CN110204620B

CN110204620B - Application of plant as host in expression of MGLP fusion protein

Info

Publication number: CN110204620B
Application number: CN201910549759.8A
Authority: CN
Inventors: 王跃驹
Original assignee: Individual
Current assignee: Ruicheng Haihui Biotechnology (Shandong) Co.,Ltd.
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2022-12-20
Anticipated expiration: 2039-06-24
Also published as: CN110204620A

Abstract

The invention relates to the field of biotechnology, in particular to application of a plant as a host in expression of MGLP fusion protein. The present invention utilizes plants such as lettuce as an efficient expression platform for recombinant protein production, and utilizes a simple and efficient Agrobacterium-mediated vacuum infiltration method to express MGLP fusion proteins. The expression system ensures that the plant foreign protein can be collected after 4 days of agrobacterium infection. And determining the successful expression of the MGLP fusion protein by using a Western Blot protein hybridization method, and successfully purifying the MGLP fusion protein by using an AKTA protein purification system. The results of biological activity tests show that the MGLP fusion protein produced by the platform technology obviously reduces the blood sugar concentration of dog blood.

Description

Application of plant as host in expression of MGLP fusion protein

Technical Field

The invention relates to the technical field of biology, in particular to application of a plant as a host in expression of MGLP fusion protein.

Background

Diabetes is a common disease or frequently encountered disease characterized by chronic hyperglycemia, and is a disorder of metabolism of sugar, fat and protein caused by defective secretion or action of insulin in vivo or by the presence of both of them. Clinically, there are two major types, insulin-dependent (IDDM, type I) and non-insulin-dependent (NIDDM, type II). With the increase in living standard, the incidence of diabetes has increased year by year both in developed and developing countries. Diabetes, a serious non-infectious chronic disease, has now become one of the major public health problems of great concern in countries around the world, and is number three killer following cardiovascular and neoplastic diseases worldwide. From the data published by the world health organization, there are only about 3000 million diabetics worldwide in 1995, and have increased to 1.35 billion in 1997, and 3 billion type II diabetics in 2030, with asia and africa being the areas where patients are most rapidly amplified. The traditional treatment modalities for type II diabetics generally follow a stepwise treatment with dietary controls, oral antidiabetic drugs and exogenous insulin. However, there are still many important problems to be solved in the field of diabetes treatment, and there are some side effects and limitations.

Glucagon-like peptide-1 (glp-1) is an incretin secreted by endocrine cells in the intestinal tract, is a post-translational product of a Glucagon gene, and exists in various forms in the body. It has the following physiological effects: acts on islet beta cells in a glucose-dependent manner, promotes the transcription of insulin genes, and increases the biosynthesis and secretion of insulin; stimulating the proliferation and differentiation of beta cells, inhibiting beta cell apoptosis, increasing the number of pancreatic islet beta cells, inhibiting glucagon secretion, inhibiting appetite and ingestion, delaying gastric content emptying, etc. These functions are all beneficial in lowering postprandial blood glucose and maintaining blood glucose at a constant level.

Although natural GLP-1 has a plurality of advantages in treating diabetes, the half-life period in vivo is only about 2 minutes, which limits the direct application of the natural GLP-1 in clinic.

Disclosure of Invention

In view of this, the present invention provides the use of plants as hosts for the expression of MGLP fusion proteins. The invention uses plant, especially lettuce, as the high efficiency platform technology of recombinant protein production to express MGLP fusion protein. And active foreign proteins are successfully separated under mild conditions, which proves that plants, particularly lettuce expression platforms can be successfully used for producing MGLP fusion protein. Short time (4 d), simple purification and convenient production. Eliminating gene pollution, eliminating potential diseases and pests infecting human body, etc. Greatly reduces the production cost and improves the product safety.

In order to achieve the above object, the present invention provides the following technical solutions:

the present invention provides MGLP fusion proteins having:

(I) an amino acid sequence shown as SEQ ID No. 1; or

(II) an amino acid sequence obtained by substituting, deleting or adding one or more amino acids in the amino acid sequence shown in the (I), and the amino acid sequence has the same or similar functions with the amino acid sequence shown in the (I); or

(III) and an amino acid sequence having at least 80% homology with the sequence of (I) or (II).

Based on the above studies, the present invention provides a nucleotide encoding the MGLP fusion protein, having

(I) a nucleotide sequence shown as SEQ ID No. 2; or

(II) a complementary nucleotide sequence of the nucleotide sequence shown as SEQ ID 2; or

(III) a nucleotide sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or

(IV) a nucleotide sequence obtained by substituting, deleting or adding one or more nucleotide sequences with the nucleotide sequence shown in the (I), (II) or (III), and the nucleotide sequence has the same or similar functions with the nucleotide sequence shown in the (I), (II) or (III); or

(V) a nucleotide sequence having at least 80% homology with the nucleotide sequence of (I), (II), (III) or (IV).

