CN110229822B

CN110229822B - Application of plant as host in expression of Albiglutide

Info

Publication number: CN110229822B
Application number: CN201910549721.0A
Authority: CN
Inventors: 王跃驹; 马磊; 唐辉
Original assignee: Individual
Current assignee: Individual
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2023-11-03
Anticipated expiration: 2039-06-24
Also published as: CN110229822A

Abstract

The invention relates to the field of biotechnology, in particular to application of plants as hosts in expression of Albiglutide. The invention uses plants such as lettuce as an effective expression platform for recombinant protein production, and uses a simple and effective agrobacterium-mediated vacuum infiltration method to express Albiglutide. The expression system determines that the plant exogenous proteins can be collected 4 days after the infection of agrobacterium. The successful expression of Albiglutide is determined by using a Western Blot protein hybridization method, and Albiglutide is successfully purified by using an AKTA protein purification system. Biological activity test results show that Albiglutide produced by the platform technology remarkably reduces blood glucose concentration in dog blood.

Description

Application of plant as host in expression of Albiglutide

Technical Field

The invention relates to the field of biotechnology, in particular to application of plants as hosts in expression of Albiglutide.

Background

Diabetes is a common and frequently occurring disease characterized by chronic hyperglycemia, and is a disorder of glucose, fat and protein metabolism caused by a defect in insulin secretion or action in the body, or both. Clinically, there are mainly two types, insulin dependent (IDDM, type I) and non-insulin dependent (NIDDM, type II). With the increasing level of living, the incidence of diabetes has increased year by year, both in developed and developing countries. Diabetes, a serious non-infectious chronic disease, has become one of the major public health problems of close concern worldwide, being the third killer worldwide after cardiovascular and oncological diseases. From the data published by the world health organization, worldwide diabetics have only about 3000 tens of thousands of people in 1995, and have increased to 1.35 million by 1997, and by 2030 there will be 3 million type II diabetics, with the fastest growing areas being asia and africa. The traditional mode of treatment for type II diabetics is generally a stepwise treatment following diet control, oral antidiabetic drugs and exogenous insulin. However, there are still a number of important problems to be solved in the field of diabetes treatment, and there are also some side effects and limitations.

Glucagon-like peptide-1 (glp-1) is incretin secreted by endocrine cells of the intestinal tract, a post-translational processing product of the Glucagon-like gene, and exists in a variety of forms in the body. It has the following physiological actions: acting on islet beta cells in a glucose-dependent manner, promoting transcription of insulin genes, and increasing biosynthesis and secretion of insulin; stimulating proliferation and differentiation of beta cells, inhibiting beta cell apoptosis, increasing islet beta cell number, inhibiting glucagon secretion, suppressing appetite and ingestion, and delaying gastric emptying etc. Both of these functions are 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 in vivo of the natural GLP-1 is only about 2 minutes, and the direct clinical application of the natural GLP-1 is limited.

Disclosure of Invention

In view of this, the present invention provides the use of plants as hosts for the expression of Albiglutide. The invention expresses Albiglutide by using plants, especially lettuce, as a high-efficiency platform technology for recombinant protein production. And the active exogenous protein is successfully separated under mild conditions, which proves that the plant, especially lettuce expression platform, can be successfully used for producing Albiglutide protein. Short time (4 d), simple purification and convenient production. Eliminating gene pollution, eliminating potential diseases and insect pests which infect human body, etc. Greatly reduces the production cost and improves the safety of the product.

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

the invention provides an application of a plant as a host in expression of Albiglutide; the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from seeds, leaves, rhizomes or whole plants.

The plant transient expression technology is a technology for transferring plasmids containing target proteins into plant cells by utilizing a plurality of different technical modes when plants grow to a certain stage, and establishing a high-efficiency and controllable expression system in the plant cells to obtain transient and controllable expression of the genes. Compared with stable expression, the transient expression requires short time, exogenous genes are not required to be integrated into host plant chromosomes, and experimental results can be obtained in a few days. Compared with a bacterial expression system, the plant expression system can carry out correct folding, processing and modification on the expressed protein, and the activity of the produced protein is higher than that of the bacterial expression system; the cost of plant expression systems is very low compared to animal cell expression systems, only one to two thousandths of it.

Based on the above research, the invention also provides an expression vector comprising the sequence of Albiglutide and a binary plant vector.

