WO2022032991A1 - 甘露聚糖酶及其同源物与glp-1重组融合蛋白的高效表达纯化方法及应用 - Google Patents

甘露聚糖酶及其同源物与glp-1重组融合蛋白的高效表达纯化方法及应用 Download PDF

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WO2022032991A1
WO2022032991A1 PCT/CN2021/073745 CN2021073745W WO2022032991A1 WO 2022032991 A1 WO2022032991 A1 WO 2022032991A1 CN 2021073745 W CN2021073745 W CN 2021073745W WO 2022032991 A1 WO2022032991 A1 WO 2022032991A1
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glp
mannanase
fusion protein
recombinant
homologue
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French (fr)
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姜伟
楼慧强
王岩
余卫雄
徐冲
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安徽新熙盟生物科技有限公司
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Priority to US18/020,429 priority Critical patent/US20230295594A1/en
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    • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
    • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
    • C12N9/2477—Hemicellulases not provided in a preceding group
    • C12N9/2488—Mannanases
    • A—HUMAN NECESSITIES
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    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
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    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
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    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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    • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
    • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
    • C12Y302/01025—Beta-mannosidase (3.2.1.25), i.e. mannanase
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2319/00—Fusion polypeptide
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/84—Pichia

Definitions

  • the invention belongs to the technical field of biomedicine, and in particular relates to a high-efficiency expression and purification method and application of mannanase and its homologue and GLP-1 recombinant fusion protein.
  • Diabetes is one of the important diseases that seriously threaten human life and health in modern society. Diabetes mellitus is an endocrine-metabolic syndrome characterized by chronic hyperglycemia. According to the National Health and Family Planning Commission, 50.1% of adults are currently in pre-diabetes, and they may become new diabetic patients every day. More data show that the prevalence of diabetes in China has increased rapidly in recent decades: from 0.67% in the 1980s to 11.6% in 2010, an increase of 17 times, and it is getting younger and younger.
  • glucagon-like peptide-1 (glucagon-likepeptide-1, GLP-1) not only has excellent hypoglycemic effect, but also has the characteristics of controlling body weight, regulating blood lipids, and improving islet ⁇ -cell function.
  • the reaction rate is low.
  • the excellent effects of GLP-1 and its analogs in the treatment of type 2 diabetes and weight loss have made it gradually occupy an important position in the diabetes treatment drug market in recent years.
  • the structure of GLP-1 is usually modified to prolong its half-life.
  • GLP-1 analogs there are currently two long-acting GLP-1 analogs on the market, both of which are fused with macromolecular proteins, namely dulaglutide and albiglutide, the former is fused with G4 immunoalbumin, and the latter is fused with serum albumin , prolongs the half-life of drug metabolism, and can achieve weekly injection.
  • both albiglutide and dulaglutide are drugs that are injected once a week and cannot achieve the limitations of oral administration. Gastrointestinal side effects such as injection site and nausea, vomiting and abdominal pain may occur after injection.
  • ⁇ -mannanase is the most important enzyme in the degradation process of mannan, and it is widely used in industry. Oligosaccharides cannot be fully degraded in animals due to the lack of corresponding enzymes, but they can be absorbed and metabolized by probiotics existing in animals, such as lactic acid bacteria and bifidobacteria.
  • the products of metabolism include short-chain fatty acids (SCFA) such as acetic acid, propionic acid and butyric acid.
  • SCFA short-chain fatty acids
  • SCFA can have important effects on the body, such as inhibiting the growth of pathogenic bacteria and supplying energy in muscles, kidneys, heart, brain, etc. .
  • the present invention provides a high-efficiency expression and purification method and application of a recombinant fusion protein of mannanase and its homologues with GLP-1.
  • the present invention uses Pichia pastoris as an engineering strain, which can efficiently express mannanase (MANNase) and its homologues.
  • MANNase mannanase
  • the mannanase and its homologues and GLP-1 form a fusion protein, and undergo high-density fermentation and culture. , which can efficiently secrete and express the fusion protein, which has both mannanase and its homolog activity, and GLP-1 activity.
  • the expression is induced by methanol, and the target protein in the supernatant has high expression and high purity.
  • the purification process is only filtration and concentration, which is a low-cost and high-efficiency preparation method.
  • the fusion protein injection and oral administration have good hypoglycemic effects.
  • a high-efficiency expression and purification method for mannanase and its homologue and GLP-1 recombinant fusion protein comprising the following steps:
  • step (1) (2) transforming the recombinant expression vector described in step (1) into Pichia pastoris competent cells, and constructing to obtain recombinant engineering bacteria;
  • step (3) The fermentation broth obtained in step (3) is centrifuged, and the supernatant is purified, concentrated and dried in sequence to obtain the recombinant fusion protein of the mannanase and its homologue and GLP-1.
  • the plasmid is any one of pPICZ ⁇ A, pPICZ ⁇ B, and pPICZ ⁇ C.
  • the strain of Pichia pastoris is any one of X-33, GS115, KM71, SMD1168, and SMD1168H.
  • step (1) the gene sequences encoding GLP-1 and mannanase are respectively shown in SEQ ID NO.1 and SEQ ID NO.2.
  • step (1) the specific operation is:
  • a primer pair was used to clone the target fragment encoding GLP-1 and mannanase or its homolog. After PCR amplification, double digestion was performed. The gene sequence is connected to the pPICZ ⁇ plasmid to complete the construction of the recombinant expression vector.
  • the primer pair includes a primer pair for amplifying the gene sequence encoding GLP-1 and mannanase, and the sequence of the primer pair amplifying the gene sequence encoding GLP-1 is shown in SEQ ID NO.3 and SEQ ID NO. .4; the sequences of the primer pairs for the amplification of the gene sequence encoding mannanase are shown in SEQ ID NO.5 and SEQ ID NO.6.
  • step (3) the amino acid sequence of the fusion protein is shown in SEQ ID NO.7.
  • step (3) the specific steps of fermenting and culturing the recombinant engineered bacteria and inducing expression of fusion protein MANNase-31P are:
  • (S1) inoculate a single colony of the recombinant engineered bacteria in a test tube of YPD liquid culture medium containing bleomycin, shake and culture for 12 hours at 30° C. and 200 rpm; pour the bacterial liquid into a medium containing YPD, Incubate at 30°C and 200rpm for 12h to obtain first-class seed liquid;
  • step (S3) the fermentation culture is high-density fermentation culture
  • the inducer is methanol, and the volume percentage of the inducer added is 0.2%-3%.
  • step (S4) when the fermentation culture is carried out, the initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the ventilation rate is 4 L/min, and the pH is 5.5.
  • step (4) the specific steps of purifying, concentrating and drying the supernatant are as follows:
  • mannanase and its homologue and GLP-1 recombinant fusion protein in the preparation of medicine for treating diabetes or metabolic syndrome induced by high-fat and high-sugar diet.
  • Pichia pastoris is a methanolotrophic yeast that can grow on a medium with methanol as the sole carbon source.