In addition, the invention also provides an expression vector, which comprises the nucleotide and a vector to be transformed.

In some embodiments of the invention, the vector to be transformed is a chloroplast expression vector.

The invention also provides a construction method of the expression vector, which comprises the following steps:

step 1: respectively optimizing codons of the MGLP fusion proteins into codons preferred by plants to obtain sequences of the optimized MGLP fusion proteins; the nucleotide sequence is shown as SEQ ID No. 2;

step 2: adding an Xbal restriction site at the 5 'end and a Sac I site at the 3' end of the optimized MGLP fusion protein sequence;

cloning into a pUC57 vector to obtain a pMGLP cloning vector;

and 3, step 3: obtaining a gene fragment from the cloning vector obtained in the step 2 through Xbal/Sacl, cloning to a binary plant vector pCam35S, and obtaining an expression vector p35S-MGLP.

The plant transient expression technology is a technology that when a plant grows to a certain stage, a plasmid containing target protein is transferred into plant cells by utilizing a plurality of different technical modes, and a high-efficiency controllable expression system is established in the plant cells to obtain the transient controllable expression of the gene. Compared with stable expression, the transient expression requires short time, does not need to integrate exogenous genes into host plant chromosomes, and can obtain experimental results only in a few days. Compared with a bacterial expression system, the plant expression system can correctly fold, process and modify the expressed protein, and the activity of the produced protein is higher than that of the bacterial expression system; plant expression systems are very low cost, only one to two thousandths of their cost, compared to animal cell expression systems.

The invention also provides the application of the expression vector or the plant in expressing the MGLP fusion protein or preparing a medicament containing the MGLP fusion protein; the plant is selected from lettuce, spinach, tomato, radish, chinese cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.

In some embodiments of the invention, the medicament is an oral hypoglycemic formulation.

The present invention also provides a host, a plant or microorganism transformed with the expression vector; the plant is selected from lettuce, spinach, tomato, radish, chinese cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.

The invention also provides a medicament which comprises the fusion protein and pharmaceutically acceptable auxiliary materials.

The invention also provides a method for expressing the MGLP fusion protein by taking the plant as a host, which comprises the steps of transforming the expression vector into agrobacterium, penetrating the agrobacterium into plant tissues through agrobacterium-mediated vacuum, extracting and separating protein to obtain the MGLP fusion protein.

In some embodiments of the invention, the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant.

In some embodiments of the invention, the agrobacterium-mediated vacuum infiltration comprises the steps of:

step 1: vacuumizing for 25-45 s;

step 2: keeping the vacuum (-95 kPa) pressure for 30-60 s;

and 3, step 3: releasing the pressure such that the permeate permeates the plant tissue;

repeating the steps for 2 to 3 times, and carrying out light-proof treatment for 4d.

Certain amino acids in natural GLP-1 are mutated to prolong the half-life period of the natural GLP-1 under the condition of ensuring the activity of the natural GLP-1, so that the normal blood sugar level can be maintained by once weekly administration. The related products on the market at present include Liraglutide, dulaglutide, semaglutide and the like. The invention fuses and expresses transferrin and GLP-1, can realize long-acting blood sugar reduction, and relieves the pain of patients caused by long-term frequent injection.

The present invention utilizes plants such as lettuce as an efficient expression platform for recombinant protein production, and utilizes a simple and efficient Agrobacterium-mediated vacuum infiltration method to express MGLP fusion proteins. The expression system ensures that the plant exogenous protein can be collected after the agrobacterium is infected for 4 days. And determining the successful expression of the MGLP fusion protein by using a Western Blot protein hybridization method, and successfully purifying the MGLP fusion protein by using an AKTA protein purification system. The results of biological activity tests show that the MGLP fusion protein produced by the platform technology obviously reduces the blood sugar concentration of dog blood.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows a schematic diagram of the cloning vector pMGLP;

FIG. 2 shows the construction of the MGLP fusion protein plant binary expression vector p35S-MGLP; cutting off MGLP fusion protein from the cloning vector in the figure 1 by utilizing restriction enzyme (Xbal/SacI) double digestion, and connecting the MGLP fusion protein into Xbal/SacI sites of pCam35S to generate a plant binary expression vector p35S-MGLP;

LB and RB are the left and right borders of the Ti plasmid; 35S, caMV 35S promoter having Tobacco Mosaic Virus (TMV) 5' UTR; NPT II, the expression of the nptII gene encoding for kanamycin resistance; nos 3', terminator;

FIG. 3 shows the result of SDS-PAGE gel electrophoresis; lane 1: non-infested lettuce; lane 2: lettuce expressed MGLP fusion protein; lane 3: MGLP fusion protein is a commercial product.

