METHOD FOR THE EFFICIENT EXPRESSION AND PURIFICATION AND APPLICATION OF A RECOMBINANT FUSION PROTEIN OF MANNASE AND HOMOLOGUES THEREOF AND GLP-1

Abstract

The invention relates to method for the efficient expression and purification and application of a recombinant fusion protein of MANNase and homologues thereof and GLP-1. Through genetic recombination technology, soluble proteins are obtained by using high density fermentation of Pichia pastoris to induce secretory expression, followed by isolation and purification by filtration and concentration to obtain high yield of target proteins. The high fermentation expression and simple isolation steps solve the limitations of the current GLP-1 analogs such as low drug yield, high cost and the need for frequent injections. More importantly, the fusion protein has hypoglycemic effect not only by injection, but also by oral administration for hypoglycemia and weight reduction, which has good application value in obese and pre-diabetic patients. It also provides a basis for further research on the mechanism of hypoglycemia and weight loss of mannanase and homologues thereof with GLP-1 recombinant fusion protein.

Claims

1. A method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1, comprising the following steps: (1) ligating a gene sequence of the gene encoding GLP-1 and mannanase or homologues thereof to a pPICZ-a plasmid, to obtain a recombinant expression vector; (2) transforming the recombinant expression vector in step (1) into Pichia pastoris competent cells to construct recombinant engineering yeast; (3) fermenting and culturing the recombinant engineering yeast, to induce expression of the fusion protein; (4) centrifuging the fermentation solution obtained from step (3), and the supernatant purified, concentrated, and dried in turn to obtain the MANNase and homologues thereof and GLP-1 recombinant fusion protein.

2. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 1, wherein in step (1), the sequences of the genes of encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively.

3. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 1, wherein in step (1), this was done as follows: the target fragments encoding GLP-1 and mannanase or homologues thereof are cloned using primer pairs; after PCR amplification and double digestion, the obtained gene sequences encoding GLP-1 and mannanase or homologues thereof are ligated to the pPICZα plasmid, which completed the construction of the recombinant expression vector.

4. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 3, wherein the primer pairs comprises a primer pair for amplifying the gene sequence encoding GLP-1 with mannanase, the sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4, and the sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6.

5. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 1, wherein in step (3), the amino acid sequence of the fusion protein is shown in SEQ ID NO. 7.

6. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 1, wherein in step (3), the specific steps for fermenting and culturing the recombinant engineering yeast and inducing the expression of the fusion protein are as follow: (S1) inoculating a single colony of the recombinant engineering yeast into a test tube of YPD liquid medium containing bleomycin, and incubating it with shaking at 30° C. and 200 rpm for 12 h; pouring the yeast solution into the YPD liquid medium and incubating it at 30° C. and 200 rpm for 12 h to obtain a primary seed solution; (S2) inoculating the primary seed solution in YPD medium at 10% inoculum and incubating for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution; and (S3) adding the secondary seed solution into the fermentation culture medium at 10% inoculum for fermentation culture, and adding the induction agent when the OD600 of the fermentation broth reaches above 60-120, and releasing the jar after induction and collecting the yeast by centrifugation.

7. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 5, wherein in step (S3), the fermentation culture is a high density fermentation culture; and the inducer is methanol, and the volume percentage of the inducer added is 0.2%-3%.

8. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 5, wherein in step (S4), the initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4 L/min, and the pH is 5.5 when carrying out the fermentation culture.

9. The method for the efficient expression and purification of a recombinant fusion protein of MANNase and homologues thereof and GLP-1 according to claim 1, wherein in step (4), the specific steps for purifying, concentrating and drying the supernatant are as follows: (SS1) taking the supernatant and filter it through a 0.8 um membrane, then a 0.2 um membrane, and collecting the filtrate; (SS2) concentrating the filtrate first 10 times with an ultrafiltration membrane pack, and then 10 times with deionized water to obtain a concentrate; and (SS3) freeze-drying the concentrate to obtain the mannanase and homologues thereof and GLP-1 recombinant fusion protein.