  • the alcohol oxidase gene AOX1 promoter on Pichia pastoris is one of the most stringent promoters known so far.
  • AOX1 plays an important role in the process of methanol oxidation. When methanol is used as the only carbon source, the alcohol oxidase protein produced It accounts for about 30% of the total soluble protein of bacteria.
  • the exogenous gene was placed under the control of the AOX1 promoter to achieve high-level expression under methanol induction.
  • Mannanase is the most important enzyme in the degradation process of mannan, and it is widely used in industry. It can hydrolyze mannan into mannose oligosaccharide. It is absorbed and metabolized by bacteria, such as lactic acid bacteria and bifidobacteria, and mannanase is also not easily degraded by digestive enzymes.
  • the inventor of the present application innovatively recombines mannanase and its homologue protein carrier with GLP-1 to construct a recombinant protein.
  • Pichia pastoris has a strict alcohol oxidase promoter regulation mechanism, can use methanol as the only carbon source, the cell grows fast, and is easy to ferment at high density, so the inventor of the present application uses Pichia pastoris (Pichia pastoris) recombinant protein for high secretion expression system to efficiently express recombinant protein MANNase-GLP-1 and its homologues. After a series of separation and purification methods, high-efficiency, stable and low-cost oral hypoglycemic peptides are obtained, breaking through the common bottleneck of the existing GLP analog fusion proteins that are not resistant to gastric acid and easily degraded by various digestive tract proteases, and realize oral hypoglycemic administration. .
  • the high-efficiency expression and purification of the recombinant fusion protein of mannanase and its homologues and GLP-1 described in the present invention is performed by first synthesizing the sequence from the existing GLP-1 and containing MANNase (mannanase) or its homologues.
  • the two target fragments were cloned on the plasmid of the compound, PCR amplified, and then double-enzyme digestion was performed, and the obtained two fragments were connected to the pPICZ ⁇ plasmid to construct a recombinant expression vector, which was transformed into Pichia
  • recombinant engineering bacteria are constructed, and the recombinant engineering bacteria are fermented and induced to express fusion proteins; the present invention uses Pichia pastoris as engineering strains, which can efficiently express mannanase.
  • the source and GLP-1 form a fusion protein, and after high-density fermentation and culture, the fusion protein can be efficiently secreted and expressed, and has both mannanase activity and GLP-1 activity; in the fermentation process of the present invention, the expression is induced by methanol to obtain
  • the target protein in the supernatant has high expression and high purity; the purification process of the present invention is only filtration and concentration, which is a low-cost and high-efficiency preparation method.
  • the mannanase and its homologue of the present invention and GLP-1 recombinant fusion protein are suitable for injection and oral administration at the same time. It has good application value and provides a basis for further research on the mechanism of the mannanase and its homologue and the GLP-1 recombinant fusion protein in reducing blood sugar and weight loss.
  • Fig. 1 is the fermentation curve diagram of recombinant engineering bacteria MANNase-31P-X-33 described in Example 1 of the present invention
  • FIG. 2 is a schematic diagram of the expression level of recombinant engineering bacteria MANNase-31P-X-33 described in Example 1 of the present invention
  • Figure 3 is a graph showing the statistical analysis and comparison of the blood glucose levels of the mice in the control group and the model group;
  • Figure 4 is a comparison chart of the statistical analysis of the weight levels of mice in the control group and the model group;
  • Figure 5 is a graph comparing the glucose tolerance curves of mice in the control group and the model group
  • Fig. 6 is the HE staining section analysis diagram of the liver and adipose tissue of the mice in the control group and the model group;
  • Fig. 7 is a schematic diagram of detecting changes in the body weight level of mice at different times
  • Fig. 8 is a schematic diagram of detecting changes in blood glucose levels in mice at different times
  • Figure 9 is a comparison chart of the effect of injection on blood glucose levels in mice.
  • Figure 10 is a schematic diagram of the expression level of the recombinant engineering bacteria MANNase-31P-X-33 described in Example 3 of the present invention.
  • YPD medium The YPD medium, fermentation medium and other involved reagents in the following examples are all commercially available products known to those skilled in the art.
  • the present embodiment provides a high-efficiency expression and purification method for a recombinant fusion protein of mannanase and GLP-1 (MANNase-GLP-1), which includes the following steps:
  • the gene sequences encoding GLP-1 and mannanase are respectively shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of the primer pair for amplifying the gene sequence encoding GLP-1 is shown in SEQ ID NO. Shown in NO.3 and SEQ ID NO.4; the sequence of the primer pair for amplifying the gene sequence encoding mannanase is shown in SEQ ID NO.5 and SEQ ID NO.6; the fusion The amino acid sequence of protein MANNase-31P is shown in SEQ ID NO.7;
  • step (1) (2) transforming the recombinant expression vector described in step (1) into Pichia pastoris X-33 competent cells, and constructing recombinant engineering bacteria MANNase-31P-X-33;
  • Seed liquid preparation pick a single colony of the recombinant engineered bacteria MANNase-31P-X-33 and inoculate it into a 5 ml YPD liquid medium test tube containing bleomycin, and shake it for 12 hours at 30°C and 200 rpm; The solution was poured into 50ml of YPD medium, and cultivated at 30°C and 200rpm for 12h to obtain first-class seed solution; the first-class seed solution was inoculated into 500ml of YPD medium at 10% of the inoculation amount, and the Cultivated at 30°C and 200rpm for 22h to obtain secondary seed solution;
  • Fermentation tank culture insert the secondary seed liquid into 4.5L fermentation medium (7.5L fermentation tank) according to 10% of the inoculum, and carry out high-density fermentation culture, the initial fermentation temperature is 30 ° C, and the stirring speed is 300 rpm, The ventilation rate was 4 L/min and the pH was 5.5.
  • the fermentation process includes four stages. The first stage is the growth stage with glycerol as the basic carbon source, which is mainly used for the initial accumulation of cell biomass. With the gradual increase of the cell density, the dissolved oxygen in the medium will increase rapidly.
  • the carbon source is provided by feeding glycerol, the cells continue to grow, and the dissolved oxygen content is kept above 20%; when the cell density reaches 60OD, after a 2h starvation period, it enters the methanol induction stage , when the OD 600 of the fermentation broth reaches more than 60, add the inducer methanol for induction.
  • the volume percentage of methanol added to the fermentation broth is 0.2%.
