WO2020259110A1

WO2020259110A1 - Application of plant-produced fast-acting oral hypoglycemic capsules of fusion protein of human cholera toxin b subunit (ctb) and proinsulin

Info

Publication number: WO2020259110A1
Application number: PCT/CN2020/089982
Authority: WO
Inventors: 王跃驹
Original assignee: 王跃驹
Priority date: 2019-06-24
Filing date: 2020-05-13
Publication date: 2020-12-30
Also published as: CN110256578A; CN110256578B

Abstract

Provided is a fusion protein of human cholera toxin B subunit (CTB) and proinsulin. The invention uses plants such as lettuce as an expression platform for recombinant protein production, and freeze-dries leaves producing the active substance so as to form a capsule. The invention can reduce blood glucose concentration in canine blood.

Description

Application of plant-produced human cholera toxin B subunit (CTB) and proinsulin fusion protein quick-acting oral hypoglycemic capsule

This application requires that it be submitted to the Chinese Patent Office on June 24, 2019, the application number is 201910549741.8, and the title of the invention is "The application of plant-produced human cholera toxin B subunit (CTB) and proinsulin fusion protein quick-acting oral hypoglycemic capsules" The priority of the Chinese patent application, the entire content of which is incorporated in this application by reference.

Technical field

The present invention relates to the field of biotechnology, in particular to the application of a plant-produced human cholera toxin B subunit (CTB) and proinsulin fusion protein quick-acting oral hypoglycemic capsule.

Background technique

Diabetes is a common and frequently-occurring disease characterized by chronic hyperglycemia. It is a disorder of sugar, fat, and protein metabolism caused by defects 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 improvement of living standards, both in developed and developing countries, the incidence of diabetes is increasing year by year. Diabetes, as a serious non-communicable chronic disease, has now become one of the major public health problems that countries around the world pay close attention to, and it is the third killer worldwide after cardiovascular and tumor diseases.

On April 8, 2016, an article titled "China's Diabetes Prevention and Control" published on the official website of the National Health and Family Planning Commission cited data from "Chinese Residents' Nutrition and Chronic Disease Report" (2015), saying that China in 2012 The prevalence of diabetes among residents over 18 years old is 9.7%, among which the prevalence rates in urban and rural areas are 12.3% and 8.4%, respectively. The number of patients is about 100 million. The awareness rate of diabetes among residents over 18 years is 36.1% and the treatment rate It is 33.4%, and the control rate is 30.6%. In addition, the newly released "Chinese Diabetes Dietary Guidelines (2017)" on May 22, 2017 quoted the 2013 "Journal of the American Medical Association" (JAMA) research findings that the prevalence of adult diabetes in China has reached 11.6%. In all cases of diabetes, insulin-dependent diabetes mellitus (IDDM, also known as type 1 diabetes, formerly known as "juvenile-onset diabetes") accounts for about 5-10%. This type of diabetes is due to endogenous Insufficient insulin secretion, which requires long-term insulin therapy; another more common type of diabetes is non-insulin-dependent diabetes mellitus (NIDDM, also known as type 2 diabetes, formerly known as "adult-onset diabetes") In the proportion of more than 90%, insulin can also be used for treatment. Therefore, although more and more oral hypoglycemic drugs are used in clinical practice, insulin still plays an important role in the treatment of diabetes.

According to the Pharmarket database, the amount of insulin glargine, insulin aspart, and protamine zinc insulin used in sample hospitals in 22 key cities across the country in 2016 were 546 million yuan, 445 million yuan and 260 million yuan, respectively. However, there are still many important problems to be solved in the field of diabetes treatment, and there are also some side effects and limitations.

Humalog is an insulin lispro developed by Eli Lilly. Insulin Lispro (Insulin Lispro) is an ultra-short-acting human insulin analogue launched by Eli Lilly in 1996 for blood sugar control in diabetes management. In 2000, the FDA extended its indications to the treatment of hyperglycemia in children over 3 years old and adults over 65 years old. In addition, insulin lispro is also used in combination with sulfonylurea drugs. At present, insulin lispro is sold in the United States, the European Union, Canada, Japan and China and other countries in the world. Insulin lispro is a genetically engineered product, that is, the strength of the human insulin β-chain exchange between the 28 proline and the 29 lysine is equivalent to that of human insulin. The hypoglycemic effect is the same, but the effect is more rapid and sustained The time is shorter. The mechanism of action of insulin lispro is the same as that of insulin aspart. It takes effect within 15-20 minutes, reaches its peak in 30-60 minutes, and its hypoglycemic effect lasts for 4-5 hours. It can be used as a substitute for conventional soluble insulin to play a quick-acting hypoglycemic effect. It is an ultra-short-acting insulin, and can also be combined with protamine as a medium-acting agent.