10. Application of the recombinant fusion protein of MANNase and homologues thereof and GLP-1 in the preparation of drugs for the treatment of diabetes or high-fat, high-glucose diet-induced metabolic syndrome.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0036] In order to more clearly illustrate the technical solutions in the embodiments or prior art of the invention, the following is a brief description of the accompanying drawings that need to be used in the description of the embodiments or prior art. Obviously, the accompanying drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained from them without creative effort by a person of ordinary skill in the art.

[0037] FIG. 1 is a fermentation curve diagram of the recombinant engineering yeast MANNase-31P-X-33 as described in Example 1 of the present invention;

[0038] FIG. 2 is a schematic representation of the expression of the recombinant engineering yeast MANNase-31P-X-33 as described in Example 1 of the present invention;

[0039] FIG. 3 is a schematic representation of the comparative statistical analysis of the blood glucose levels in the control and model mice;

[0040] FIG. 4 is a schematic representation of the comparative statistical analysis of the body weight levels in the control and model mice;

[0041] FIG. 5 is a schematic representation of the comparison of glucose tolerance curves between control and model mice.

[0042] FIG. 6 is a schematic representation of the HE stained sections of liver and adipose tissue of control and model mice;

[0043] FIG. 7 is a schematic representation of the changes in body weight levels of mice tested at different times;

[0044] FIG. 8 is a schematic representation of the changes in blood glucose levels of mice tested at different times;

[0045] FIG. 9 is a schematic representation of the comparison of the effects of injectable administration on blood glucose levels in mice;

[0046] FIG. 10 is a schematic representation of the expression of the recombinant yeast MANNase-31P-X-33 as described in Example 3 of the present invention.

DESCRIPTION OF THE EMBODIMENTS

[0047] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are described in detail below. Obviously, the described embodiments are only a part of the embodiments of the invention, and not all of them. Based on the embodiments in the present invention, all other embodiments obtained by a person of ordinary skill in the art without making creative labor fall within the scope of protection of the present invention.

[0048] The YPD medium, fermentation medium and other reagents involved in the following examples are commercially available products known to those skilled in the art.

Embodiment 1

[0049] This embodiment provides a method for the efficient expression and purification of a recombinant fusion protein of mannanase and GLP-1 (MANNase-GLP-1), including the steps as follow. [0050] (1) The target fragments encoding GLP-1 and mannanase were cloned using primer pairs, amplified by PCR and then double digested, after which the obtained gene sequences encoding GLP-1 and mannanase were ligated to the pPICZαA plasmid to construct a recombinant expression vector pPICZαA-MANNase-31P.

[0051] The sequences of the genes encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of said fusion protein MANNase-31P is shown in SEQ ID NO.7. [0052] (2) the recombinant expression vector in step (1) was transformed into Pichia pastoris X-33 competent cells to construct recombinant engineering yeast MANNase-31P-X-33. [0053] (3) The reorganized engineering yeast was fermented and cultured to induce expression of the fusion protein MANNase-31P. The specific steps were as follow.

[0054] Seed solution preparation: A single colony of the recombinant engineering yeast MANNase-31P-X-33 was inoculated into a test tube of 5 ml of YPD liquid medium containing bleomycin, incubating which with shaking at 30° C. and 200 rpm for 12 h. The yeast solution was poured into 50 ml of YPD liquid medium, incubating which at 30° C. and 200 rpm for 12 h to obtain a primary seed solution. The primary seed solution was inoculated in 500 ml of YPD medium at 10% inoculum, incubating which for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution.

[0055] Fermentation tank culture: The secondary seed solution was added to 4.5 L fermentation medium (7.5 L fermentation tank) at 10% inoculum for high density fermentation culture. The initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4 L/min, and the pH is 5.5. The fermentation process includes four stages, the first stage was the growth stage with glycerol as the basic carbon source, mainly for the initial accumulation of cell biomass. With the gradual increase in the density of the yeast, the dissolved oxygen of the medium would rise rapidly, at this time to enter the glycerol replenishment stage, through the flow of glycerol to provide a carbon source, the yeast continued to grow, the dissolved oxygen was maintained at more than 20%. When the density of the yeast reaches 60 OD, after a 2 h starvation period, it entered the methanol induction phase. When the OD.sub.600 of the fermentation broth reached above 60, the induction agent methanol was added and the percentage of methanol in the volume of the fermentation broth was 0.2%. During the period, the OD.sub.600 value of the fermentation broth was measured by sampling every four hours, and the samples were kept for SDS-PAGE electrophoresis to detect the protein expression, as shown in FIG. 1 and FIG. 2 (indicated by arrows). As can be seen in the figures, the expression of the target protein was substantially increased. After 5 days of induction, the target protein was secreted into the extracellular, and the fermentation broth was collected from the tank after induction, and the supernatant was collected after centrifugation at 6000 rpm for 15 min. The content of the target protein in the supernatant was about 1-2 mg/ml. [0056] (4) The supernatant obtained from step (3) was purified, concentrated, and dried in turn to obtain the recombinant MANNase-GLP-1.