  • the expression level as shown in Figure 1 and Figure 2 (indicated by arrows), can be seen in the figures: the expression of the target protein is greatly increased. After induction for 5 consecutive days, the target protein is secreted to the outside of the cell. After the induction, the fermented liquid is collected and centrifuged at 6000 rpm for 15 min, and the supernatant is collected; the content of the target protein in the supernatant is about 1-2 mg/ml;
  • step (3) after successively purifying, concentrating and drying the supernatant obtained in step (3), the recombinant MANNase-GLP-1 is obtained;
  • the present embodiment provides a high-efficiency expression and purification method for recombinant fusion protein of mannanase and GLP-1, which comprises the following steps:
  • the gene sequences encoding GLP-1 and mannanase are respectively shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of the primer pair for amplifying the gene sequence encoding GLP-1 is shown in SEQ ID NO. As shown in NO.3 and SEQ ID NO.4, the sequences of the primer pairs for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO.5 and SEQ ID NO.6; the fusion The amino acid sequence of protein MANNase-31P is shown in SEQ ID NO.7;
  • step (2) transforming the recombinant expression vector described in step (1) into Pichia pastoris GS115 competent cells to construct a recombinant engineering strain MANNase-31P-GS115;
  • Seed liquid preparation Pick a single colony of the recombinant engineering bacteria MANNase-31P-GS115 and inoculate it into a 5 ml YPD liquid medium test tube containing bleomycin, and shake it for 12 hours at 30 °C and 200 rpm; pour the bacterial liquid into Put it into 50ml YPD medium, and cultivate it at 30°C and 200rpm for 12h to obtain the first-grade seed liquid; inoculate the first-grade seed liquid in 500ml of YPD medium at 10% of the inoculation amount, at 30°C 22h under the condition of 200rpm to obtain secondary seed liquid;
  • Fermentation tank culture insert the secondary seed liquid into 4.5L fermentation medium (7.5L fermentation tank) according to 10% of the inoculum, and carry out high-density fermentation culture, the initial fermentation temperature is 30 ° C, and the stirring speed is 300 rpm, The ventilation rate was 4 L/min and the pH was 5.5.
  • the fermentation process includes four stages. The first stage is the growth stage with glycerol as the basic carbon source, which is mainly used for the initial accumulation of cell biomass. With the gradual increase of the cell density, the dissolved oxygen in the medium will increase rapidly.
  • the carbon source is provided by feeding glycerol, the cells continue to grow, and the dissolved oxygen content is kept above 20%; when the cell density reaches 120OD, after a 5h starvation period, it enters the methanol induction stage , when the OD 600 of the fermentation broth reaches above 120, add the inducer methanol for induction.
  • the volume percentage of methanol added to the fermentation broth is 3%.
  • samples are taken every four hours to measure the OD600 value of the bacteria, and the samples are retained for SDS-PAGE electrophoresis to detect the protein. expression.
  • the target protein is secreted to the outside of the cell.
  • the fermented liquid is collected and centrifuged at 6000 rpm for 15 min, and the supernatant is collected; the content of the target protein in the supernatant is about 1-2 mg/ml;
  • step (3) after successively purifying, concentrating and drying the supernatant obtained in step (3), the recombinant MANNase-GLP-1 is obtained;
  • the present embodiment provides a high-efficiency expression and purification method for recombinant fusion protein of mannanase and GLP-1, which comprises the following steps:
  • the gene sequences encoding GLP-1 and mannanase are respectively shown in SEQ ID NO.1 and SEQ ID NO.2; the sequence of the primer pair for amplifying the gene sequence encoding GLP-1 is shown in SEQ ID NO. Shown in NO.3 and SEQ ID NO.4; the sequence of the primer pair for amplifying the gene sequence encoding mannanase is shown in SEQ ID NO.5 and SEQ ID NO.6; the fusion The amino acid sequence of protein MANNase-31P is shown in SEQ ID NO.7;
  • step (2) transforming the recombinant expression vector described in step (1) into Pichia pastoris KM71 competent cells to construct a recombinant engineering strain MANNase-31P-KM71;
  • Seed liquid preparation pick a single colony of the recombinant engineering bacteria MANNase-31P-KM71 and inoculate it into a 5 ml YPD liquid medium test tube containing bleomycin, and shake it for 12 hours at 30 ° C and 200 rpm; pour the bacterial liquid into Put it into 50ml YPD medium, and cultivate it at 30°C and 200rpm for 12h to obtain the first-grade seed liquid; inoculate the first-grade seed liquid in 500ml of YPD medium at 10% of the inoculation amount, at 30°C 22h under the condition of 200rpm to obtain secondary seed liquid;
  • Fermentation tank culture insert the secondary seed liquid into 4.5L fermentation medium (7.5L fermentation tank) according to 10% of the inoculum, and carry out high-density fermentation culture, the initial fermentation temperature is 30 ° C, and the stirring speed is 300 rpm, The ventilation rate was 4 L/min and the pH was 5.5.
  • the fermentation process includes four stages. The first stage is the growth stage with glycerol as the basic carbon source, which is mainly used for the initial accumulation of cell biomass. With the gradual increase of the cell density, the dissolved oxygen in the medium will increase rapidly.
  • the glycerol feeding stage and the carbon source is provided by feeding glycerol, the cells continue to grow, and the dissolved oxygen content is kept above 20%; when the cell density reaches 90OD, after a 3.5h starvation period, it enters methanol induction.
  • the stage when the OD600 of the fermentation broth reaches above 90, the inducer methanol is added for induction, and the volume percentage of methanol added to the fermentation broth is 1.6%.
  • samples are taken every four hours to measure the OD600 value of the bacteria, and the samples are retained for SDS-PAGE electrophoresis detection.
  • the amount of protein expression can be seen in the figures: the expression of the target protein is greatly increased. After induction for 5 consecutive days, the target protein is secreted to the outside of the cell. After the induction, the fermented liquid is collected and centrifuged at 6000 rpm for 15 min, and the supernatant is collected; the content of the target protein in the supernatant is about 1-2 mg/ml;
  • step (3) after successively purifying, concentrating and drying the supernatant obtained in step (3), the recombinant MANNase-GLP-1 is obtained;
  • Example 4 The only difference between this Example 4 and Example 1 is that in step (3), the strain of Pichia pastoris is SMD1168, and the others are the same as Example 1.
  • Example 5 The only difference between this Example 5 and Example 1 is that in step (3), the strain of Pichia pastoris is SMD1168H, and the others are the same as Example 1.
  • Example 6 The difference between Example 6 and Example 1 is only that: in step (3), the steps of fermenting and inducing expression of the recombinant engineered bacteria are different; The specific steps of protein MANNase-31P are:
  • mice 100 6-week-old (18-20g) C57-6J mice (4-6 weeks old, male) were kept in separate cages, the temperature of the animal room was controlled at 25 ⁇ 2°C, the humidity was 50 ⁇ 10%, and the light was dark for 12 hours. 12h cycle, adapt to the environment for a week. Mice were randomly divided into cages of 5-6 mice/group. All mice were fasted for 12 h and their body weight and fasting blood-glucose (FBG) were measured. The control group was fed with standard feed, and the model group was fed with high-fat and high-sugar (HFSD) feed for 24 weeks. After the end, the body weight and FBG of the mice in each group were measured.
  • FBG blood-glucose
  • mice on high-fat and high-sugar diet is about 42.5g, and the weight of mice on normal diet is about 30g. (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • Figures 3 and 4 below it can be seen that after 24 weeks of induction with a high-fat and high-sugar diet, the C57-6J mice had a body weight of 41.67% over the normal diet, which was in line with the obesity model standard (20%); In C57-6J mice induced by sugar diet, fasting blood glucose was 22.73% higher than normal diet, and blood glucose was significantly increased.