Although Humalog has many advantages in the treatment of diabetes, due to the nature of peptide drugs and various barriers created by the human body, injection has always been the main route of conventional administration. The present invention expresses CTB and Humalog fusion, can realize oral administration, and alleviate the pain caused by long-term frequent injection of patients.

Summary of the invention

In view of this, the present invention provides the application of a plant to produce a quick-acting oral hypoglycemic capsule of a fusion protein of human cholera toxin B subunit (CTB) and proinsulin. The present invention carries out structural transformation and modification of the active polypeptide with hypoglycemic effect, so that it can be absorbed through the intestinal tract and reach an effective therapeutic concentration in the body, and the active substance is produced by plants. The invention uses plants, especially lettuce, as an efficient platform technology for recombinant protein production, and expresses a fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin. And make oral hypoglycemic capsules.

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

The present invention provides a fusion protein of human cholera toxin B subunit (CTB) and Humalog proinsulin, which has:

(Ⅰ) The amino acid sequence shown in SEQ ID No. 1; or

(II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same or similar function as the amino acid sequence shown in (I); or

(III) An amino acid sequence that has at least 80% homology with the sequence described in (I) or (II).

The present invention also provides nucleotides encoding the fusion protein, having

(Ⅰ) The nucleotide sequence shown in SEQ ID No. 2; or

(II) The complementary nucleotide sequence of the nucleotide sequence shown in SEQ ID 2; or

(Ⅲ). A nucleotide sequence that encodes the same protein as the nucleotide sequence of (I) or (II), but is different 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 to the nucleotide sequence shown in (I), (II) or (III), and with (I), (II) or (III) nucleotide sequences with the same or similar functions; or

(V), a nucleotide sequence that has at least 80% homology with the nucleotide sequence described in (I), (II), (III) or (IV).

On the basis of the above research, the present invention also provides an expression vector, including the nucleotide and the vector to be transformed.

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

In addition, the present invention also provides the construction method of the expression vector, which includes the following steps:

Step 1: The codons of the fusion protein of the human cholera toxin B subunit and Humalog proinsulin are optimized to plant-preferred codons, and the nucleotide sequence is shown in SEQ ID No. 2;

Step 2: Cloning the nucleotide sequence into the pUC57 vector to obtain pHuma.

In addition, the present invention also provides the application of the expression vector or plant in expressing the fusion protein of human cholera toxin B subunit and Humalog proinsulin or preparing a medicine containing the fusion protein; the plant is selected from lettuce and spinach , Tomato, radish, cabbage, corn, soybean, wheat or tobacco; the organ of the plant is selected from seeds, leaves, rhizomes or whole plants.

In some specific embodiments of the present invention, the drug is a hypoglycemic oral preparation.

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, cabbage, corn, soybean, wheat or tobacco; the organ of the plant is selected from seeds, Leaves, rhizomes or whole plants.

On the basis of the above research, the present invention also provides drugs, including the fusion protein and pharmaceutically acceptable excipients.

The present invention also provides a method for expressing the fusion protein of human cholera toxin B subunit and Humalog proinsulin in a plant as a host. The expression vector is bombarded with a gene gun to the leaves, and the regenerated plant is obtained after expression in the plant chloroplast. The plant leaves were freeze-dried, crushed and extracted to obtain the fusion protein of human cholera toxin B subunit and Humalog proinsulin.

In some specific embodiments of the present invention, the gene gun bombardment includes the following steps:

Step 1: Prepare vector for transformation;

Step 2: Prepare particle bullets;

Step 3: Gene gun bombardment;

Step 4: After conversion, cultivate and regenerate into plants.

The present invention also provides a method for preparing a hypoglycemic drug by using a plant as a host. The expression vector is bombarded with a gene gun to the leaves and expressed in plant chloroplasts to obtain regenerated plants. The plant leaves are freeze-dried, crushed, and extracted to obtain The fusion protein of human cholera toxin B subunit and Humalog proinsulin, filling.

Step 1: Prepare vector for transformation;

Step 2: Prepare particle bullets;

Step 3: Gene gun bombardment;

Step 4: After conversion, cultivate and regenerate into plants.

Plant chloroplast expression technology is the use of gene gun bombardment and homologous recombination to transfer a plasmid containing the target protein to plant chloroplasts to obtain high-efficiency expression of the gene in plant chloroplasts. Compared with animal cell expression systems, the cost of plant expression systems is very low, only one thousandth to two thousandths.