In some embodiments of the invention, the sequence of Albiglutide is one that optimizes the codons of Albiglutide to plant-preferred codons, resulting in optimized Albiglutide.

In some embodiments of the invention, the nucleotide sequence of the optimized Albiglutide is shown in SEQ ID No. 1; the amino acid sequence of the optimized Albiglutide is shown as SEQ ID No. 2.

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

step 1: optimizing the codon of Albiglutide into a plant preferred codon to obtain an optimized nucleotide sequence of Albiglutide;

step 2: adding an Xbal restriction enzyme cutting site at the 5 'end of the optimized Albiglutide sequence, adding a Sac I site at the 3' end, and cloning into a pUC57 vector to obtain a pAlbi cloning vector;

step 3: the gene fragment was obtained from the pAlbi cloning vector obtained in step 2 by Xbal/Sacl, and cloned into the binary plant vector pCam35S to obtain the expression vector p35S-Albi.

The invention also provides application of the expression vector in expression of Albiglutide.

The invention also provides a method for expressing Albiglutide by taking a plant as a host, the expression vector is transformed into agrobacterium, and after the agrobacterium-mediated vacuum infiltration into plant tissues, proteins are extracted and separated to obtain Albiglutide.

In some embodiments of the invention, the plant is selected from lettuce, spinach, tomatoes, radishes, cabbages, corns, soybeans, wheat or tobacco; the plant organ is selected from seeds, leaves, rhizomes or whole plants.

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

step 1: vacuumizing for 25-45 s;

step 2: vacuum (-95 kPa) pressure is maintained for 30-60 s;

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

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

The invention adopts a plant system to produce Abililutide fusion protein, which is an original novel drug for fusing mutated GLP short peptide with HAS to prolong the benefit period.

The invention uses plants such as lettuce as an effective expression platform for recombinant protein production, and uses a simple and effective agrobacterium-mediated vacuum infiltration method to express Albiglutide. The expression system determines that the plant exogenous proteins can be collected 4 days after the infection of agrobacterium. The successful expression of Albiglutide is determined by using a Western Blot protein hybridization method, and Albiglutide is successfully purified by using an AKTA protein purification system. Biological activity test results show that Albiglutide produced by the platform technology remarkably reduces blood glucose concentration in 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 representation of cloning vector pAlbi;

FIG. 2 shows the construction flow of Albiglutide plant binary expression vector p35S-Albi; cutting Albiglutide from the cloning vector of FIG. 1 by using restriction enzyme (Xbal/SacI) double digestion, and connecting the Albiglutide with the Xbal/SacI site of pCam35S to generate a plant binary expression vector p35S-Albi;

LB and RB, ti plasmid left and right borders; 35S, caMV 35S promoter with Tobacco Mosaic Virus (TMV) 5' utr; NPT II, expression of the NPT II-encoding gene for kanamycin resistance; nos3', terminator;

FIG. 3 shows the result of SDS-PAGE gel electrophoresis; lane 1: non-infesting lettuce; lane 2: lettuce expressed Albiglutide; lane 3: albiglutide commercial products.

Detailed Description

The invention discloses an application of a plant as a host in expression of Albiglutide, and a person skilled in the art can refer to the content of the plant and properly improve the technological parameters. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that variations and modifications can be made in the methods and applications described herein, and in the practice and application of the techniques of this invention, without departing from the spirit or scope of the invention.

the present invention provides the use of plants as hosts for the expression of Albiglutide. Preferably, the plant is selected from lettuce, spinach, tomatoes, radishes, cabbages, corns, soybeans, wheat or tobacco; the plant organ is selected from seeds, leaves, rhizomes or whole plants. The invention also provides an expression vector comprising the sequence of Albiglutide and the vector.

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

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

step 1: the codons of Albiglutide were optimized as plant preferred codons to obtain:

optimized Albiglutide sequence;

step 2: an Xbal restriction enzyme cutting site is added at the 5 'end of the optimized Albiglutide sequence, and a Sac I site is added at the 3' end;

cloning the gene into a pUC57 vector from gold to obtain a pAlbi cloning vector;

step 3: the gene fragment was obtained from the cloning vector obtained in step 2 by Xbal/Sacl, and cloned into the binary plant vector pCam35S to obtain the expression vector p35S-Albi.