  • mice on a high-fat and high-sugar diet had obvious fatty liver , and the size of adipocytes in the two groups of mice was also significantly different.
  • mice were randomly divided into 5 groups: fusion protein high-dose group (0.7 mg/kg d), fusion protein low-dose group (0.14 mg/kg d), 30 mg/kg orlistat as the positive control group, normal diet
  • the control group and the high-fat and high-sugar diet negative control group were given the same volume of water, and all the mice were given gavage respectively without changing their diet. Changes in body weight were measured weekly and changes in fasting blood glucose were measured every two weeks. The results are shown in Figures 7 and 8. It can be concluded that the MANNase-GLP-1 fusion protein described in Example 1 of the present invention has obvious weight loss and hypoglycemic effects.
  • mice Fifty 6-week-old (18-20 g) BALB/C mice (male) were randomized into cages of 8/group. All mice were fasted for 12 hours and their body weight and fasting blood-glucose (FBG) were measured. The control group was fed with standard feed for 4 weeks, and the modeling group was fed with high-fat feed for 4 weeks. Mouse body weight and FBG. After fasting for 12 hours, STZ was injected intraperitoneally at a dose of 60 mg/kg ⁇ Bw for 3 consecutive days.
  • FBG blood-glucose
  • mice with FBG ⁇ 11.1 mmol/L and stable for one week were selected as the type II diabetes model mice, with 8 mice/group.
  • the rats were randomly divided into cages for the hypoglycemic experiment by injection administration.
  • mice were randomly divided into three groups: diabetes negative control group (normal saline), diabetes positive control group (Lir), diabetes treatment group (MANNase-GLP-1), 8 mice in each group. After overnight fasting for 12 h, all mice were given D-glucose by gavage at 1.5 g/kg (body weight). MANNase-GLP-1 (250ug/50g) was then injected subcutaneously (s.c.). After 0, 15, 30, 60, 120, 180 min, and 300 min after injection, blood was collected by tail docking, and blood glucose levels were measured with Roche blood glucose test strips. The results are shown in Figure 9.
  • the purpose of the present invention is to provide a method for preparing a recombinant fusion protein of mannanase and its homologue and GLP-1 that can improve the body weight and blood sugar of patients with metabolic syndrome induced by high-fat and high-sugar diet.
  • the high-yield target protein is obtained by the separation and purification method of filtration and concentration.
  • the fermentation expression is high and the separation steps are simple, which solves the problem of the current drug yield of GLP-1 analogs.
  • the fusion protein not only has the effect of hypoglycemic injection, but also can reduce blood sugar and weight through oral administration, and has a good effect in obese and prediabetic patients. application value. At the same time, it provides a basis for further research on the mechanism of mannanase and its homologue and GLP-1 recombinant fusion protein in reducing blood sugar and weight loss.
  • the invention provides a high-efficiency expression and purification method and application of mannanase and its homologue and GLP-1 recombinant fusion protein.
  • the recombinant fusion protein of mannanase and its homologues and GLP-1 was obtained by high-density fermentation of Pichia pastoris, and the recombinant fusion protein of GLP-1 was obtained.