[0057] The specific steps for purification, concentration and drying of the supernatant were as follows: [0058] (SS1) taking the supernatant and filter it through a 0.8 um membrane, then a 0.2 um membrane, and collecting the filtrate; [0059] (SS2) concentrating the filtrate (about 3 L) first 10 times with an 10 kd ultrafiltration membrane pack, and then 10 times with 3 L of deionized water to obtain a concentrate; [0060] (SS3) freeze-drying the concentrate to obtain the recombinant MANNase-GLP-1.

Embodiment 2

[0061] This embodiment provides a method for the efficient expression and purification of a recombinant fusion protein of mannanase and GLP-1, including the steps as follow. [0062] (1) The target fragments encoding GLP-1 and mannanase were cloned using primer pairs, amplified by PCR and then double digested, after which the obtained gene sequences encoding GLP-1 and mannanase were ligated to the pPICZαB plasmid to construct a recombinant expression vector pPICZαB-MANNase-31P.

[0063] The sequences of the genes encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of said fusion protein MANNase-31P is shown in SEQ ID NO.7. [0064] (2) the recombinant expression vector in step (1) was transformed into Pichia pastoris GS115 competent cells to construct recombinant engineering yeast MANNase-31P-GS115. [0065] (3) The recombinant engineering yeast was fermented and cultured to induce expression of the fusion protein MANNase-31P. The specific steps were as follow.

[0066] Seed solution preparation: A single colony of the recombinant engineering yeast MANNase-31P-GS115 was inoculated into a test tube of 5 ml of YPD liquid medium containing bleomycin, incubating which with shaking at 30° C. and 200 rpm for 12 h. The yeast solution was poured into 50 ml of YPD liquid medium, incubating which at 30° C. and 200 rpm for 12 h to obtain a primary seed solution. The primary seed solution was inoculated in 500 ml of YPD medium at 10% inoculum, incubating which for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution.

[0067] Fermentation tank culture: The secondary seed solution was added to 4.5 L fermentation medium (7.5L fermentation tank) at 10% inoculum for high density fermentation culture. The initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4L/min, and the pH is 5.5. The fermentation process includes four stages, the first stage was the growth stage with glycerol as the basic carbon source, mainly for the initial accumulation of cell biomass. With the gradual increase in the density of the yeast, the dissolved oxygen of the medium would rise rapidly, at this time to enter the glycerol replenishment stage, through the flow of glycerol to provide a carbon source, the yeast continued to grow, the dissolved oxygen was maintained at more than 20%. When the density of the yeast reaches 120 OD.sub.600, after a 5 h starvation period, it entered the methanol induction phase. When the OD.sub.600 of the fermentation broth reached above 120, the induction agent methanol was added and the percentage of methanol in the volume of the fermentation broth was 3%. During the period, the OD.sub.600 value of the fermentation broth was measured by sampling every four hours, and the samples were kept for SDS-PAGE electrophoresis to detect the protein expression. After 5 days of induction, the target protein was secreted into the extracellular, and the fermentation broth was collected from the tank after induction, and the supernatant was collected after centrifugation at 6000 rpm for 15 min. The content of the target protein in the supernatant was about 1-2 mg/ml. [0068] (4) The supernatant obtained from step (3) was purified, concentrated, and dried in turn to obtain the recombinant MANNase-GLP-1.

[0069] The specific steps for purification, concentration and drying of the supernatant were as follows: [0070] (SS1) taking the supernatant and filter it through a 0.8 um membrane, then a 0.2 um membrane, and collecting the filtrate; [0071] (SS2) concentrating the filtrate (about 3 L) first 10 times with an 10 kd ultrafiltration membrane pack, and then 10 times with 3L of deionized water to obtain a concentrate; [0072] (SS3) freeze-drying the concentrate to obtain the recombinant MANNase-GLP-1.