Specifically, in order to provide efficient expression of foreign proteins in plants, the present invention synthesizes human Albiglutide amino acid sequences using reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss_backtraneq /) to obtain nucleotide sequences, and optimizing their codons to plant-preferred codons, synthesized by Kingshi corporation (Nanjin, china). An Xbal restriction site was added to the 5 'end of the optimized Albiglutide sequence, and a Sacl site was added to the 3' end. And cloned from gold into pUC57 vector to obtain pAlbi cloning vector (FIG. 1). The gene fragment was isolated from the cloning vector by XbaI/Sacl and cloned into the binary plant vector pCam35S, yielding the plant expression vector p35S-Albi (FIG. 2).

In addition, the invention also provides a method for expressing Albiglutide by taking plants as hosts, the expression vector provided by the invention is transformed into agrobacterium, and proteins are extracted and separated after the agrobacterium-mediated vacuum infiltration into plant tissues, so that Albiglutide is obtained. The plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the plant organ is selected from seeds, leaves, rhizomes or whole plants. The invention also provides an expression vector comprising the sequence of Albiglutide and the vector.

Specifically, the plant expression vector p35S-Albi was transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, hamburg, germany). The resulting strain was spread uniformly on a selective LB plate containing kanamycin antibiotic (50 mg/L). After incubation in the dark at 28℃for 2d, single colonies were picked and inoculated into 0.5L of YEB (Yeast extract broth, 5g/L sucrose, 5g/L tryptone, 6g/L Yeast extract, 0.24g/L MgSO) ₄ pH 7.2) and supplemented with antibiotic broth (50 mg/L kanamycin). The inoculated culture was incubated at 25-28℃for 72h in a shaker (220 rpm). OD600 was measured by adding 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 osmotic medium (10mM MES,10mM MgSO4) to an O.D.600 of 0.5.

Uniformly mixing the prepared agrobacterium containing p35S-Albi until the O.D.600 is 0.5; the culture suspension was placed in a 2L beaker and placed in a desiccator. The lab kept lettuce was inverted (core up) and gently swirled in bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, niles, IL, USA) was turned on to evacuate and the permeate was seen in the leaf tissue. The pressure state is maintained for 30 to 60 seconds. The system is opened quickly to release the pressure, allowing the permeate to penetrate the space within the tissue. The process is repeated for 2 to 3 times until the clear and visible penetrating fluid is obviously diffused in the raw vegetable tissue. Lettuce tissue was then gently removed from the permeate and rinsed three times in succession with distilled water and transferred to plastic film covered containers. The treated samples were kept in the dark for 4d.

step 1: vacuumizing for 25-45 s;

step 2: vacuum (-95 kPa) pressure is maintained for 30-60 s;

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

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

The cloning pAlbi gene fragment and constructing a binary plant expression vector p35S-Albi (FIG. 2) verify that the gene fragment is complete by digestion with specific restriction enzymes after the construct is completed. After infiltration, most lettuce tissue was submerged during the vacuum infiltration process, with the remainder showing yellowish brown areas after 4 days of vacuum infiltration, except for firm mid-rib areas.

The extraction and separation of protein are specifically as follows: the lettuce samples which are infiltrated by the agrobacterium vacuum are stirred by a stirrer, and are homogenized for 1 to 2 minutes at a high speed by an extraction buffer (100mM KPi,pH7.8;5mM EDTA;10mM beta-mercaptoethanol) stirrer with the volume ratio of 1:1. The homogenate was adjusted to pH8.0, filtered with gauze and the filtrate 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 with shaking for 60min. The mixture was separated again by means of a centrifuge (10,000 g) at 4℃for 15min. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended by shaking on ice for 60min, and centrifuged again at 10,000g for 15min at 4 ℃. The supernatant was then discarded and the treated sample precipitated protein was dissolved in 5mL buffer (20mM KPi,pH 7.8;2mM EDTA;10m M beta-mercaptoethanol) and stored at 4 ℃.