  • the recombinant fusion protein of its homologue and GLP-1 has high fermentation expression and simple separation steps, and solves the limitations of the current GLP-1 analogs such as low yield, high cost, and frequent injection.
  • the mannanase and its homologue and the GLP-1 recombinant fusion protein of the present invention not only have the effect of lowering blood sugar, but also can lower blood sugar and reduce body weight through oral administration, and have great potential in obese and prediabetic patients. good application value. At the same time, it provides a basis for further research on the mechanism of mannanase and its homologues and GLP-1 recombinant fusion protein in reducing blood sugar and weight loss, and has good economic value and application prospect.

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Abstract

提供了一种甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法及应用。通过基因重组技术，利用毕赤酵母高密度发酵诱导分泌表达获得甘露聚糖酶及其同源物与GLP-1重组融合蛋白，经过滤和浓缩的分离纯化方法获得所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白。该甘露聚糖酶及其同源物与GLP-1重组融合蛋白，不仅注射剂具有降血糖效果，还可以通过口服给药进行降血糖和减轻体重，在肥胖及糖尿病前期患者中具有很好的应用价值。

Description

甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法及应用
交叉引用
本申请要求2020年8月10日提交的专利名称为“重组MANNase-GLP-1及同源物的制备及应用”的第202010795389.9号中国专利申请的优先权，其全部公开内容通过引用整体并入本文。
技术领域
本发明属于生物医药技术领域，具体涉及一种甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法及应用。
背景技术
糖尿病是现代社会严重威胁全球人类生命健康的重要疾病之一。糖尿病是一种以慢性高血糖为特征的内分泌代谢综合征。据国家卫计委，目前有50.1％的成年人正处于糖尿病前期，每天都有可能变成新的糖尿病患者。更有数据显示，中国的糖尿病患病率近几十年来快速增长：从80年代的0.67％升高到2010年的11.6％，增加了17倍，而且越来越呈年轻化趋势。
近年来胰高血糖素样肽-1(glucagon-likepeptide-1，GLP-1)不仅具有优异的降糖效果，还有控制体重，调节血脂，改善胰岛β细胞功能等特点，同时低血糖的不良反应发生率较低。GLP-1及其类似物在治疗2型糖尿病及减重方面的优良效果，使其近年来在糖尿病治疗药物市场中逐渐占据重要地位。通常对GLP-1的结构进行修饰，延长其半衰期。目前已上市的长效GLP-1类似物有两种，均融合了大分子蛋白，分别为度拉糖肽和阿必鲁肽，前者是融合了G4免疫白蛋白，后者融合了血清白蛋白，延长了药物代谢的半衰期，可实现每周注射一次。但是阿必鲁肽和度拉糖肽均为每周注射一次的药物，无法实现口服给药的局限性。注射后会出现注射部位及恶心呕吐腹痛等胃肠道不良反应。
甘露聚糖广泛存在于自然界的各种植物中，β-甘露聚糖酶是甘露聚糖降解过程中最重要的酶，在工业中应用十分广泛，可将甘露聚糖水解成甘露寡糖，在动物体内由于缺少相应的酶而不能被充分降解的寡糖，但是它们却能够被动物体内存在的益生菌吸收代谢，例如乳酸菌和双歧杆菌等。代谢的产物包括乙酸、丙酸、丁酸等短链的脂肪酸(SCFA)，反过来SCFA能够对机体产生重要的作用，例如抑制病原细菌的生长，在肌肉、肾脏、心脏、脑等部位供能量。
发明内容
为了解决现有技术存在的上述问题，本发明提供了一种甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法及应用。本发明以毕赤酵母为工程菌株，能高效表达甘露聚糖酶(MANNase)及其同源物，通过将甘露聚糖酶及其同源物与GLP-1构成融合蛋白，经过高密度发酵培养，能够高效分泌表达该融合蛋白，既具有甘露聚糖酶及其同源物活性，又具有GLP-1活性，发酵过程中通过甲醇诱导表达，上清液中目的蛋白表达量高，纯度高，且纯化工艺仅仅是过滤浓缩，是一种低成本高效率的制备方法，与此同时，该融合蛋白注射和口服给药均具有良好的降血糖效果。
本发明所采用的技术方案为：
一种甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，包括如下步骤：
(1)将编码GLP-1与甘露聚糖酶或其同源物的基因序列连接到pPICZα质粒上，得到重组表达载体；
(2)将步骤(1)所述重组表达载体转化到毕赤酵母感受态细胞中，构建得到重组工程菌；
(3)将所述重组工程菌进行发酵培养，诱导表达融合蛋白；
(4)步骤(3)得到发酵液进行离心，取上清液依次进行纯化、浓缩、干燥后，即得所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白。
步骤(1)中，所述质粒为：pPICZαA、pPICZαB、pPICZαC中的任一种。
步骤(3)中，所述毕赤酵母的菌株为X-33、GS115、KM71、SMD1168、SMD1168H中的任一种。
步骤(1)中，所述编码GLP-1与甘露聚糖酶的基因序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示。
步骤(1)中，具体操作为：
采用引物对克隆编码GLP-1和甘露聚糖酶或其同源物的目的片段，PCR扩增后进行双酶切，之后将得到的编码GLP-1与甘露聚糖酶或其同源物的基因序列连接到pPICZα质粒上，即完成重组表达载体的构建。
所述引物对包括扩增编码GLP-1与甘露聚糖酶的基因序列的引物对，所述扩增编码GLP-1的基因序列引物对的序列如SEQ ID NO.3所示和SEQ ID NO.4所示；所述扩增编码甘露聚糖酶的基因序列引物对的序列如SEQ ID NO.5所示和SEQ ID NO.6所示。
步骤(3)中，所述融合蛋白的氨基酸序列如SEQ ID NO.7所示。
步骤(3)中，所述重组工程菌发酵培养、诱导表达融合蛋白MANNase-31P的具体步骤为：
(S1)将所述重组工程菌的单菌落接种于含有博来霉素的YPD液体培养基试管中，在30℃、200rpm条件下振荡培养12h；将菌液倒入装有YPD培养基中，在30℃、200rpm条件下培养12h，即得一级种子液；
(S2)将所述一级种子液按10％的接种量接种于YPD培养基中，在30℃、200rpm条件下培养22h，即得二级种子液；
(S3)将所述二级种子液按10％的接种量接入发酵培养基中，进行发酵培养，当发酵液OD 600达到60-120以上时加入诱导剂进行诱导，诱导完成后放罐，离心收集菌体。
步骤(S3)中，所述发酵培养为高密度发酵培养；
所述诱导剂为甲醇，加入所述诱导剂的体积百分数为0.2％-3％。
步骤(S4)中，进行所述发酵培养时，初始发酵温度为30℃，搅拌速度为300rpm，通气量为4L/min，pH为5.5。
步骤(4)中，对所述上清液进行纯化、浓缩、干燥的具体步骤如下：
