaya 4 cultivar) to undergo somatic embryogenesis was studied by suspension culture on MSM medium with different combinations of growth regulators such as auxin (2,4-D, IAA) and cytokinin (kinetin and Benzylaminopurine (BAP)). The Nantskaya 4 calli produced from zygotic embryos were used as grafts and the cell suspensions were cultured in 100ml vials at 80ml RPM on a shaker and the results of the study are listed in Table 4.

TABLE 4 average number of embryos obtained by suspension culture of Nantskaya 4 in media with different combinations of growth regulators

The table shows that embryo production can occur satisfactorily on medium without 2, 4-D.

To produce callus, different types of explants were used: main root tissue, stem and leaf fragments, petioles, cotyledons, hypocotyls and zygotic embryos of Nantskaya 4 carrot. Explants were cultured on MSM nutrient medium containing 0.2mg/L2,4-D, and then for induction of embryogenesis, the resulting callus was transferred to medium of the following composition: MSM with 0.2mg/L2,4-D and kinetin and MSM with 0.2mg/L IAA and kinetin. The cell suspension was cultured on a shaker at 80rpm in 100ml vials and the results of the study are listed in Table 5.

TABLE 5 number of embryos produced by calli of different explants of Nantskaya 4 carrot

Table 5 demonstrates that zygotic embryos are the best explants for embryogenic callus generation.

The possibility of producing and using plant food based on human AP is demonstrated by the following examples.

For ease of use, the produced callus cells or plant somatic embryos having the human AP gene may be dried by any suitable method.

The disclosed plant based food products containing human AP can be used in dosage forms for internal use, including but not limited to as capsules, tablets, sachets and other dosage forms.

The disclosed food products based on plants comprising human AP can be used as ingredients for food products for large-scale consumption, such as dairy products, beverages, confectionery, and medical and functional food products.

The disclosed products based on plant callus cells and somatic embryos comprising human AP are useful for health maintenance, including maintaining gastrointestinal (GI tract) homeostasis and preventing diseases associated with disruption of GI tract homeostasis. In particular, the product can be used for:

-modulating the gastrointestinal microflora, including maintaining a normal gastrointestinal microflora and preventing the growth of pathogenic microflora in the gastrointestinal tract;

-modulating the immune system of an organism, including maintaining normal immune system activity and restoring its viability during immune system disorders associated with various negative factors, including stress, environmental factors, use of drugs and other harmful substances;

-reducing toxic effects, including those associated with bacterial infection or inflammatory bowel disease;

prevention of gastrointestinal disorders associated with the use of different xenobiotics, including drugs.

Both healthy persons and persons suffering from various inflammatory and autoimmune diseases, including bowel diseases (ulcerative colitis, crohn's disease, enterocolitis), arthritis, eczema and other systemic diseases, diseases associated with increased cell wall permeability, can use plant based food products containing human AP. The food can also be used by people suffering from obesity and various cosmetic problems.

The production of food products by the disclosed method is illustrated by the following examples.

Example 1Production of carrot based food products containing human AP.

In order to introduce the human intestinal AP gene into a plant expression vector, a nucleotide sequence of a human AP recombinant gene having a size of 1587 base pairs which is completely identical to the nucleotide sequence of the natural mRNA of the human alkaline phosphatase gene was produced by synthesis. Then, a sequence (agatact) having a restriction site Bgl II was added to the sequence of the recombinant AP gene at the 5 'end before the translation initiation site (atg), and a sequence (atctagaat) having a restriction site Xba I was added after the stop codon (tga) at the 3' end. The resulting nucleotide sequence with 1602 base pairs was cloned into the plasmid pAL-T vector. A nucleotide sequence of 1600 base pairs in size was excised from the restriction sites Bgl II and Xba I of the resulting pAL-T-AP plasmid and ligated into the earlier generated plasmid p35S-NLS-recA-licBM3 to generate plasmid p35S-AP, in which the recombinant AP gene was controlled by the cauliflower mosaic virus 35S promoter. The accuracy of the gene construction of the p35S-AP splice was verified by sequencing.

Agrobacterium strain AGL0p35S-AP having a human AP gene was produced using E.coli cells harboring a p35S-AP plasmid as a donor, E.coli cells of HB101 pRK2013 strain as a promoter of conjugative transfer, and Agrobacterium tumefaciens cells of AGL0 strain as an acceptor.

Callus cells from carrot seed zygotic embryos were used as the target for agrobacterium transformation and were produced as follows: carrot seeds were sterilized by adding 1 drop of Tween-20 in 50% aqueous solution of a commercial disinfectant containing chlorine per 100ml of the solution, and then washed 3 times for 10 minutes each in sterile distilled water. Embryos are then extracted from sterile seeds under sterile conditions and then transferred to vials with MSM nutrient medium, which is rich in growth regulator 2,4-D and kinetin. The vial was placed in a thermostat and incubated at 23 ℃ in the dark until callus formation occurred.

The production process of agrobacterium strain AGL0 is as follows: in a laminar flow cabinet, bacterial colonies isolated from fresh bacterial cultures with selective antibiotics are placed in vials (using sterile inoculating loops) containing 3 sterile liquid media LB of the above composition. Agrobacterium is grown for 24-48 hours at 28 ℃ on a rotating shaker (amplitude 5-10 cm, rate 150-200 RPM) equipped with a thermostat.

Transformation and growth of carrot callus cells were performed according to the following procedure: carrot callus cells from zygotic embryos were placed on sterile filter paper in petri dishes in a laminar flow cabinet and 10-25 mcl of agrl 0 agrobacterium with alkaline phosphatase gene cultured overnight was used for each graft. Thereafter, the callus was dried slightly and transferred to a vial with 0.2-0.5 mg/L2,4-D MSM nutrient medium added. After 3 days, the calli were transplanted on an agar medium having the same composition but supplemented with 500mg/L cefotaxime. The callus was cultured in the dark at 22-24 ℃ for 10 days. Then 1-2 additional transfers were performed on medium with the same composition until a new callus cell population appeared. To propagate them, callus cells were transferred onto MSM nutrient medium with 0.2-0.5 mg/L2,4-D and 200mg/L cefotaxime. Transgenic carrot cells were selected for their ability to dephosphorylate p-nitrophenyl phosphate with alkaline phosphatase, resulting in dephosphorylation of yellow p-nitrophenol.

The carrot cells containing the human AP gene were then washed with distilled water from the remaining nutrient medium and freeze-dried at-55 ℃. Determining the activity of alkaline phosphatase in the dried cell pellet by the ability of alkaline phosphatase to dephosphorylate p-nitrophenyl phosphate; the dried pieces were placed in a small bag.

The obtained food is used for maintaining normal gastrointestinal microflora and normal state of immune system by adding it to dairy products or beverages (0.5-1 sachet per person per day).