Detailed Description

The invention discloses application of a plant as a host in expression of MGLP fusion protein, and a person skilled in the art can use the content for reference and appropriately improve process parameters to realize the MGLP fusion protein. It is specifically noted that all such substitutions and modifications will be apparent to those skilled in the art and are intended to be included herein. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.

the invention provides the use of plants as hosts for the expression of MGLP fusion proteins. Preferably, the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from the group consisting of seed, leaf, rhizome, or whole plant. The invention also provides an expression vector, which comprises the sequence of the MGLP fusion protein and a vector.

In some embodiments of the invention, the sequence of the MGLP fusion protein is an optimized MGLP fusion protein sequence obtained by optimizing codons of the MGLP fusion protein to plant-preferred codons.

In some embodiments of the invention, the optimized MGLP fusion protein has the nucleotide sequence shown in SEQ ID No. 1; the amino acid sequence of the optimized MGLP fusion protein is shown as SEQID No. 2.

In some embodiments of the invention, the vector is a binary plant vector.

In some embodiments of the present invention, the method for constructing the expression vector comprises the following steps:

step 1: optimizing codons of the MGLP fusion protein into codons preferred by plants to obtain a sequence of the optimized MGLP fusion protein;

cloning Kinseri into pUC57 vector to obtain pMGLP cloning vector;

Specifically, in order to provide efficient expression of foreign proteins in plants, the human MGLP fusion protein amino acid sequence is subjected to nucleotide sequence obtaining by utilizing reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss _ backstrans eq /), and codon optimization is optimized to plant-preferred codon, and the codon is synthesized by Kinsruit corporation (Nanjing, china). Xbal restriction sites were added at the 5 'end and Sacl sites at the 3' end of the optimized MGLP fusion protein sequence. And cloned from Kinseri into pUC57 vector to obtain pMGLP cloning vector (FIG. 1). The gene fragment was isolated from the cloning vector by XbaI/Sacl and cloned into the binary plant vector pCam35S, resulting in the plant expression vector p35S-MGLP (FIG. 2).

The invention also provides application of the expression vector in expression of MGLP fusion protein.

In addition, the invention also provides a method for expressing the MGLP fusion protein by taking the plant as a host, which comprises the steps of transforming the expression vector provided by the invention into agrobacterium, extracting and separating protein after the agrobacterium-mediated vacuum infiltration into plant tissues, and obtaining the MGLP fusion protein.

Specifically, the plant expression vector p35S-MGLP was transformed into Agrobacterium tumefaciens GV3101 by electroporation with a multipolator (Eppendorf, hamburg, germany). The resulting strains were spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28 ℃ for 2 days, a single colony was picked and inoculated into 0.5L YEB (yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L MgSO ₄ pH7.2) and supplemented with antibiotic liquid medium (50 mg/L kanamycin). The inoculated culture was incubated at 25-28 ℃ for 72h in a shaker (220 rpm). OD600 values were measured by addition of YEB medium and adjusted to 3.5-4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10min. The Agrobacterium cells were resuspended in permeation medium (10mM MES,10mM MgSO4) to an O.D.600 of 0.5.

Uniformly mixing the prepared p 35S-MGLP-containing agrobacterium tumefaciens in equal quantity until the O.D.600 is 0.5; the culture suspension was placed in a 2L beaker and placed in a desiccator. Lettuce stored in this laboratory was inverted (core up) and gently swirled into the bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, niles, IL, USA) was turned on to evacuate and the permeate was visible in the leaf tissue. Keeping the pressure state for 30-60 s. The system is rapidly opened to release the pressure and allow the permeate to permeate the spaces within the tissue. The process is repeated for 2-3 times until the clear visible penetrating fluid is obviously diffused in the tissue of the lettuce. The lettuce tissues were then gently removed from the permeate and rinsed three times in succession with distilled water before being transferred to a plastic film covered container. The treated sample was kept in the dark for 4d.

step 1: vacuumizing for 25-45 s;

step 2: keeping the vacuum (-95 kPa) pressure for 30-60 s;

and step 3: releasing the pressure such that the permeate permeates the plant tissue;

In some embodiments of the invention, the agrobacterium is specifically agrobacterium tumefaciens GV3101.

