HIGH-ACTIVITY LONG-ACTING HYPOGLYCEMIC FUSION PROTEIN AS WELL AS PREPARATION METHOD AND MEDICAL APPLICATION THEREOF
20190322717 ยท 2019-10-24
Assignee
Inventors
- Shuhua Tan (Nanjing, CN)
- Lili Gu (Nanjing, CN)
- Jian Fu (Nanjing, CN)
- Yongbo Zhang (Nanjing, CN)
- Qinghua Tian (Nanjing, CN)
- Yue Wang (Nanjing, CN)
- Xiaojian Gong (Nanjing, CN)
Cpc classification
C07K19/00
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C07K14/57563
CHEMISTRY; METALLURGY
International classification
C12N15/70
CHEMISTRY; METALLURGY
Abstract
The disclosure provides a high-activity long-acting hypoglycemic fusion protein, which is formed by connecting, via a linker peptide or directly, a high-activity Exendin-4 mutant with an optimally mutated Fc fragment of a human immunoglobulin IgG1. The optimally mutated Fc fragment of the human immunoglobulin IgG1 comprises an optimally mutated human IgG1 hinge region and human IgG1 constant regions CH2 and CH3.
Claims
1. A high-activity long-acting hypoglycemic fusion protein, wherein the hypoglycemic fusion protein is formed by connecting, via a linker peptide or directly, a high-activity Exendin-4 mutant with an optimally mutated Fc fragment of a human immunoglobulin IgG1.
2. The hypoglycemic fusion protein according to claim 1, wherein, the optimally mutated Fc fragment of the human immunoglobulin IgG1 comprises an optimally mutated human IgG1 hinge region and human IgG1 constant regions CH2 and CH3, and the amino acid sequence of the optimally mutated human IgG1 hinge region is as shown in SEQ ID NO: 6.
3. The hypoglycemic fusion protein according to claim 2, wherein, the amino acid sequences of the human IgG1 constant regions CH2 and CH3 are as shown in SEQ ID NO: 7.
4. The hypoglycemic fusion protein according to claim 1, wherein, the linker peptide is a flexible peptide rich in Gly and/or Ala and/or Ser, having 150 amino acid residues in length.
5. The hypoglycemic fusion protein according to claim 4, wherein, the amino acid sequence of the linker peptide is as shown in SEQ ID NO: 8.
6. The hypoglycemic fusion protein according to claim 1, wherein, the amino acid sequence of the high-activity Exendin-4 mutant is as shown in SEQ ID NO:2, SEQ ID NO: 3, or SEQ ID NO: 4.
7. The fusion protein according to claim 1, wherein, the amino acid sequence of the fusion protein is as shown in SEQ ID NO: 11, SEQ ID NO: 15 or SEQ ID NO: 16.
8. A method for preparing the fusion protein according to claim 1, wherein the fusion protein is capable of being expressed in E. coli in a soluble form, and the method comprises the following steps: a) designing to synthesis and clone a coding gene of the fusion protein; b) constructing as an expression plasmid to be transformed into an E. coli host cell for expression; and c) collecting bacterial sludge and broken walls, collecting broken wall supernatant, and separating and purifying to obtain the soluble fusion protein.
9. An application of the hypoglycemic fusion protein according to claim 1 in preparation of a drug for reducing blood glucose.
Description
DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS
[0036] Specific steps of the disclosure will be illustrated through examples below, but are not limited thereto.
[0037] The terms used in the disclosure generally have their ordinary meanings in the art, unless otherwise stated.
[0038] The disclosure will be further described in detail in combination with embodiments and with reference to data in the following. It should be understood that these embodiments are only for illustrating the disclosure as examples but not limiting the scope of the disclosure in any manners.
[0039] In the following examples, various processes and methods that are not described in detail are well-known conventional methods in the art.
[0040] The disclosure will be illustrated in combination with embodiments in the following.
[0041] All of materials, reagents and the like used in the following examples, unless otherwise specified herein, are commercially available.
Example 1 Design of a Long-Acting Fusion Protein of Wild Exendin-4 and a Long-Acting Fusion Protein of a High-Activity Exendin-4 Mutant
[0042] 1. The amino acid sequence (SEQ ID NO: 1) of wild Exendin-4, and Leu at the 21.sup.st position is mutated into Lys to obtain the amino acid sequence (SEQ ID NO: 2) of the high-activity Exendin-4 mutant (EX-L21K).
[0043] The amino acid sequence of wild Exendin-4:
TABLE-US-00001 (SEQIDNO:1) HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS.