The invention uses plant leaves to produce oral hypoglycemic capsules. The hypoglycemic product does not require injections, which reduces the suffering of patients. Lettuce does not contain plant toxic substances, and this product does not require a protein purification process, which can greatly shorten the production cycle and production costs.

The present invention found through experiments that the plant system, especially the lettuce system, is a more economical and efficient expression platform, and the chloroplast can efficiently express active proteins. Because lettuce is easy to grow and can be produced in large quantities commercially, it is easier to obtain and cheaper than other plants, such as tobacco, and because it does not require complex special production equipment, the cost can be significantly reduced. In summary, the present invention can use the lettuce system to produce large-scale fusion protein sequences of human cholera toxin B subunit (CTB) and Humalog proinsulin.

Description of the drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following will briefly introduce the drawings that need to be used in the description of the embodiments or the prior art.

Figure 1 shows a schematic diagram of the vector pHuma;

Figure 2 shows the western-blot results.

Detailed ways

The invention discloses an application of a plant-produced quick-acting oral hypoglycemic capsule of a fusion protein of human cholera toxin B subunit (CTB) and proinsulin. Those skilled in the art can learn from the content of this article and appropriately improve the process parameters. In particular, it should be pointed out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention. The method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of the present invention.

The invention provides the application of plant production of oral hypoglycemic capsules. The invention uses plants, especially lettuce, as an efficient platform technology for recombinant protein production, and expresses a fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin. And make oral hypoglycemic capsules.

The present invention provides the use of plants as hosts to express the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin. Preferably, the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; and the organ of the plant is selected from seeds, leaves, rhizomes or whole plants. The present invention also provides an expression vector, including the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin and the vector.

In some specific embodiments of the present invention, the codons of the fusion protein sequence of the human cholera toxin B subunit (CTB) and Humalog proinsulin are optimized to plant-preferred codons.

In some specific embodiments of the present invention, the sequence of the fusion protein sequence of the optimized human cholera toxin B subunit (CTB) and Humalog proinsulin is shown in SEQ ID No. 1; the optimized human cholera toxin B subunit The nucleotide sequence of the fusion protein sequence of CTB and Humalog proinsulin is shown in SEQ ID No.2.

In some specific embodiments of the present invention, the vector is a plant chloroplast vector.

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

Step 1: Optimize the codons of the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin to plant-preferred codons;

Step 2: Gene synthesis and cloning into pUC57 vector by GenScript to obtain pHuma cloning vector;

Specifically, in order to provide high-efficiency expression of foreign proteins in plants, the present invention uses the amino acid sequence of the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin using reverse translation software (https://www.ebi. ac.uk/Tools/st/emboss_backtranseq/) to obtain the nucleotide sequence and optimize its codons to plant-preferred codons, synthesized by GenScript (Nanjing, China). And cloned into the pUC57 vector from GenScript to obtain the pHuma vector (Figure 1).

The invention also provides the application of the expression vector in expressing the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin.

The expression vector provided by the present invention is bombarded with gene gun to plant leaves, regenerated into plants, and then harvested and made into oral hypoglycemic capsules.

The raw materials and reagents used in the application of the fusion protein quick-acting oral hypoglycemic capsule of human cholera toxin B subunit (CTB) and proinsulin produced by the plant provided by the present invention can be purchased from the market.

The following examples further illustrate the present invention:

Example 1 Construction of chloroplast expression vector

In order to express the foreign protein efficiently in plants, the amino acid sequence of the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin was used reverse translation software (https://www.ebi.ac.uk/Tools) /st/emboss_backtranseq/) to obtain the nucleotide sequence and optimize its codons to plant-preferred codons, synthesized by GenScript (Nanjing, China).

Example 2 Preparation of transformation materials

Soak the plant seeds in sterile water overnight, soak in 70% ethanol for 1 minute, and then rinse once with sterile water; then treat them with 2% NaClO (with 0.1% Tween-20) for 15 minutes, and gently mix every 5 minutes. Rinse 4-5 times with sterile water; blot dry with sterile filter paper and plant on 1/2MS medium (containing 3% sucrose, 0.7% agar powder, pH 5.8), and place in a light incubator 25 ℃, 16h light for 8h dark culture, about 3 weeks can be used for transformation.