The pMGLP gene fragment is cloned and a binary plant expression vector p35S-MGLP (figure 2) is constructed, and after the construction is completed, the gene fragment is confirmed to be complete by digestion with specific restriction enzymes. After infiltration, most lettuce tissues were submerged during vacuum infiltration, except for the firm mid-rib areas, which all showed a light tan area 4 days after vacuum infiltration.

The specific steps of extracting and separating the protein are as follows: the lettuce samples subjected to vacuum infiltration by the agrobacterium are stirred by a stirrer and are homogenized for 1 to 2min at high speed in an extraction buffer (100mM KPi, pH7.8, 5mM EDTA, 10mM beta-mercaptoethanol) stirrer with the volume ratio of 1. The homogenate was adjusted to pH8.0, filtered through gauze, and the filtrate was centrifuged at 10,000g for 15min at 4 ℃ to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated on ice for 60min with shaking. The separation was again carried out by means of a centrifuge (10,000g) at 4 ℃ for 15min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended with shaking on ice for 60min, and centrifuged again at 10,000g for 15min at 4 ℃. Then, the supernatant was discarded, and the treated sample precipitated protein was dissolved in 5mL of a buffer (20mM KPi, 20mM EDTA, 10mBeta-mercaptoethanol) and stored at 4 ℃.

The SDS-PAGE gel electrophoresis specifically comprises the following steps: the purified protein from the Agrobacterium vacuum osmosed lettuce was collected and a sample (5. Mu.L) was heat denatured (95 ℃) loading buffer (Biorad, hercules, calif., USA) at 4-12%

Bis-Tris Plus SDS-denaturing gels (ThermoFisher Scientific, waltham, MA, USA) were run. Similarly, the degree of affinity of the antibody was examined in non-denaturing gel electrophoresis. The gel was then photographed again after staining with coomassie blue G250 (Biorad).

Downstream processing of recombinant proteins of plant origin is often difficult and expensive because of the difficulty of lysis of the cellulose cell wall and secondary plant metabolites. The homogenizer is used for stirring and homogenizing, so that the homogenizing cost and the homogenizing process are greatly saved. Recombinant MGLP fusion protein separation by denaturing gel SDS-PAGE A band with an estimated molecular weight of approximately 92kDa (FIG. 3) was observed in the lanes, corresponding to the MGLP fusion protein molecular weight. The protein content of the purified sample was determined to be approximately 1.78mg/g based on the Bradford assay and densitometry controls.

The invention uses lettuce to express MGLP fusion protein instantly, and can produce high content protein in a short time (4 d). Lettuce is a higher plant and can undergo post-translational modification processes, i.e., the expressed protein is automatically active. Moreover, this approach minimizes bio-safety issues, as the treated lettuce tissue is typically developed in a completely enclosed facility or container, without bio-contamination issues. The lettuce does not contain plant toxic substances, has low protein content, and is beneficial to downstream protein purification. The MGLP fusion protein is produced by utilizing a lettuce system, so that the production period and the production cost can be greatly shortened.

Experiments show that a plant system, particularly a lettuce system is a more economic and efficient expression platform and is a method for quickly and instantaneously expressing recombinant protein. The vacuum agrobacterium infiltration method described in the invention is simple and rapid, and can improve the yield of recombinant protein. Lettuce can increase protein production by withstanding vacuum pressure and allow for more complete penetration of each leaf. Lettuce is easier to grow and commercially mass-produced, and therefore is more readily available and less expensive than other transiently expressed plants, such as tobacco, and the cost can be significantly reduced since complicated special production facilities are not required. In conclusion, the present invention can produce MGLP fusion protein in a large scale in a short time using lettuce system.

The raw materials and reagents used in the application of the plant provided by the invention as a host in expressing the MGLP fusion protein are all commercially available.

The invention is further illustrated by the following examples:

example 1 construction of plant transient expression vectors

To achieve high efficiency of expression of foreign proteins in plantsTo achieve, the MGLP fusion protein amino acid sequence was synthesized by Kinshire (Nanjing, china) using the reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss _ backstrans eq /) to obtain the nucleotide sequence and optimizing the codon to the codon preferred by the plant. Xbal restriction sites were added at the 5 'end and Sacl sites at the 3' end of the optimized MGLP fusion protein sequence. And cloned into pUC57 vector by Kinry to obtain pMGLP cloning vector (FIG. 1), the gene fragment was isolated from the cloning vector by Xbal/Sacl and cloned into binary plant vector, pCam35S, to generate plant expression vector p35S-MGLP (FIG. 2). The plant expression vector was transformed into Agrobacterium tumefaciens GV3101 by electroporation with a multipolator (Eppendorf, hamburg, germany). The resulting strain was spread evenly on selective LB plates containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28 ℃ for 2 days, a single colony was picked and inoculated into 0.5L YEB (yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L yeast extract, 0.24g/L MgSO4, pH 7.2) and supplemented with antibiotic liquid medium (50 mg/L kanamycin). The inoculated culture was incubated for 72h at 25-28 ℃ in a shaker (220 rpm). OD600 values were measured by addition of YEB medium and adjusted to 3.5-4.5. The culture broth was then collected and centrifuged (4500 rpm) for 10min. Agrobacterium cells were resuspended in permeation medium (10mM MES,10mM MgSO ₄ ) The neutral to o.d.600 is 0.5.