(SS1)取上清液，先用0.8um的滤膜过滤，再用0.2um的滤膜过滤，收集滤液；
(SS2)将所述滤液先用超滤膜包浓缩10倍，加去离子水后再浓缩10倍，得到浓缩液；
(SS3)将所述浓缩液进行冷冻干燥，即得所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白。
所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白在制备治疗糖尿病或高脂高糖饮食诱导的代谢综合症药物中的应用。
毕赤酵母是一种甲醇营养型酵母，能在以甲醇为唯一碳源的培养基上生长。毕赤酵母上的醇氧化酶基因AOX1启动子是目前已知的最严格的启动子之一，AOX1在甲醇氧化过程中起重要作用，当以甲醇为唯一碳源时，产生的醇氧化酶蛋白约占菌体可溶性蛋白总量的30％。将外源基因置于AOX1启动子的控制之下，在甲醇诱导下可实现高水平表达。
甘露聚糖酶是甘露聚糖降解过程中最重要的酶，在工业中应用十分广泛，可将甘露聚糖水解成甘露寡糖，甘露寡糖作为一种益生元，能够被动物体内存在的益生菌吸收代谢，例如乳酸菌和双歧杆菌等，同时甘露聚糖酶还具有不易被消化酶降解的特性。本申请发明人创新性的将甘露聚糖酶及其同源物蛋白载体与GLP-1重组，构建重组蛋白。毕赤酵母具有严格的醇氧化酶启动子调控机制，可以利用甲醇作为唯一碳源，细胞生长快，易于高密度发酵，因此本申请发明人通过采用毕赤酵母(Pichia pastoris)重组蛋白高分泌表达体系，高效表达重组蛋白MANNase-GLP-1及其同源物。经过一系列分离纯化方法，获得高效、稳定、低成本的口服降糖肽，突破现有GLP类似物融合蛋白不耐胃酸且易被各种消化道蛋白酶降解的共性瓶颈，实现降糖口服给药。
本发明的有益效果为：
本发明所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化，通过先从已有的GLP-1合成序列和含有MANNase(甘露聚糖酶)或其同源物的质粒上将两个目的片段分别进行克隆，PCR扩增，之后进行双酶切，得到的两个片段连接到pPICZα质粒上，构建得到重组表达载体，将所述重组表达载体转化到毕赤酵母感受态细胞中，构建得到重组工程菌，所述重组工程菌发酵诱导表达融合蛋白；本发明以毕赤酵母为工程菌株，能高效表达甘露聚糖酶，通过将甘露聚糖酶及其同源物与GLP-1构成融合蛋白，经过高密度发酵培养，能够高效分泌表达该融合蛋白，既具有甘露聚糖酶活性，又具有GLP-1活性；本发明发酵过程中通过甲醇诱导表达，获得上清液中目的蛋白表达量高，纯度高；本发明纯化工艺仅仅是过滤浓缩，是一种低成本高效率的制备方法。本发明所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白，同时适合于注射和口服给药，均具有良好的降血糖和减轻体重效果，在肥胖及糖尿病前期患者中具有很好的应用价值，同时为进一步研究所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白的降血糖及减重的机理提供基础。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明实施例1所述重组工程菌MANNase-31P-X-33的发酵曲线图；
图2为本发明实施例1所述重组工程菌MANNase-31P-X-33的表达量示意图；
图3为对照组和模型组小鼠的血糖水平统计分析对比图；
图4为对照组和模型组小鼠的体重水平统计分析对比图；
图5为对照组和模型组小鼠的糖耐量曲线对比图；
图6为对照组和模型组小鼠的的肝脏和脂肪组织的HE染色切片分析图；
图7为不同时间检测小鼠体重水平变化示意图；
图8为不同时间检测小鼠血糖水平变化示意图；
图9为注射给药对小鼠血糖水平的影响对比图；
图10为本发明实施例3所述重组工程菌MANNase-31P-X-33的表达量示意图。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚，下面将对本发明的技术方案进行详细的描述。显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动的前提下所得到的所有其它实施方式，都属于本发明所保护的范围。
下述实施例中的YPD培养基、发酵培养基以及其他涉及到的试剂等均为本领域技术人员所知晓的市售产品。
实施例1
本实施例提供一种甘露聚糖酶与GLP-1重组融合蛋白(MANNase-GLP-1)的高效表达纯化方法，其包括如下步骤：
(1)采用引物对克隆编码GLP-1和甘露聚糖酶的目的片段，PCR扩增后进行双酶切，之后将得到的编码GLP-1与甘露聚糖酶的基因序列连接到pPICZαA质粒上，构建得到重组表达载体pPICZαA-MANNase-31P；
所述编码GLP-1与甘露聚糖酶的基因序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示；所述扩增编码GLP-1的基因序列引物对的序列如SEQ ID NO.3所示和SEQ ID NO.4所示；所述扩增编码甘露聚糖酶的基因序列引物对的序列如SEQ ID NO.5所示和SEQ ID NO.6所示；所述融 合蛋白MANNase-31P的氨基酸序列如SEQ ID NO.7所示；
(2)将步骤(1)所述重组表达载体转化到毕赤酵母X-33感受态细胞中，构建得到重组工程菌MANNase-31P-X-33；
(3)将所述重组工程菌进行发酵培养，诱导表达融合蛋白MANNase-31P；具体步骤为：
种子液制备：挑取所述重组工程菌MANNase-31P-X-33的单菌落接种于5ml含有博来霉素的YPD液体培养基试管中，在30℃、200rpm条件下振荡培养12h；将菌液倒入装有50mlYPD培养基中，在30℃、200rpm条件下培养12h，即得一级种子液；将所述一级种子液按10％的接种量接种于500ml的YPD培养基中，在30℃、200rpm条件下培养22h，即得二级种子液；
发酵罐培养：将所述二级种子液按10％的接种量接入4.5L发酵培养基(7.5L发酵罐)中，进行高密度发酵培养，初始发酵温度为30℃，搅拌速度为300rpm，通气量为4L/min，pH为5.5。发酵过程包括四个阶段，第一阶段是以甘油为基础碳源的生长阶段，主要是用于细胞生物量的初步积累，随着菌体密度的逐渐增加，培养基的溶氧量会迅速升高，此时进入甘油补料阶段，通过流加甘油提供碳源，菌体继续生长，溶氧量保持在20％以上；当菌体密度达到60OD后，经过2h的饥饿期，进入甲醇诱导阶段，当发酵液OD 600达到60以上时加入诱导剂甲醇进行诱导，加入甲醇占发酵液的体积百分数为0.2％，期间每隔四小时取样测菌体OD600值，留样进行SDS-PAGE电泳检测蛋白表达量，如图1和图2中所示(箭头所指)，图中可以看出：目标蛋白表达大幅度提高。经过连续5天诱导后，目的蛋白分泌到胞外，诱导结束后下罐，收集发酵液经6000rpm离心15min后，收集上清液；上清液中目的蛋白的含量约为1-2mg/ml；
(4)对步骤(3)得到上清液依次进行纯化、浓缩、干燥后，即得所述重组MANNase-GLP-1；
对所述上清液进行纯化、浓缩、干燥的具体步骤如下：
(SS1)取上清液，先用0.8um的滤膜过滤，再用0.2um的滤膜过滤，收集滤液；
(SS2)将所述滤液(约3L)先用10kd超滤膜包浓缩10倍，加3L去离子水后再浓缩10倍，得到浓缩液；
(SS3)将所述浓缩液进行冷冻干燥，即得所述重组MANNase-GLP-1。
实施例2
本实施例提供一种甘露聚糖酶与GLP-1重组融合蛋白的高效表达纯化方法，其包括如下步骤：
(1)采用引物对克隆编码GLP-1和甘露聚糖酶的目的片段，PCR扩增后进行双酶切，之后将得到的编码GLP-1与甘露聚糖酶的基因序列连接到pPICZαB质粒上，构建得到重组表达载体pPICZαB-MANNase-31P；
所述编码GLP-1与甘露聚糖酶的基因序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示；所述扩增编码GLP-1的基因序列引物对的序列如SEQ ID NO.3所示和SEQ ID NO.4所示，所述扩增编码甘露聚糖酶的基因序列引物对的序列如SEQ ID NO.5所示和SEQ ID NO.6所示；所述融合蛋白MANNase-31P的氨基酸序列如SEQ ID NO.7所示；