SDS-PAGE gel electrophoresis is specifically: purified protein extracted from Agrobacterium vacuum-infiltrated lettuce was collected, and samples (5. Mu.L) were heat denatured (95 ℃) loaded with buffer (Biorad, hercules, calif., USA) at 4-12%Bis-Tris Plus SDS-denaturing gels (ThermoFisher Scientific, waltham, mass., USA)Running electrophoresis. Also, the degree of affinity of the antibodies was detected 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, as the cellulose cell wall is difficult to lyse and secondary plant metabolites. The stirrer is used for stirring and homogenizing, so that the homogenizing cost and the process are greatly saved. Separation of recombinant Albiglutide by denaturing gel SDS-PAGE we observed a band in the lane estimated to be about 75kDa (FIG. 3), consistent with the molecular weight of Albiglutide protein. The protein content of the purified samples was approximately 1.75mg/g based on the Bradford assay and densitometry control.

The invention uses lettuce to express Albiglutide transiently, and can produce high protein content in a short time (4 d). Lettuce is a higher plant that can undergo post-translational modification, i.e. the expressed protein is automatically active. Moreover, this approach minimizes biosafety problems, as the treated lettuce tissue is typically developed in a completely enclosed facility or container, without the problem of biological contamination. Lettuce does not contain plant toxic substances, has low protein content, and is favorable for downstream protein purification. The production cycle and the production cost can be greatly shortened by utilizing the lettuce system to produce Albiglutide.

Experiments show that the plant system, especially lettuce system, is a more economical and efficient expression platform and is a rapid method for transiently expressing recombinant protein. The vacuum agrobacterium infiltration method described by the invention is simple and quick, and can improve the yield of recombinant protein. Lettuce can increase protein production by being subjected to vacuum pressure and allow more complete penetration of each leaf. Lettuce is easier to grow and commercially produced in large quantities, and is therefore more readily available and cheaper than other transiently expressed plants, such as tobacco, and costs can be significantly reduced as no complex special production equipment is required. In conclusion, the invention can be used for mass production of Albiglutide in a short time by using lettuce system.

The plant provided by the invention can be used as a host for the application of Albiglutide expression, and raw materials and reagents used in the application can be purchased from the market.

The invention is further illustrated by the following examples:

EXAMPLE 1 construction of plant transient expression vectors

In order to efficiently express the foreign protein in plants, the Albiglutide amino acid sequence was synthesized by Kirsrui company (Nanjing, china) using reverse translation software (https:// www.ebi.ac.uk/Tools/st/emboss_backtraneq /) to obtain a nucleotide sequence, and optimizing its codon to a plant-preferred codon. An Xbal restriction site was added to the 5 '-end of the optimized Albiglutide nucleotide sequence (shown in SEQ ID No. 1), and a Sacl site was added to the 3' -end. And cloned into pUC57 vector from Kirschner, to obtain pAlbi cloning vector (FIG. 1), the gene fragment was isolated from the cloning vector by Xbal/Sacl, and cloned into binary plant vector, pCam35S, to yield plant expression vector p35S-Albi (FIG. 2). Plant expression vectors were transformed into Agrobacterium tumefaciens GV3101 by electroporation with a Multiporator (Eppendorf, hamburg, germany). The resulting strain was spread uniformly on a selective LB plate containing kanamycin antibiotic (50 mg/L). After incubation for 2d at 28℃in the dark, single colonies were picked and inoculated into 0.5L of 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 broth (50 mg/L kanamycin). The inoculated culture was incubated at 25-28℃for 72h in a shaker (220 rpm). OD600 was measured by adding 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 osmotic medium (10mM MES,10mM MgSO ₄ ) Medium to o.d.600 is 0.5.

EXAMPLE 2 Agrobacterium-mediated vacuum infiltration

And uniformly mixing the prepared agrobacterium containing p35S-Albi until the O.D.600 is 0.5. The culture suspension was placed in a 2L beaker and placed in a desiccator. The lab kept lettuce was inverted (core up) and gently swirled in bacterial suspension and the desiccator was sealed. The Vacuum pump (Welch Vacuum, niles, IL, USA) was turned on to evacuate and the permeate was seen in the leaf tissue. The pressure state is maintained for 30 to 60 seconds. The system is opened quickly to release the pressure, allowing the permeate to penetrate the space within the tissue. This process was repeated 2 to 3 times until the clear visible diffusion of the permeate in the raw vegetable tissue was evident. Lettuce tissue was then gently removed from the permeate and rinsed three times in succession with distilled water and transferred to plastic film covered containers. The treated samples were kept in the dark for 4 days.