[0044] The amino acid sequence (SEQ ID NO: 2) of a high-activity Exendin-4 mutant (EX-L21K): HGEGTFTSDLSKQMEEEAVRKFIEWLKNGGPSSGAPPPS (SEQ ID NO: 2).
[0045] 2. The amino acid sequence of an optimally mhIgG1 hinge region is seen in SEQ ID NO: 6, which is mutated by a nhIgG1 hinge region (the amino acid sequence is seen in SEQ ID NO: 5) (Edelman G M. Proc Natl Acad Sci USA. 1969; 63: 78-85). Compared with the nhIgG1 hinge region, amino acid at the N terminal region of the mhIgG1 hinge region is rich in Gly and Ser, so that the flexibility and hydrophilia of the N terminal of the hinge region are enhanced, a possibility that the first Cys at the N terminal of the original hinge region forms a mispairing disulfide bond during the protein expression is eliminated. The underline portion is a mutation region.
[0046] The amino acid sequence of the nhIgG1 hinge region (hIgG1 hinge region):
TABLE-US-00002 (SEQ.IDNO:5) VEPKSCDKTHTCPPCP.
[0047] The amino acid sequence of the optimally mutated human hIgG1 hinge region (mutated human IgG1 hinge, called mhIgG1 hinge region for short):
TABLE-US-00003 (SEQ.IDNO:6) SGGGGSDKTHTCPPCP.
[0048] Design of a linker peptide sequence: the linker peptide is a flexible peptide rich in Gly and/or Ala and/or Ser, and has 150 amino acid residues in length, and the amino acid sequence of the preferred linker peptide is as shown in SEQ ID NO: 8.
[0049] 3. The long-acting fusion protein EX-mhIgG1Fc of wild Exendin-4 consists of the following parts: wild Exendin-4, a linker peptide, and an optimally mhIgG1 hinge region and constant regions CH2 and CH3 of human IgG1. Its structure diagram is seen in
[0050] 4. The long-acting fusion protein EX-L21K-mhIgG1Fc of the high-activity Exendin-4 mutant EX-L21K consists of the following parts: a high-activity Exendin-4 mutant EX-L21K, a linker peptide, and a mhIgG1 hinge region and constant regions CH2 and CH3 of an optimally mhIgG1. Its structure diagram is seen in
[0051] 5. The long-acting fusion protein EX-hIgG1Fc of wild Exendin-4 consists of the following parts: wild Exendin-4, a linker peptide, and a nhIgG1 hinge region and constant regions CH2 and CH3 of nhIgG1. Its structure diagram is seen in
Example 2 Cloning and Expression of a Long-Acting Fusion Protein EX-mhIgG1Fc of Wild Exendin-4
[0052] The coding gene (SEQ ID NO: 10) of the long-acting fusion protein EX-mhIgG1Fc of wild Exendin-4 is synthesized and cloned by Nanjing GenScript Biotech Co., Ltd. The gene is subjected to double-enzyme digestion with Nde I and Hind III and then sub-cloned to a prokaryotic expression vector pET21b to construct an expression plasmid pET-EX-mhIgG1Fc (shown in part A of
[0053] A single bacterial colony is inoculated into a 50 ml LB liquid culture medium (containing 100 g/ml ampicillin) and then subjected to shake culture for 14 h at 200 rpm and 37 C. The cultured product is transferred to a 200 ml TB culture medium (tryptone 1.2%, yeast powder 2.4%, glycerinum 0.4% (v/v), 17 mM KH.sub.2PO.sub.4, 72 mM K.sub.2HPO.sub.4.3H.sub.2O, and 100 g/ml ampicillin) in an inoculation amount of 1% (V/V) and then subjected to shake culture at 37 C. until OD.sub.600 nm is up to about 1.0, lactose is added to 1% (v/v), the above mixture is shaken at 25 C. with a speed of 200 rpm to induce expression for 15 h, and meanwhile a negative control is set (namely, lactose is not added for induction).