Example 2 Agrobacterium-mediated vacuum infiltration

The prepared p 35S-MGLP-containing agrobacterium is uniformly mixed in equal quantity until the O.D.600 is 0.5. The culture suspension was placed in a 2L beaker and placed in a desiccator. Lettuce stored in this laboratory was inverted (core up) and gently swirled into the bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, niles, IL, USA) was turned on to evacuate and permeate was visible in the leaf tissue. Keeping the pressure state for 30-60 seconds. The system is rapidly opened to release pressure and allow the permeate to penetrate into the space within the tissue. This process was repeated 2 to 3 times until the clear visible permeate diffused significantly in the lettuce tissue. The lettuce tissues were then gently removed from the permeate and rinsed three times in succession with distilled water before being transferred to a plastic film covered container. The treated samples were kept in the dark for 4 days.

Example 3 protein extraction and isolation

The lettuce samples infiltrated by the agrobacterium under vacuum were stirred with a stirrer and homogenized for 1-2 minutes at high speed in an extraction buffer (100mM kpi, ph7.8, 5mm edta, 10m M β -mercaptoethanol) mixer at a volume ratio of 1. The homogenate was adjusted to pH8.0, filtered through gauze, and the filtrate was centrifuged at 10,000g for 15 minutes at 4 ℃ to remove cell debris. The supernatant was collected, mixed with ammonium sulfate (50%) and incubated on ice for 60 minutes with shaking. The separation was again carried out by means of a centrifuge (10,000g) at 4 ℃ for 15 minutes. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended with shaking on ice for 60 minutes, and centrifuged again at 10,000g for 15 minutes at 4 ℃. Then, the supernatant was discarded, and the treated sample precipitated protein was dissolved in 5mL of a buffer (20mM KPi, 2mM EDTA, 10mM. Beta. -mercaptoethanol) and stored at 4 ℃.

Downstream processing of recombinant proteins of plant origin is often difficult and expensive because of the difficulty of lysis of the cellulose cell wall and secondary plant metabolites. The homogenizer is used for stirring and homogenizing, so that the homogenizing cost and the homogenizing process are greatly saved.

Example 4 SDS-PAGE gel electrophoresis

The purified protein from the Agrobacterium vacuum osmosed lettuce was collected and a sample (5. Mu.L) was heat denatured (95 ℃) loading buffer (Biorad, hercules, calif., USA) at 4-12%

Bis-TrisPlus SDS-denaturing gels (ThermoFisher Scientific, waltham, MA, USA) were run. Similarly, the degree of affinity of the antibody was examined in non-denaturing gel electrophoresis. The gel was then photographed again after staining with coomassie blue G250 (Biorad). Recombinant MGLP fusion protein separation by denaturing gel SDS-PAGE A band with an estimated molecular weight of approximately 92kDa (FIG. 3) was observed in the lanes, corresponding to the MGLP fusion protein molecular weight. The protein content of the purified sample was determined to be approximately 1.81mg/g based on the Bradford assay and densitometry controls.

Example 5 detection of protein Activity of MGLP fusion proteins

After a stabilization period lasting seven weeks, dogs were randomized into two treatment groups of 3 dogs, and received one of two experimental capsules containing a hypoglycaemic protein (MGLP fusion protein prepared in example 4) and no hypoglycaemic protein, for the first repetition. The dogs were randomized again and received a different experimental diet for a second repeat. Repeats I and II were continued for at least 2 weeks and the glycemic response was measured after the end of each repeat.