(2)将步骤(1)所述重组表达载体转化到毕赤酵母GS115感受态细胞中，构建得到重组工程菌MANNase-31P-GS115；
(3)将所述重组工程菌进行发酵培养，诱导表达融合蛋白MANNase-31P；具体步骤为：
种子液制备：挑取所述重组工程菌MANNase-31P-GS115的单菌落接种于5ml含有博来霉素的YPD液体培养基试管中，在30℃、200rpm条件下振荡培养12h；将菌液倒入装有50mlYPD培养基中，在30℃、200rpm条件下培养12h，即得一级种子液；将所述一级种子液按10％的接种量接种于500ml的YPD培养基中，在30℃、200rpm条件下培养22h，即得二级种子液；
发酵罐培养：将所述二级种子液按10％的接种量接入4.5L发酵培养基(7.5L发酵罐)中，进行高密度发酵培养，初始发酵温度为30℃，搅拌速度为300rpm，通气量为4L/min，pH为5.5。发酵过程包括四个阶段，第一阶段是以甘油为基础碳源的生长阶段，主要是用于细胞生物量的初步积累，随着菌体密度的逐渐增加，培养基的溶氧量会迅速升高，此时进入甘油补料阶段，通过流加甘油提供碳源，菌体继续生长，溶氧量保持在20％以上；当菌体密度达到120OD后，经过5h的饥饿期，进入甲醇诱导阶段，当发酵液OD 600达到120以上时加入诱导剂甲醇进行诱导，加入甲醇占发酵液的体积百分数为3％，期间每隔四小时取样测菌体OD600值，留样进行SDS-PAGE电泳检测蛋白表达量。经过连续5天诱导后，目的蛋白分泌到胞外，诱导结束后下罐，收集发酵液经6000rpm离心15min后，收集上清液；上清液中目的蛋白的含量约为1-2mg/ml；
(4)对步骤(3)得到上清液依次进行纯化、浓缩、干燥后，即得所述重组MANNase-GLP-1；
对所述上清液进行纯化、浓缩、干燥的具体步骤如下：
(SS1)取上清液，先用0.8um的滤膜过滤，再用0.2um的滤膜过滤，收集滤液；
(SS2)将所述滤液(约3L)先用10kd超滤膜包浓缩10倍，加3L去离子水后再浓缩10倍，得到浓缩液；
(SS3)将所述浓缩液进行冷冻干燥，即得所述重组MANNase-GLP-1。
实施例3
本实施例提供一种甘露聚糖酶与GLP-1重组融合蛋白的高效表达纯化方法，其包括如下步骤：
(1)采用引物对克隆编码GLP-1和甘露聚糖酶的目的片段，PCR扩增后进行双酶切，之后将得到的编码GLP-1与甘露聚糖酶的基因序列连接到pPICZαC质粒上，构建得到重组表达载体pPICZαC-MANNase-31P；
所述编码GLP-1与甘露聚糖酶的基因序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示；所述扩增编码GLP-1的基因序列引物对的序列如SEQ ID NO.3所示和SEQ ID NO.4所示；所述扩增编码甘露聚糖酶的基因序列引物对的序列如SEQ ID NO.5所示和SEQ ID NO.6所示；所述融合蛋白MANNase-31P的氨基酸序列如SEQ ID NO.7所示；
(2)将步骤(1)所述重组表达载体转化到毕赤酵母KM71感受态细胞中，构建得到重组工程菌MANNase-31P-KM71；
(3)将所述重组工程菌进行发酵培养，诱导表达融合蛋白MANNase-31P；具体步骤为：
种子液制备：挑取所述重组工程菌MANNase-31P-KM71的单菌落接种于5ml含有博来霉素的YPD液体培养基试管中，在30℃、200rpm条件下振荡培养12h；将菌液倒入装有50mlYPD培养基中，在30℃、200rpm条件下培养12h，即得一级种子液；将所述一级种子液按10％的接种量接种于500ml的YPD培养基中，在30℃、200rpm条件下培养22h，即得二级种子液；
发酵罐培养：将所述二级种子液按10％的接种量接入4.5L发酵培养基(7.5L发酵罐)中，进行高密度发酵培养，初始发酵温度为30℃，搅拌速度为300rpm，通气量为4L/min，pH为5.5。发酵过程包括四个阶段，第一阶段是以甘油为基础碳源的生长阶段，主要是用于细胞生物量的初步积累，随着菌体密度的逐渐增加，培养基的溶氧量会迅速升高，此时进入甘油补料阶段，通过流加甘油提供碳源，菌体继续生长，溶氧量保持在20％以上；当菌体密度达到90OD后，经过3.5h的饥饿期，进入甲醇诱导阶段，当发酵液OD 600达到90以上时加入诱导剂甲醇进行诱导，加入甲醇占发酵液的体积百分数为1.6％，期间每隔四小时取样测菌体OD600值，留样进行SDS-PAGE电泳检测蛋白表达量，如图1和图2中所示(箭头所指)，图中可以看出：目标蛋白表达大幅度提高。经过连续5天诱导后，目的蛋白分泌到胞外，诱导结束后下罐，收集发酵液经6000rpm离 心15min后，收集上清液；上清液中目的蛋白的含量约为1-2mg/ml；
(4)对步骤(3)得到上清液依次进行纯化、浓缩、干燥后，即得所述重组MANNase-GLP-1；
对所述上清液进行纯化、浓缩、干燥的具体步骤如下：
(SS1)取上清液，先用0.8um的滤膜过滤，再用0.2um的滤膜过滤，收集滤液；
(SS2)将所述滤液(约3L)先用10kd超滤膜包浓缩10倍，加3L去离子水后再浓缩10倍，得到浓缩液；
(SS3)将所述浓缩液进行冷冻干燥，即得所述重组MANNase-GLP-1。
实施例4
本实施例4与实施例1的区别仅在于，步骤(3)中，所述毕赤酵母的菌株为SMD1168，其他均与实施例1相同。
实施例5
本实施例5与实施例1的区别仅在于，步骤(3)中，所述毕赤酵母的菌株为SMD1168H，其他均与实施例1相同。
实施例6
本实施例6与实施例1的区别仅在于：步骤(3)中，对所述重组工程菌进行发酵培养、诱导表达的步骤不同；本实施例中所述重组工程菌发酵培养、诱导表达融合蛋白MANNase-31P的具体步骤为：
将构建成功的毕赤酵母表达工程菌MANNase-31P-X-33挑取单菌落，接种到5ml的YPD液体培养基，于30℃、250rpm摇床过夜培养。将培养好的菌种按1％的比例接种于BMGY培养基，继续震荡培养24h，之后再将菌体转入BMMY培养基中，调整OD600为1.0左右，继续于30℃、200rpm摇床中培养中诱导培养，每24h按照体积百分比0.5％加入甲醇，连续诱导4天。上清液中目的蛋白的含量约为0.2mg/ml。SDS-PAGE电泳检测蛋白表达量如图10所示。
实验例
一、实施例1所得重组MANNase-GLP-1在高脂高糖饮食诱导的代谢综合症中的应用
1-1)高脂高糖饮食诱导代谢综合征小鼠模型的构建
100只6周龄(18-20g)C57-6J小鼠(4-6周龄，雄性)，将小鼠分笼饲养，控制动物房温度25±2℃，湿度50±10％，光照12h黑暗12h循环，适应环境一周。将小鼠以5-6只/组随机分笼。所有小鼠禁食12h后测定体重及空腹血糖(Fasting blood-glucose，FBG)，对照组以标准饲料继续饲喂，模型组以高脂高糖(HFSD)饲料持续饲喂24周。结束后，测定各组小鼠体重及FBG。
高脂高糖饮食小鼠的体重约为42.5g左右，正常饮食小鼠体重约30g左右，高脂高糖饮食小鼠的空腹血糖约5.67，正常饮食小鼠的空腹血糖4.62，具有统计学差异(*P<0.05，**P<0.01，***P<0.001)。如下图3和图4所示，可以看出：C57-6J小鼠经过高脂高糖饮食诱导24周后，体重超过正常饮食41.67％，符合肥胖模型的标准(20％)；经过高脂高糖饮食诱导的C57-6J小鼠，空腹血糖超过正常饮食22.73％，血糖显著升高。
普通饮食小鼠和高脂高糖小鼠过夜禁食12h后，按2g/kg(体重)灌胃D-葡萄糖，分别于0、0.5、1.0、1.5、2.0、2.5h的血糖，得到口服葡萄糖耐量(OGTT)曲线，如图5所示，可以看出：经过高脂高糖饮食诱导24周后，其葡萄糖耐量明显受损。口服葡萄糖耐量试验是一种葡萄糖负荷试验，糖耐量受损表明胰岛β细胞功能和机体对血糖的调节能力下降，用以诊断糖尿病前期的轻度高血糖。
对高脂高糖饮食小鼠和正常饮食小鼠的肝脏和脂肪组织进行HE染色切片分析，如图6所示，从图中可以看出：高脂高糖饮食的小鼠有明显的脂肪肝，且两组小鼠脂肪细胞的大小也有显著差异。