Embodiment 3

[0073] This embodiment provides a method for the efficient expression and purification of a recombinant fusion protein of mannanase and GLP-1, including the steps as follow. [0074] (1) The target fragments encoding GLP-1 and mannanase were cloned using primer pairs, amplified by PCR and then double digested, after which the obtained gene sequences encoding GLP-1 and mannanase were ligated to the pPICZαC plasmid to construct a recombinant expression vector pPICZαC-MANNase-31P.

[0075] The sequences of the genes encoding GLP-1 and mannanase are shown in SEQ ID NO. 1 and SEQ ID NO. 2, respectively. The sequences of the primer pair for amplifying the gene sequence encoding GLP-1 are shown in SEQ ID NO. 3 and shown in SEQ ID NO. 4. The sequences of the primer pair for amplifying the gene sequence encoding mannanase are shown in SEQ ID NO. 5 and SEQ ID NO. 6. The amino acid sequence of said fusion protein MANNase-31P is shown in SEQ ID NO.7. [0076] (2) the recombinant expression vector in step (1) was transformed into Pichia pastoris KM71 competent cells to construct recombinant engineering yeast MANNase-31P- KM71. [0077] (3) The recombinant engineering yeast was fermented and cultured to induce expression of the fusion protein. The specific steps were as follow.

[0078] Seed solution preparation: A single colony of the recombinant engineering yeast MANNase-31P-KM71 was inoculated into a test tube of 5 ml of YPD liquid medium containing bleomycin, incubating which with shaking at 30° C. and 200 rpm for 12 h. The yeast solution was poured into 50 ml of YPD liquid medium, incubating which at 30° C. and 200 rpm for 12 h to obtain a primary seed solution. The primary seed solution was inoculated in 500 ml of YPD medium at 10% inoculum, incubating which for 22 h at 30° C. and 200 rpm to obtain a secondary seed solution.

[0079] Fermentation tank culture: The secondary seed solution was added to 4.5 L fermentation medium (7.5 L fermentation tank) at 10% inoculum for high density fermentation culture. The initial fermentation temperature is 30° C., the stirring speed is 300 rpm, the aeration rate is 4L/min, and the pH is 5.5. The fermentation process includes four stages, the first stage was the growth stage with glycerol as the basic carbon source, mainly for the initial accumulation of cell biomass. With the gradual increase in the density of the yeast, the dissolved oxygen of the medium would rise rapidly, at this time to enter the glycerol replenishment stage, through the flow of glycerol to provide a carbon source, the yeast continued to grow, the dissolved oxygen was maintained at more than 20%. When the density of the yeast reaches 90 OD.sub.600, after a 3.5 h starvation period, it entered the methanol induction phase. When the OD.sub.600 of the fermentation broth reached above 90, the induction agent methanol was added and the percentage of methanol in the volume of the fermentation broth was 1.6%. During the period, the OD.sub.600 value of the fermentation broth was measured by sampling every four hours, and the samples were kept for SDS-PAGE electrophoresis to detect the protein expression, as shown in FIG. 1 and FIG. 2 (indicated by arrows). As can be seen in the figures, the expression of the target protein was substantially increased. After 5 days of induction, the target protein was secreted into the extracellular, and the fermentation broth was collected from the tank after induction, and the supernatant was collected after centrifugation at 6000 rpm for 15 min. The content of the target protein in the supernatant was about 1-2 mg/ml. [0080] (4) The supernatant obtained from step (3) was purified, concentrated, and dried in turn to obtain the recombinant MANNase-GLP-1.

[0081] The specific steps for purification, concentration and drying of the supernatant were as follows: [0082] (SS1) taking the supernatant and filter it through a 0.8 um membrane, then a 0.2 um membrane, and collecting the filtrate; [0083] (SS2) concentrating the filtrate (about 3 L) first 10 times with an 10 kd ultrafiltration membrane pack, and then 10 times with 3 L of deionized water to obtain a concentrate; [0084] (SS3) freeze-drying the concentrate to obtain the recombinant MANNase-GLP-1.