EXAMPLE 3 protein extraction and separation

The lettuce samples which are infiltrated by the agrobacterium vacuum are stirred by a stirrer and homogenized for 1-2 minutes at high speed by an extraction buffer (100mM KPi,pH7.8;5mM EDTA;10m M beta-mercaptoethanol) stirrer with a volume ratio of 1:1. The homogenate was adjusted to pH8.0, filtered with gauze and the filtrate 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 with shaking for 60 minutes. The mixture was separated again by means of a centrifuge (10,000 g) at 4℃for 15 minutes. The resulting supernatant was subjected to a second round of ammonium sulfate (70%) precipitation, suspended by shaking on ice for 60 minutes, and centrifuged again at 10,000g for 15 minutes at 4 ℃. The supernatant was then discarded and the treated sample precipitated protein was dissolved in 5mL buffer (20mM KPi,pH 7.8;2mM EDTA;10mM beta-mercaptoethanol) and stored at 4 ℃.

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

EXAMPLE 4 SDS-PAGE gel electrophoresis

Collecting purified protein extracted from Agrobacterium vacuum-infiltrated lettuce, and loading the sample (5. Mu.L) with heat-denatured (95 ℃) buffer (Biorad, hercules, calif., USA) at 4-12%Bis-Tris Plus SDS-denaturing gels (ThermoFisher Scientific, waltham, mass., USA) were run. Also, the degree of affinity of the antibodies was detected in non-denaturing gel electrophoresis. Then with Coomassie blue G250 (Biorad) staining and photographing the gel again. Separation of recombinant Albiglutide by denaturing gel SDS-PAGE we observed a band in the lane estimated to be about 75kDa (FIG. 3), consistent with the molecular weight of Albiglutide protein. The protein content of the purified samples was approximately 1.75mg/g based on the Bradford assay and densitometry control.

Example 5 Albiglutide protein Activity assay

After a stabilization period of seven weeks, dogs were randomized into two treatment groups of 3 animals each, receiving one of two experimental capsules containing a glycoprotein (Albiglutide protein prepared in example 4) and no glycoprotein, and repeated for the first time. Dogs were again randomized and received a different experimental diet for a second repeat. Repeat I and II for at least 2 weeks, blood glucose response was measured after the end of each repeat.

Dogs fasted for 24 hours before blood glucose testing began. Shaving the catheterization site, aseptically treating, and catheterizing the right head vein. Two baseline samples were collected 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 dogs did not eat the experimental diet for 15 minutes, their glycemic response was not detected the day, and re-detected the next day. Additional blood samples were collected 10, 20, 30, 45, 60, 120, 180 and 240 minutes after feeding. Blood samples were centrifuged at 1300 Xg for 15 minutes and two aliquots of 1ml plasma at each time point were frozen within two hours after collection. Plasma glucose concentration (mg/dl) was measured using hexokinase method.

TABLE 1 Experimental results of sugar concentration in dog blood

Note that: ^* shows significant differences (P < 0.05) compared to the no-hypoglycemic control; ^** shows a very significant difference (P < 0.01) compared with the control group without blood glucose reduction.

The present invention utilizes lettuce to transiently express antibodies and can produce high levels of protein in a relatively short period of time (4 d). Lettuce is a higher plant that can undergo post-translational modification, i.e. the expressed protein is automatically active. Moreover, this approach minimizes biosafety problems, as the treated lettuce tissue is typically developed in a completely enclosed facility or container, without the problem of biological contamination. Lettuce does not contain plant toxic substances, has low protein content, and is favorable for downstream protein purification. The lettuce system is utilized for production, and the production period and the production cost can be greatly shortened.

EXAMPLE 6 animal toxicity test

Mice for experiments of 7 weeks size were randomized into three treatment groups of 10 animals each, each receiving one of two experimental capsules containing a glycoprotein (Albiglutide protein obtained according to the present invention) and no glycoprotein, fed 500ng/g according to body weight, and the same experimental diet. The feeding is continuously carried out for 10 days, the observation is carried out after each feeding, more than 6 hours are needed for daily continuous observation, the mice are not observed to be in an excited state or an inhibited state, phenomena such as slow action and the like are not caused, and the conditions such as diarrhea and the like are not caused. The Albiglutide protein has high oral safety.