Dogs fasted 24 hours before blood glucose testing began. The catheterized site was shaved, aseptically treated, and catheterized into the right cephalic vein. Two baseline samples were taken approximately 5 minutes apart. Immediately after the last baseline sample was taken, the dogs were fed a diet equivalent to 1% of their body weight and contained 1 or 3 hypoglycemic capsules, which were allowed to eat for up to 15 minutes. If the dog did not eat the experimental diet within 15 minutes, his glycemic response was not detected the same day and was re-detected the following day. Additional blood samples were collected at 10, 20, 30, 45, 60, 120, 180 and 240 minutes after the meal. Blood samples were centrifuged at 1300 Xg for 15 minutes and two aliquots of 1ml plasma at each time point were cryopreserved within two hours after collection. Plasma glucose concentration (mg/dl) was determined using the hexokinase method.

TABLE 1 test results for sugar concentration in dog blood

Note: ^* shows significant difference (P is less than 0.05) compared with a control group without reducing the sugar; ^** shows very significant difference (P is less than 0.01) compared with a control group without sugar reduction.

The invention uses lettuce to express antibody instantaneously, and can produce high content protein in shorter time (4 d). Lettuce is a higher plant and can undergo a post-translational modification process, i.e., the expressed protein is automatically active. Moreover, this approach minimizes biosafety issues, as the treated lettuce tissue is typically developed in a completely enclosed facility or container, without the problem of biological contamination. The lettuce does not contain plant toxic substances, has low protein content, and is beneficial to downstream protein purification. The production by using the lettuce system can greatly shorten the production period and the production cost

Example 6 animal toxicity test

Experimental mice of 7 weeks size were randomly divided into three treatment groups of 10 mice each receiving a diet containing the hypoglycemic protein (500 ng/g fed by body weight) (MGLP fusion protein obtained in accordance with the present invention), commercially available somaglutid (500 ng/g fed by body weight) as a positive control, and one of two experimental capsules without the hypoglycemic protein, receiving the same experimental diet. Continuously feeding for 10 days, observing after each feeding, continuously observing for more than 6 hours every day, and not seeing whether the mouse is in an excited state or a suppressed state, and not appearing phenomena such as bradykinesia and diarrhea. Proves that the oral administration safety of the fusion protein capsule of the somaglutide and the lumbrokinase is high.

Example 7

Control group: producing MGLP fusion protein by using animal cells;

experimental group 1: the plant provided by the invention produces MGLP fusion protein;

experimental group 2: utilizing tobacco leaves to produce MGLP fusion protein;

TABLE 2 MGLP fusion proteins

^* Shows that P is less than or equal to 0.05 compared with the control group; ^** shows that P is less than or equal to 0.01 compared with the control group;

^# shows that P is less than or equal to 0.05 compared with the experimental group 2; ^## shows that P is less than or equal to 0.01 compared with the experimental group 2;

as can be seen from Table 2, compared with animal systems of a control group, in the experimental group 1, the MGLP fusion protein expressed instantly by lettuce provided by the invention has the advantages that the production period is remarkably shortened (P is less than or equal to 0.01), the protein content is remarkably increased (P is less than or equal to 0.01), the difficulty of protein purification is simplified, and the production cost is remarkably reduced (P is less than or equal to 0.01).

Compared with the tobacco leaf system of the experimental group 1 and the experimental group 2, the lettuce instantly expresses the MGLP fusion protein, obviously (P is less than or equal to 0.05) shortens the production period, obviously (P is less than or equal to 0.05) improves the protein content, simplifies the difficulty of protein purification, and greatly (P is less than or equal to 0.01) reduces the production cost.

Compared with a control group, the tobacco leaf transient expression MGLP fusion protein in the experimental group 2 has the advantages that the production period is shortened, the difficulty of protein purification is simplified, and the production cost is remarkably reduced (P is less than or equal to 0.05) compared with an animal system. The combination of the test results shows that the plant system, especially the lettuce system, is a more economic and efficient expression platform. Can express recombinant protein rapidly and transiently, and can produce MGLP fusion protein in large scale in short time.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

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agggatcagt acgagcttct gtgccttgac aacaccagaa agcctgtgga tgagtacaag 1200