Embodiment 4

[0085] The only difference between Embodiment 4 and Embodiment 1 is that in step (3), the strain of Pichia pastoris is SMD1168, but the rest is the same as in Embodiment 1.

Embodiment 5

[0086] The only difference between Embodiment 5 and Embodiment 1 is that in step (3), the strain of Pichia pastoris is SMD1168H, but the rest is the same as in Embodiment 1.

Embodiment 6

[0087] The only difference between Embodiment 6 and Embodiment 1 is that in step (3), the steps of fermentation and culture and induction of expression of the recombinant engineering yeast are different. The specific steps for fermentation and culture of recombinant engineering yeast and induction of expression of the fusion protein MANNase-31P in this embodiment were as follow.

[0088] A single colony of the successfully constructed engineering yeast for expression of Pichia pastoris MANNase-31P-X-33 were picked and inoculated into 5 ml of YPD liquid medium and incubated overnight at 30° C. and 250 rpm in a shaker. The cultured strain was inoculated into BMGY medium at 1% and continued to shake for 24 h, after which the yeast was transferred into BMMY medium and the OD.sub.600 was adjusted to about 1.0, and the culture was continued to be induced in a shaker at 30° C. and 200 rpm, with methanol added at 0.5% by volume every 24h for 4 days. The amount of target protein in the supernatant was about 0.2 mg/ml. The protein expression by SDS-PAGE electrophoresis is shown in FIG. 10.

EXPERIMENTAL EXAMPLE

1. Application of Recombinant MANNase-GLP-1 Obtained in Embodiment 1 in High-Fat and High-Sugar Diet-Induced Metabolic Syndrome

[0089] 1-1) Construction of a Mouse Model of Metabolic Syndrome Induced by High-Fat and High-Sugar Diet

[0090] One hundred 6-week-old (18-20 g) C57-6J mice (4-6 weeks old, male) were housed in separate cages with controlled animal room temperature of 25±2° C., humidity of 50±10%, and a 12-h light and 12-h dark cycle to acclimatize to the environment for one week. The mice were randomly divided into cages of 5-6 mice/group. Body weight and fasting blood-glucose (FBG) were measured after 12 h of fasting. The control group was fed with standard chow, and the model group was fed with high-fat, high-sugar (HFSD) chow for 24 weeks. After finishing, the body weight and FBG of each group of mice were measured.

[0091] The body weight of mice on high-fat and high-sugar diet was about 42.5 g and that of mice on normal diet was about 30 g. The fasting blood glucose of mice on high-fat and high-sugar diet was about 5.67 and that of mice on normal diet was 4.62, with statistical differences (*P<0.05, **P<0.01, ***P<0.001). As shown in FIGS. 3 and 4 below, it can be seen that: After C57-6J mice induced by high-fat and high-sugar diet for 24 weeks, their body weight exceeded normal diet by 41.67%, meeting the criteria of obesity model (20%). C57-6J mice induced by high-fat and high-sugar diet had significantly higher fasting blood glucose than normal diet by 22.73%.

[0092] The oral glucose tolerance (OGTT) curves were obtained from regular diet mice and high-fat and high-sugar mice after overnight fasting for 12 h and gavage of D-glucose at 2 g/kg (body weight) at 0, 0.5, 1.0, 1.5, 2.0, and 2.5 h, respectively, as shown in FIG. 5. It can be seen that the glucose tolerance was significantly impaired after 24 weeks of high-fat and high-sugar diet induction. The oral glucose tolerance test is a glucose stress test. Impaired glucose tolerance indicates a decrease in pancreatic islet beta cell function and the body's ability to regulate blood glucose, and is used to diagnose mild hyperglycemia in prediabetes.

[0093] HE-stained sections of liver and adipose tissue from mice on a high-fat, high-sugar diet and mice on a normal diet were analyzed. As shown in FIG. 6, it can be seen from the figure: mice on high-fat and high-sugar diets had significant fatty liver and there was also a significant difference in the size of adipocytes between the two groups.

[0094] After successful modeling, 11 animals/group were randomly grouped, plus the normal feed group, for subsequent gavage experiments.