建模成功后，以11只/组进行随机分组，加上正常饲料组，进行后续灌胃实验。
1-2)MANNase-GLP-1口服给药对高脂高糖饮食诱导的代谢综合症的影响
将小鼠随机分成5组：融合蛋白高剂量组(0.7mg/kg·d)、融合蛋白低剂量组(0.14mg/kg·d)、30mg/kg奥利司他作为正对照组，正常饲料对照组和高脂高糖饲料负对照组均给予相同体积的水，所有小鼠在不改变饮食的情况下，分别进行灌胃。测定每周体重变化，每两周测定空腹血糖的变化。结果如图7和图8所示。可以得出：本发明实施例1所述MANNase-GLP-1融合蛋白具有明显的减重和降血糖效果。
二、实施例1所得重组MANNase-GLP-1注射给药对Ⅱ型糖尿病的影响
2-1)Ⅱ型糖尿病小鼠模型的构建：
将50只6周龄(18-20g)BALB/C小鼠(雄)以8只/组随机分笼。所有小鼠禁食12h后测定体重及空腹血糖(Fasting blood-glucose，FBG)，对照组以标准饲料继续饲喂4周，建模组以高脂饲料持续饲喂4周后，测定各组小鼠体重及FBG。禁食12h后，按60mg/kg·Bw的剂量腹腔注射STZ，连续注射3天。
分别于建模后第3、7、10、14天测定建模组小鼠体重及FBG，选取FBG≥11.1mmol/L且稳定一周的小鼠作为Ⅱ型糖尿病模型小鼠，以8只/组随机分笼进行注射给药降血糖实验。
将小鼠随机分三组：糖尿病负对照组(生理盐水)，糖尿病正对照组(Lir)，糖尿病治疗组(MANNase-GLP-1)，每组8只小鼠。所有小鼠过夜禁食12h后，按1.5g/kg(体重)灌胃D-葡萄糖。然后皮下注射(s.c.)MANNase-GLP-1(250ug/50g)。注射后0、15、30、60、120、180min、300min后断尾取血，用罗氏血糖试纸测定血糖水平。结果如图9所示。
本发明的目的在于提供一种能够改善高脂高糖饮食诱导的代谢综合症患者体重和血糖甘露聚糖酶及其同源物与GLP-1重组融合蛋白的制备方法，通过基因重组技术，利用毕赤酵母高密度发酵诱导分泌表达获得可 溶性蛋白后，经过滤和浓缩的分离纯化方法获得高收率的目的蛋白，发酵表达量高、分离步骤简单，解决了目前GLP-1类似物药物收率低、成本高、需要频繁注射等局限性，更重要的是，该融合蛋白不仅注射剂具有降血糖效果，还可以通过口服给药进行降血糖和减轻体重，在肥胖及糖尿病前期患者中具有很好的应用价值。同时为进一步研究甘露聚糖酶及其同源物与GLP-1重组融合蛋白的降血糖及减重的机理提供基础。
以上所述，仅为本发明的具体实施方式，但本发明的保护范围并不局限于此，任何熟悉本技术领域的技术人员在本发明揭露的技术范围内，可轻易想到变化或替换，都应涵盖在本发明的保护范围之内。因此，本发明的保护范围应以所述权利要求的保护范围为准。
工业实用性
本发明提供一种甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法及应用。通过基因重组技术，利用毕赤酵母高密度发酵诱导分泌表达获得甘露聚糖酶及其同源物与GLP-1重组融合蛋白，经过滤和浓缩的分离纯化方法获得高收率的甘露聚糖酶及其同源物与GLP-1重组融合蛋白，发酵表达量高、分离步骤简单，解决了目前GLP-1类似物药物收率低、成本高、需要频繁注射等局限性。本发明所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白，不仅注射剂具有降血糖效果，还可以通过口服给药进行降血糖和减轻体重，在肥胖及糖尿病前期患者中具有很好的应用价值。同时为进一步研究甘露聚糖酶及其同源物与GLP-1重组融合蛋白的降血糖及减重的机理提供基础，具有较好的经济价值和应用前景。

Claims (10)

  1. 一种甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，包括如下步骤：
    (1)将编码GLP-1与甘露聚糖酶或其同源物的基因序列连接到pPICZα质粒上，得到重组表达载体；
    (2)将步骤(1)所述重组表达载体转化到毕赤酵母感受态细胞中，构建得到重组工程菌；
    (3)将所述重组工程菌进行发酵培养，诱导表达融合蛋白；
    (4)步骤(3)得到发酵液进行离心，取上清液依次进行纯化、浓缩、干燥后，即得所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白。
  2. 根据权利要求1所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，步骤(1)中，所述编码GLP-1与甘露聚糖酶的基因序列分别如SEQ ID NO.1所示和SEQ ID NO.2所示。
  3. 根据权利要求1所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，步骤(1)中，具体操作为：
    采用引物对克隆编码GLP-1和甘露聚糖酶或其同源物的目的片段，PCR扩增后进行双酶切，之后将得到的编码GLP-1与甘露聚糖酶或其同源物的基因序列连接到pPICZα质粒上，即完成重组表达载体的构建。
  4. 根据权利要求3所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，所述引物对包括扩增编码GLP-1与甘露聚糖酶的基因序列的引物对，所述扩增编码GLP-1的基因序列引物对的序列如SEQ ID NO.3所示和SEQ ID NO.4所示；
    所述扩增编码甘露聚糖酶的基因序列引物对的序列如SEQ ID NO.5所示和SEQ ID NO.6所示。
  5. 根据权利要求1所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，步骤(3)中，所述融合蛋白的氨基酸序列如SEQ ID NO.7所示。
  6. 根据权利要求1所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，步骤(3)中，所述重组工程菌发酵培养、诱导表达融合蛋白的具体步骤为：
    (S1)将所述重组工程菌的单菌落接种于含有博来霉素的YPD液体培养基试管中，在30℃、200rpm条件下振荡培养12h；将菌液倒入装有YPD培养基中，在30℃、200rpm条件下培养12h，即得一级种子液；
    (S2)将所述一级种子液按10％的接种量接种于YPD培养基中，在30℃、200rpm条件下培养22h，即得二级种子液；
    (S3)将所述二级种子液按10％的接种量接入发酵培养基中，进行发酵培养，当发酵液OD 600达到60-120以上时加入诱导剂进行诱导，诱导完成后放罐，离心收集菌体。
  7. 根据权利要求5所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，步骤(S3)中，所述发酵培养为高密度发酵培养；
    所述诱导剂为甲醇，加入所述诱导剂的体积百分数为0.2％-3％。
  8. 根据权利要求5所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，步骤(S4)中，进行所述发酵培养时，初始发酵温度为30℃，搅拌速度为300rpm，通气量为4L/min，pH为5.5。
  9. 根据权利要求1所述的甘露聚糖酶及其同源物与GLP-1重组融合蛋白的高效表达纯化方法，其特征在于，步骤(4)中，对所述上清液进行纯化、浓缩、干燥的具体步骤如下：
    (SS1)取上清液，先用0.8um的滤膜过滤，再用0.2um的滤膜过滤，收集滤液；
    (SS2)将所述滤液先用超滤膜包浓缩10倍，加去离子水后再浓缩10倍，得到浓缩液；
    (SS3)将所述浓缩液进行冷冻干燥，即得所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白。
  10. 所述甘露聚糖酶及其同源物与GLP-1重组融合蛋白在制备治疗糖尿病或高脂高糖饮食诱导的代谢综合症药物中的应用。
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