gattgccacc ttgctcaggt gccatctcat actgtggtgg ctagatccat gggtggcaaa 1260

gaggatctta tctgggagct tctgaaccag gctcaagagc acttcggcaa ggacaagtct 1320

aaagagttcc agctgttcag cagccctcac ggtaaggatc tgctgttcaa ggattctgct 1380

cacggcttcc ttaaggtgcc acctagaatg gacgctaaga tgtacctggg ctacgagtac 1440

gttaccgcca ttaggaatct tagagagggg acttgtccag aggctcctac tgatgaatgc 1500

aagccagtta agtggtgtgc cttgtctcat cacgagaggc tgaagtgtga tgagtggtct 1560

gtgaacagcg tgggcaagat tgagtgtgtg tctgctgaaa ctaccgagga ctgcattgcc 1620

aagatcatga acggtgaggc tgacgctatg tctctggatg gtggattcgt gtacattgct 1680

ggtaagtgcg gtcttgtgcc tgtgcttgct gagaactacg agaagtctga taactgcgag 1740

gatacccctg aggctggtta ctttgctgtt gcagtggtga agaagtccgc ttctgatctg 1800

acctgggata acctgaaggg caagaagtca tgtcacaccg ctgttggtag aactgctggc 1860

tggaatatcc caatgggcct cctgtacaac aagatcaacc actgcaggtt cgacgagttc 1920

ttctcagaag gttgcgctcc tggcagcaag aaagatagct ctttgtgcaa gctgtgcatg 1980

ggctctggtc ttaatctttg cgagccgaac aacaaagagg gctactacgg ttacactggg 2040

gcttttagat gcctggtcga gaagggtgat gtcgcctttg ttaagcacca gactgtgcct 2100

cagaataccg gtggtaagaa tcctgatcct tgggccaaga acctgaacga gaaggattac 2160

gagctgcttt gcctggatgg tactcgtaag ccagttgaag agtacgctaa ctgccatctt 2220

gctagggctc ctaatcatgc tgtggtgacc agaaaggaca aagaggcttg cgtccacaag 2280

attcttaggc agcagcagca cctgttcggt tctgaagtta ctgattgcag cggcaacttc 2340

tgcctgttca ggtctgagac taaggacctg cttttcaggg acgataccgt gtgcttggct 2400

aagcttcatg accggaacac ctatgagaag tacctcggtg aggaatacgt gaaggctgtt 2460

ggcaatctga ggaagtgcag cacctcttca cttttggagg cttgcacttt ccgtcggcct 2520

tga 2523

<210> 2

<211> 840

<212> PRT

<213> MGLP fusion protein (MGLP fusion protein)