[0095] 1-2) Effect of Oral Administration of MANNase-GLP-1 on High-Fat and High-Sugar Diet-Induced Metabolic Syndrome

[0096] Mice were randomly divided into five groups, wherein fusion protein high-dose group (0.7 mg/kg-d), fusion protein-affected low-dose group (0.14 mg/kg-d), and 30 mg/kg orlistat were as positive control groups, and the normal feed control group and the high-fat, high-sugar feed as a negative control group were given the same volume of water. All mice were gavaged separately without any change in diet. Weekly weight changes were measured, and changes in fasting blood glucose were measured every two weeks. The results are shown in FIG. 7 and FIG. 8. It can be concluded that the MANNase-GLP-1 fusion protein described in Example 1 of the present invention has a significant weight reduction and hypoglycemic effect.

2. Effect of the Recombinant MANNase-GLP-1 Injection Administration on Type II Diabetes Mellitus Obtained in Embodiment 1

[0097] 2-1) Construction of a Mouse Model of Type II Diabetes Mellitus.

[0098] Fifty 6-week-old (18-20 g) BALB/C mice (males) were randomly divided into cages in groups of 8 mice each. Body weight and fasting blood-glucose (FBG) were measured after 12 h of fasting. The control group was fed with standard chow for 4 weeks and the modeling group was fed with high-fat chow for 4 weeks, and then the body weight and FBG of each group were measured. After 12 h of fasting, STZ was injected intraperitoneally at a dose of 60 mg/kg-Bw for 3 days.

[0099] The body weight and FBG of the modeled mice were measured on the 3rd, 7th, 10th and 14th days after modeling, and mice with FBG≥11.1 mmol/L and stable for one week were selected as type II diabetic model mice, and 8 mice/group were randomly divided into cages for hypoglycemia experiments.

[0100] Mice were randomly divided into three groups: diabetic negative control group (saline), diabetic positive control group (Lir), and diabetic treatment group (MANNase-GLP-1), with 8 mice in each group. All mice were gavaged with D-glucose at 1.5 g/kg (body weight) after overnight fasting for 12 h, and then subcutaneously injected with (s.c.) MANNase-GLP-1 (250 ug/50 g), and blood was collected from the tail after 0, 15, 30, 60, 120, 180 min and 300 min after injection, and the blood glucose level was measured by Roche blood glucose test paper. The results are shown in FIG. 9.

[0101] The purpose of the present invention is to provide a method for the preparation of mannanase and homologues thereof and GLP-1 recombinant fusion protein capable of improving body weight and blood glucose in patients with high-fat and high-glucose diet-induced metabolic syndrome. Through genetic recombination technology, soluble proteins are obtained by using high density fermentation of Pichia pastoris to induce secretory expression, followed by isolation and purification by filtration and concentration to obtain high yield of target proteins. The high fermentation expression and simple isolation steps solve the limitations of the current GLP-1 analogs such as low drug yield, high cost and the need for frequent injections. More importantly, the fusion protein has hypoglycemic effect not only by injection, but also by oral administration for hypoglycemia and weight reduction, which has good application value in obese and pre-diabetic patients. It also provides a basis for further research on the mechanism of hypoglycemia and weight loss of mannanase and homologues thereof with GLP-1 recombinant fusion protein.

[0102] The above described is only a specific implementation of the present invention, but the scope of protection of the present invention is not limited to it, and any changes or substitutions that can be readily thought of by any person skilled in the art within the technical scope disclosed by the present invention shall be covered by the scope of protection of the present invention. Therefore, the scope of protection of the present invention shall be subject to the scope of protection of the said claims.

INDUSTRIAL PRACTICALITY

[0103] This application proposes an efficient expression and purification method and application of MANNase and homologues thereof and GLP-1 recombinant fusion protein. Through genetic recombination technology, soluble proteins are obtained by using high density fermentation of Pichia pastoris to induce secretory expression, followed by isolation and purification by filtration and concentration to obtain high yield of target proteins. The high fermentation expression and simple isolation steps solve the limitations of the current GLP-1 analogs such as low drug yield, high cost and the need for frequent injections. More importantly, the fusion protein has hypoglycemic effect not only by injection, but also by oral administration for hypoglycemia and weight reduction, which has good application value in obese and pre-diabetic patients. It also provides a basis for further research on the mechanism of hypoglycemia and weight loss of mannanase and its homologues with GLP-1 recombinant fusion protein.