Example 3 Preparation of gene gun

Weigh 50-60mg gold powder (0.6μm) in a dry 1.5mL sterile EP centrifuge tube. Add 1 mL of absolute ethanol and vortex for 2 minutes. Add 1 mL of sterile water, vortex for 1 minute, place at room temperature for 1 minute, centrifuge at 10,000 rpm for 2 minutes, and remove the supernatant. Add 1mL of 50% glycerin, resuspend the gold powder, and freeze at -20°C.

The gold powder suspension in the glycerol state was vortexed for 5 minutes to resuspend the gold powder. Take 50μL of gold powder suspension in a sterile 1.5mL centrifuge tube and vortex for 1 minute. Add 10 μg plasmid DNA and vortex for 30 seconds. Add 50μL 2.5M CaCl2 and vortex for 30 seconds. Add 20 μL 0.1M spermidine, vortex the mixture for 5 minutes, and let stand on ice for 2 minutes. Add 60 μL of pre-cooled absolute ethanol, flick your fingers to resuspend, centrifuge at 14,000 rpm for 10 seconds, remove the supernatant, and repeat. Add 50μL of absolute ethanol to resuspend and set aside.

Example 4 Gene gun bombardment

Take a certain number of carrier membranes, splittable membranes, and barrier nets according to the number of samples (note: carrier membranes and splittable membranes need to be replaced every gun, and the barrier net can be shared with the same sample) soak in absolute ethanol for 15 minutes, and use sterile Rinse twice with water, let it dry naturally, and set aside. Put the dried carrier film into a sterile iron ring and flatten it. The prepared bullets were vortexed to mix well, and 10 μL bullets were placed in the center of the carrier film and dried naturally. Move the particle launcher out of the bombardment chamber, unscrew the cover, add the blocking net, install the particle slide in the fixed groove (the side with the particles is facing down), screw on the cover, and put the particle launcher back into the bombardment chamber.

Example 5 Cultivation and screening after transformation

1. Dark culture: Cut the bombarded lettuce leaves, cut into 10-20mm2 leaf discs, and place them in RMOL medium (without antibiotics) at 25°C for 2 days in the dark.

2. Screening culture: transfer the materials after dark culture to the screening medium (antibiotic concentration of 50μg/mL) for screening culture.

3. Rooting culture: transfer the buds to a rooting medium (antibiotic concentration of 100μg/mL) to induce rooting.

Example 6 Western blot detection of target protein expression

Use liquid nitrogen grinding, denaturation and lysis to extract vegetable protein, mix the lysis supernatant and 5× loading buffer (add β-mercaptoethanol to a final concentration of 5% before use) in a 4:1 ratio (such as 200μl protein lysis supernatant) Mix with 50μl 5× loading buffer), mix well, heat at 95℃ for 6min, and process negative control and positive control at the same time; electrophoresis voltage is 80V for laminated gel and 120V for separation gel. After the target protein runs to the middle of the separation gel, stop electrophoresis , Recover the lower tank electrophoresis solution, disassemble the electrophoresis device, follow the negative electrode (black), sponge, filter paper, gel, PVDF membrane (previously activated with methanol for 15s, washed with ddH2O and soaked in 1× transfer buffer) or NC membrane ( No activation), filter paper, sponge, and positive electrode (transparent) are placed in order. After removing the bubbles, assemble it and put it into the electrophoresis tank (the black side corresponds to the black side of the electrophoresis tank), fill up the transfer buffer, and place the entire electrophoresis tank Put it into the ice-water mixture, electrophoresis at 90V for 1.0h; at the end of the electrophoresis, prepare 5% skimmed milk powder (blocking solution), put the transferred membrane into the blocking solution and block at room temperature for at least 1h, and incubate the primary antibody overnight at 4℃ (primary antibody) Diluted in 5% skimmed milk powder, the dilution ratio refers to the instructions); wash with PBST or TBST for 15min×3 times, incubate the secondary antibody at room temperature for 1～2h, wash with PBST or TBST for 15min×3 times, use DAB kit for color development and take pictures Analyze the expression of the target protein, and the results show that the target protein is normally expressed in lettuce, as shown in Figure 2.

Example 7 Activity detection of the fusion protein sequence of human cholera toxin B subunit (CTB) and Humalog proinsulin

After a stabilization period lasting seven weeks, the dogs were randomly divided into two treatment groups, 3 in each group, and received the human cholera toxin B subunit (CTB) prepared in Example 5 and Humalog proinsulin respectively. The fusion protein) and one of the two experimental capsules without hypoglycemic protein, do the first repetition. Randomly group the dogs again, accept a different experimental diet, and do a second repetition. Repeat I and II for at least 2 weeks, and check the blood glucose response after each repetition.