<400> 2

Met Gly Lys Gln Met Ala Ala Leu Cys Gly Phe Leu Leu Val Ala Leu

1 5 10 15

Leu Trp Leu Thr Pro Asp Val Ala His Ala His Gly Glu Gly Thr Phe

20 25 30

Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe

35 40 45

Ile Ala Trp Leu Val Asp Gly Arg His Gly Glu Gly Thr Phe Thr Ser

50 55 60

Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala

65 70 75 80

Trp Leu Val Asp Gly Arg His Gly Glu Gly Thr Phe Thr Ser Asp Val

85 90 95

Ser Ser Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu

100 105 110

Val Asp Gly Arg His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser

115 120 125

Tyr Leu Glu Gly Gln Ala Ala Gln Glu Phe Ile Ala Trp Leu Val Asp

130 135 140

Gly Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly

145 150 155 160

Ser Val Pro Asp Lys Thr Val Arg Trp Cys Ala Val Ser Glu His Glu

165 170 175

Ala Thr Lys Cys Gln Ser Phe Arg Asp His Met Lys Ser Val Ile Pro

180 185 190

Ser Asp Gly Pro Ser Val Ala Cys Val Lys Lys Ala Ser Tyr Leu Asp

195 200 205

Cys Ile Arg Ala Ile Ala Ala Asn Glu Ala Asp Ala Val Thr Leu Asp

210 215 220

Ala Gly Leu Val Tyr Asp Ala Tyr Leu Ala Pro Asn Asn Leu Lys Pro

225 230 235 240

Val Val Ala Glu Phe Tyr Gly Ser Lys Glu Asp Pro Gln Thr Phe Tyr

245 250 255

Tyr Ala Val Ala Val Val Lys Lys Asp Ser Gly Phe Gln Met Asn Gln

260 265 270

Leu Arg Gly Lys Lys Ser Cys His Thr Gly Leu Gly Arg Ser Ala Gly

275 280 285

Trp Asn Ile Pro Ile Gly Leu Leu Tyr Cys Asp Leu Pro Glu Pro Arg

290 295 300

Lys Pro Leu Glu Lys Ala Val Ala Asn Phe Phe Ser Gly Ser Cys Ala

305 310 315 320

Pro Cys Ala Asp Gly Thr Asp Phe Pro Gln Leu Cys Gln Leu Cys Pro

325 330 335

Gly Cys Gly Cys Ser Thr Leu Asn Gln Tyr Phe Gly Tyr Ser Gly Ala

340 345 350

Phe Lys Cys Leu Lys Asp Gly Ala Gly Asp Val Ala Phe Val Lys His

355 360 365

Ser Thr Ile Phe Glu Asn Leu Ala Asn Lys Ala Asp Arg Asp Gln Tyr

370 375 380

Glu Leu Leu Cys Leu Asp Asn Thr Arg Lys Pro Val Asp Glu Tyr Lys

385 390 395 400

Asp Cys His Leu Ala Gln Val Pro Ser His Thr Val Val Ala Arg Ser

405 410 415

Met Gly Gly Lys Glu Asp Leu Ile Trp Glu Leu Leu Asn Gln Ala Gln

420 425 430

Glu His Phe Gly Lys Asp Lys Ser Lys Glu Phe Gln Leu Phe Ser Ser

435 440 445

Pro His Gly Lys Asp Leu Leu Phe Lys Asp Ser Ala His Gly Phe Leu

450 455 460

Lys Val Pro Pro Arg Met Asp Ala Lys Met Tyr Leu Gly Tyr Glu Tyr

465 470 475 480

Val Thr Ala Ile Arg Asn Leu Arg Glu Gly Thr Cys Pro Glu Ala Pro

485 490 495

Thr Asp Glu Cys Lys Pro Val Lys Trp Cys Ala Leu Ser His His Glu

500 505 510

Arg Leu Lys Cys Asp Glu Trp Ser Val Asn Ser Val Gly Lys Ile Glu

515 520 525

Cys Val Ser Ala Glu Thr Thr Glu Asp Cys Ile Ala Lys Ile Met Asn

530 535 540

Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Phe Val Tyr Ile Ala

545 550 555 560

Gly Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn Tyr Glu Lys Ser

565 570 575

Asp Asn Cys Glu Asp Thr Pro Glu Ala Gly Tyr Phe Ala Val Ala Val

580 585 590

Val Lys Lys Ser Ala Ser Asp Leu Thr Trp Asp Asn Leu Lys Gly Lys

595 600 605

Lys Ser Cys His Thr Ala Val Gly Arg Thr Ala Gly Trp Asn Ile Pro

610 615 620

Met Gly Leu Leu Tyr Asn Lys Ile Asn His Cys Arg Phe Asp Glu Phe

625 630 635 640

Phe Ser Glu Gly Cys Ala Pro Gly Ser Lys Lys Asp Ser Ser Leu Cys

645 650 655

Lys Leu Cys Met Gly Ser Gly Leu Asn Leu Cys Glu Pro Asn Asn Lys

660 665 670

Glu Gly Tyr Tyr Gly Tyr Thr Gly Ala Phe Arg Cys Leu Val Glu Lys

675 680 685

Gly Asp Val Ala Phe Val Lys His Gln Thr Val Pro Gln Asn Thr Gly

690 695 700

Gly Lys Asn Pro Asp Pro Trp Ala Lys Asn Leu Asn Glu Lys Asp Tyr

705 710 715 720

Glu Leu Leu Cys Leu Asp Gly Thr Arg Lys Pro Val Glu Glu Tyr Ala

725 730 735

Asn Cys His Leu Ala Arg Ala Pro Asn His Ala Val Val Thr Arg Lys

740 745 750

Asp Lys Glu Ala Cys Val His Lys Ile Leu Arg Gln Gln Gln His Leu

755 760 765

Phe Gly Ser Glu Val Thr Asp Cys Ser Gly Asn Phe Cys Leu Phe Arg

770 775 780

Ser Glu Thr Lys Asp Leu Leu Phe Arg Asp Asp Thr Val Cys Leu Ala

785 790 795 800

Lys Leu His Asp Arg Asn Thr Tyr Glu Lys Tyr Leu Gly Glu Glu Tyr

805 810 815

Val Lys Ala Val Gly Asn Leu Arg Lys Cys Ser Thr Ser Ser Leu Leu

820 825 830

Glu Ala Cys Thr Phe Arg Arg Pro

835 840

Claims

1. Use of an expression vector or a plant comprising the expression vector to express or prepare a medicament comprising an MGLP fusion protein; the plant is lettuce; the organ of the plant is selected from seeds, leaves or rhizomes;

the construction method of the expression vector comprises the following steps:

step 1: respectively optimizing codons of the MGLP fusion proteins into codons preferred by plants to obtain sequences of the optimized MGLP fusion proteins; the nucleotide sequence is shown as SEQ ID No. 1;

step 2: adding an Xbal restriction site at the 5 'end and a SacI site at the 3' end of the sequence of the optimized MGLP fusion protein;

cloning into a pUC57 vector to obtain a pMGLP cloning vector;

and step 3: obtaining a gene fragment from the cloning vector obtained in the step 2 through Xbal/Sacl, cloning to a binary plant vector pCam35S, and obtaining an expression vector p35S-MGLP.

2. The use of claim 1, wherein the medicament is an oral hypoglycemic formulation.