Example 7

Control group: producing Albiglutide by using animal cells;

experiment group 1: the plant provided by the invention produces Albiglutide;

experiment group 2: producing Albiglutide by utilizing tobacco leaves;

TABLE 2 Albiglutide

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

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

as can be seen from Table 2, compared with the animal system of the control group, the lettuce transient expression Albiglutide provided by the invention has the advantages of extremely remarkable (P is less than or equal to 0.01) shortening the production period, extremely remarkable (P is less than or equal to 0.01) improving the protein content, simplifying the difficulty of protein purification and extremely remarkable (P is less than or equal to 0.01) reducing the production cost.

Compared with the tobacco leaf system of the experiment group 2, the lettuce transient expression Albiglutide obviously shortens the production period (P is less than or equal to 0.05), obviously improves the protein content (P is less than or equal to 0.05), obviously improves the protein activity (P is less than or equal to 0.05), simplifies the difficulty of protein purification, and extremely obviously reduces the production cost (P is less than or equal to 0.01).

Compared with a control group, the experimental group 2 has the advantages that compared with an animal system, the instantaneous expression of the tobacco leaf Albiglutide is obviously (P is less than or equal to 0.05), the production period is shortened, the difficulty of protein purification is simplified, and the production cost is obviously reduced (P is less than or equal to 0.05). The test results show that the plant system, especially lettuce system, is a more economical and efficient expression platform. The recombinant protein can be rapidly and transiently expressed, and Albiglutide can be produced in a large scale in a short time.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Sequence listing

<110> Wang Yueju

<120> use of plants as hosts for expression of Albiglutide

<130> MP1906699

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2010

<212> DNA

<213> Albiglutide(Albiglutide)