Before the blood glucose test begins, the dog fasts for 24 hours. Shave the hair at the catheter insertion site, aseptically process, and insert the catheter into the right cephalic vein. Take two baseline samples approximately 5 minutes apart. After the last baseline sample was collected, the dog was immediately fed a diet equivalent to 1% of its body weight and containing 1 or 3 hypoglycemic capsules, and allowed to eat for up to 15 minutes. If the dog does not eat the experimental diet within 15 minutes, the blood glucose response will not be tested on the same day, and the test will be repeated the next day. At 10, 20, 30, 45, 60, 120, 180, and 240 minutes after eating, additional blood samples were collected. The blood samples were centrifuged at 1300×g for 15 minutes, and two aliquots of 1ml plasma at each time point were cryopreserved within two hours after collection. The hexokinase method was used to determine the plasma glucose concentration (mg/dl).

Table 1 Experimental results of sugar concentration in dog blood

Note: * indicates a significant difference (P<0.05); ** indicates a very significant difference (P<0.01).

Example 8 Animal toxicity test

The 7-week-old experimental mice were randomly divided into three treatment groups, with 10 mice in each group, and received glycoproteins (fed 500ng/g according to body weight) (human cholera toxin B subunit (CTB) obtained in the present invention). Fusion protein with Humalog proinsulin), and one of the two experimental capsules without hypoglycemic protein, received the same experimental diet. Feeding was continued for 10 days, and observations were made after each feeding. Continuous observation was required for more than 6 hours a day. It did not see whether the mice were excited or inhibited, did not appear to be slow or diarrhea. It proves that the fusion protein of human cholera toxin B subunit (CTB) and Humalog proinsulin has high oral safety.

The application of the fast-acting oral hypoglycemic capsule for producing the fusion protein of human cholera toxin B subunit (CTB) and proinsulin provided by the present invention has been described in detail above. This article uses specific examples to illustrate the principle and implementation of the present invention. The description of the above examples is only used to help understand the method and core idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

The fusion protein of human cholera toxin B subunit and Humalog proinsulin, characterized in that it has:

(Ⅰ) The amino acid sequence shown in SEQ ID No. 1; or

(II) An amino acid sequence obtained by substituting, deleting or adding one or more amino acids to the amino acid sequence described in (I), and having the same or similar function as the amino acid sequence shown in (I); or

(III) An amino acid sequence that has at least 80% homology with the sequence described in (I) or (II).
The nucleotide encoding the fusion protein of claim 1, characterized in that it has

(Ⅰ) The nucleotide sequence shown in SEQ ID No. 2; or

(II) The complementary nucleotide sequence of the nucleotide sequence shown in SEQ ID 2; or

(Ⅲ). A nucleotide sequence that encodes the same protein as the nucleotide sequence of (I) or (II), but is different 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 to the nucleotide sequence shown in (I), (II) or (III), and with (I), (II) or (III) nucleotide sequences with the same or similar functions; or

(V), a nucleotide sequence that has at least 80% homology with the nucleotide sequence described in (I), (II), (III) or (IV).
The expression vector is characterized by comprising the nucleotide according to claim 2 and the vector to be transformed.
8. The expression vector of claim 3, wherein the vector to be transformed is a chloroplast expression vector.
The method for constructing an expression vector according to claim 3 or 4, characterized in that it comprises the following steps:

Step 1: The codons of the fusion protein of the human cholera toxin B subunit and Humalog proinsulin are optimized to plant-preferred codons, and the nucleotide sequence is shown in SEQ ID No. 2;

Step 2: Cloning the nucleotide sequence into the pUC57 vector to obtain pHuma.
The use of the expression vector or plant according to claim 3 or 4 in expressing a fusion protein of human cholera toxin B subunit and Humalog proinsulin or preparing a medicine containing the fusion protein; the plant is selected from lettuce, spinach, Tomato, radish, cabbage, corn, soybean, wheat or tobacco; the organs of the plant are selected from seeds, leaves, rhizomes or whole plants.
The application according to claim 6, wherein the drug is an oral preparation for reducing blood sugar.
A host, characterized in that a plant or microorganism transformed with the expression vector of claim 3 or 4; the plant is selected from lettuce, spinach, tomato, radish, cabbage, corn, soybean, wheat or tobacco; the organ of the plant It is selected from seeds, leaves, rhizomes or whole plants.
The medicine is characterized by comprising the fusion protein according to claim 1 and pharmaceutically acceptable excipients.
The medicine according to claim 9, wherein the medicine is an oral preparation for reducing blood sugar.