<400> 1

atgggcatca agatggaaag ccagatccag gtgttcgttt tcgtgttcct ttggctgtct 60

ggtgtggatg gtcatggtga gggaaccttt acctccgacg tgtcatctta ccttgaaggc 120

caagctgcca aagagttcat tgcttggctt gtgaagggta gacacggcga gggaactttt 180

actagcgacg tgagttctta cctcgagggg caagcagcta aagaattcat agcctggttg 240

gtcaagggcc gtgatgctca taagtctgag gttgcacaca ggttcaagga tctgggcgaa 300

gagaacttca aggctcttgt gcttatcgct ttcgcccagt atcttcagca gtgccctttc 360

gaggatcacg tgaagcttgt taatgaggtg accgagttcg ccaagacctg tgttgctgat 420

gagtctgctg agaactgcga caagtctctt cacaccctgt tcggtgataa gttgtgcact 480

gtggctaccc tgagggaaac ttatggtgag atggctgatt gctgcgctaa gcaagagcct 540

gagaggaatg agtgcttcct gcagcacaag gatgacaacc ctaaccttcc taggcttgtg 600

aggcctgagg ttgacgttat gtgcactgct ttccacgaca acgaggaaac cttcctgaag 660

aagtacctgt acgagatcgc tcggaggcac ccttactttt atgctccaga gcttctgttc 720

ttcgccaagc gttacaaggc tgcttttact gagtgttgcc aggctgctga taaggctgca 780

tgtcttttgc ctaagctgga tgagctgcgt gatgaaggca aggcttcttc tgctaagcag 840

aggcttaagt gcgccagcct tcaaaagttt ggtgagaggg cttttaaggc ttgggctgtt 900

gctagactgt ctcagaggtt tcctaaggct gagttcgctg aggtgtcaaa gcttgtgacc 960

gatcttacta aggtgcacac cgagtgctgc cacggtgatc ttttggaatg cgctgatgat 1020

agggctgacc tggctaagta catttgcgag aaccaggaca gcatctccag caagcttaaa 1080

gagtgttgcg agaagccgct gctcgagaag tctcattgca ttgcagaggt tgagaacgac 1140

gagatgcctg ctgatcttcc ttctctcgct gctgacttcg ttgagtctaa ggacgtgtgc 1200

aagaactacg ctgaggccaa ggatgtgttc cttggcatgt tcctttacga gtacgctaga 1260

aggcacccgg attactctgt ggtgcttctt ttgaggctcg ctaagaccta cgagactacc 1320

cttgagaagt gttgcgctgc tgctgatcct catgagtgct acgctaaggt gttcgacgag 1380

ttcaagcctc tggttgagga acctcagaac ctgatcaagc agaattgcga gctgttcgag 1440

cagctgggag agtacaagtt ccagaacgct cttttggtgc ggtacaccaa gaaggtgcca 1500

caggtttcaa ctccgactct ggtggaagtg tctaggaacc ttggtaaggt gggaagcaag 1560

tgttgtaagc accctgaggc taagagaatg ccttgcgctg aggattacct gagcgtggtg 1620

cttaatcagc tttgcgtgtt gcacgaaaag acccctgtgt ctgatagggt taccaagtgc 1680

tgcactgagt ctctggtgaa tagacggcct tgcttctctg ctcttgaagt ggacgagact 1740

tacgtgccga aagagtttaa cgccgagact ttcaccttcc acgctgatat ctgcaccctg 1800

tctgagaaag agcggcagat taagaagcag accgctcttg ttgagctggt gaagcacaaa 1860

cctaaggcca ccaaagagca gctgaaggct gttatggatg acttcgctgc cttcgtggaa 1920

aagtgttgca aggccgatga caaagagaca tgcttcgctg aagagggcaa gaagttggtt 1980

gctgcttctc aggctgctct tggtctttga 2010

<210> 2

<211> 669

<212> PRT

<213> Albiglutide(Albiglutide)

<400> 2

Met Gly Ile Lys Met Glu Ser Gln Ile Gln Val Phe Val Phe Val Phe

1 5 10 15

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

20 25 30

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

35 40 45

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

50 55 60

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

65 70 75 80

Val Lys Gly Arg Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys

85 90 95

Asp Leu Gly Glu Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala

100 105 110

Gln Tyr Leu Gln Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn

115 120 125

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

130 135 140

Asn Cys Asp Lys Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr

145 150 155 160

Val Ala Thr Leu Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala

165 170 175

Lys Gln Glu Pro Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp

180 185 190

Asn Pro Asn Leu Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys

195 200 205

Thr Ala Phe His Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr

210 215 220

Glu Ile Ala Arg Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe

225 230 235 240

Phe Ala Lys Arg Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala

245 250 255

Asp Lys Ala Ala Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu

260 265 270

Gly Lys Ala Ser Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln

275 280 285

Lys Phe Gly Glu Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser

290 295 300

Gln Arg Phe Pro Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr

305 310 315 320

Asp Leu Thr Lys Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu

325 330 335

Cys Ala Asp Asp Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln

340 345 350

Asp Ser Ile Ser Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu

355 360 365

Glu Lys Ser His Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala

370 375 380

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

385 390 395 400

Lys Asn Tyr Ala Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr

405 410 415

Glu Tyr Ala Arg Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg

420 425 430

Leu Ala Lys Thr Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala

435 440 445

Asp Pro His Glu Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu

450 455 460

Val Glu Glu Pro Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Glu

465 470 475 480

Gln Leu Gly Glu Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr

485 490 495

Lys Lys Val Pro Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg

500 505 510

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

515 520 525

Arg Met Pro Cys Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu

530 535 540

Cys Val Leu His Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys

545 550 555 560

Cys Thr Glu Ser Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu

565 570 575

Val Asp Glu Thr Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr

580 585 590

Phe His Ala Asp Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys

595 600 605

Lys Gln Thr Ala Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr

610 615 620

Lys Glu Gln Leu Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu

625 630 635 640

Lys Cys Cys Lys Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly

645 650 655

Lys Lys Leu Val Ala Ala Ser Gln Ala Ala Leu Gly Leu

660 665

Claims

1. A method for expressing Albiglutide by using a plant as a host is characterized in that an expression vector is transformed into agrobacterium, and after the agrobacterium-mediated vacuum infiltration into plant tissues, proteins are extracted and separated to obtain Albiglutide;

the plant is lettuce;

the expression vector comprises an Albiglutide sequence and a binary plant vector;

the sequence of Albiglutide is obtained by optimizing the codon of Albiglutide into a codon preferred by plants;

the nucleotide sequence of the optimized Albiglutide is shown as SEQ ID No. 1; the amino acid sequence of the optimized Albiglutide is shown as SEQ ID No. 2;

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

step 2: adding an Xbal restriction enzyme cutting site at the 5 'end of the optimized Albiglutide sequence, adding a SacI site at the 3' end, and cloning into a pUC57 vector to obtain a pAlbi cloning vector;

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

the agrobacterium-mediated vacuum infiltration comprises the following steps:

step 1: vacuumizing for 25-45 s;

step 2: maintaining the vacuum-95 kPa for 30-60 s;

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

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