[0054] Fermentation broth is collected and centrifuged for 10 min at 10000 rpm to collect bacterial sludge, wet bacterial sludge is weighed, the bacterial sludge is resuspended with PBS in a ratio of 1:15 (g/ml), bacteria are broken three times with a homogenizer (AH100B, ATS Engineering Inc., Canada) at low temperature, and the pressure of the homogenizer is maintained to be 800900 bar in the process of broking. After thallus is broken, broken cell solution is centrifuged (12000 rpm, 20 min) at 4 C., and supernatant is taken and subjected to 12% SDS-PAGE electrophoretic analysis. A result shows that there is an obvious expression band at the molecular weight of about 30 KD (shown in part A of
Example 3 Cloning and Expression of a Long-Acting Fusion Protein EX-L21K-mhIgG1Fc of a High-Activity Exendin-4 Mutant
[0055] With the coding gene (SEQ ID NO: 10) of the long-acting fusion protein EX-mhIgG1Fc of wild Exendin-4 in Example 2 as a template, it is mutated using an overlap extension site-directed mutagenesis method (Ho S N. Gene. 1989; 77: 51-9) to obtain the coding gene (SEQ ID NO: 12) of the long-acting fusion protein EX-L21K-mhIgG1Fc of the high-activity Exendin-4 mutant.
[0056] The following primers are synthesized by Nanjing GenScript Biotech Co., Ltd:
TABLE-US-00004 (1)forwardmutationprimer(EX-L21K-F): (SEQ.IDNO:17) 5GGAAGAAGAAGCGGTTCGTAAATTCATCGAATGGCTGAAAAAC3; (2)reversemutationprimer(EX-L21K-R): (SEQIDNO:18) 5GTTTTTCAGCCATTCGATGAATTTACGAACCGCTTCTTCTTCC3; (3)externalforwardprimer(NdeI-EX-F): (SEQIDNO:19) 5AGATATACATATGCACGGTGAAGGTACCTTCACCTCTGAC3; (4)externalreverseprimer(HindIII-Fc-R): (SEQIDNO:20) 5CGTCGACAAGCTTCTATTATTTACCCGGAGACAGAGACAGAG3.
[0057] Amplification of upstream fragment A: with the coding gene (SEQ ID NO: 10) of EX-mhIgG1Fc as a template, PCR is carried out under the action of Fastpfu DNA Polymerase (TransGen Biotech product). A 25 l reaction system consists of an external forward primer (NdeI-EX-F) 10 pmol; a reverse mutation primer (EX-L21K-R) 10 pmol; Fast pfu DNA Polymerase 2.5 units; 5reaction buffer 5 l; dNTP (10 mM each) 0.5 l; template plasmid DNA 0.5 l (about 2.5 ng); and sterile water used for supplementation until the total volume is 25 l. PCR conditions are as follows: denaturation is carried out for 2 minutes at 95 C.; cycle reaction of 30 cycles is then carried out: denaturation for 20 seconds at 95 C., annealing for 20 seconds at 50 C., and extension for 10 seconds at 72 C.; extension finally is carried out for 5 minutes at 72 C. After the reaction is ended, the product is identified with 1% agarose gel electrophoresis (AGE) and recovered with a TaKaRa gel recovery kit.
[0058] Amplification of downstream fragment B: with the coding gene (SEQ ID NO: 10) of EX-mhIgG1Fc as a template, and PCR is carried out under the action of Fastpfu DNA Polymerase (TransGen Biotech product). A 25 l reaction system consists of a forward mutation primer (EX-L21K-F) 10 pmol; an external reverse primer (HindIII-Fc-R) 10 pmol; Fast pfu DNA Polymerase 2.5 units; 5reaction buffer 5 l; dNTP (10 mM each) 0.5 l; template plasmid DNA 0.5 l (about 2.5 ng); sterile water is used for supplementation until the volume is 25 l. PCR conditions are as follows: denaturation is carried out for 2 minutes at 95 C.; cycle reaction of 30 cycles is then carried out: denaturation for 20 seconds at 95 C., annealing for 20 seconds at 50 C., and extension for 30 seconds at 72 C.; extension is finally carried out for 5 minutes at 72 C. The PCR product is identified with 1% agarose gel electrophoresis (AGE) and recovered with a TaKaRa gel recovery kit.
[0059] A complete mutant gene is obtained by overlap-extension PCR amplification: with a mixed solution of the upstream fragment A and the downstream fragment B as a template, PCR is carried out under the action of Taq plus DNA Polymerase (a product from TAKARA company). A 50 l reaction system consists of an external forward primer (NdeI-EX-F) 10 pmol; an external reverse primer (HindIII-Fc-R) 10 pmol; Taq plus DNA Polymerase 2.5 units; 10Taq plus buffer (with MgCl.sub.2) 5 l; dNTP (10 mM each) 1 l; the mixed solution of the upstream fragment A and the downstream fragment B 1 l (about 5 ng); sterile water is used for supplementation until the volume is 5 l. PCR conditions are as follows: denaturation is carried out for 3 minutes at 95 C.; cycle reaction of 30 cycles is then carried out: denaturation for 30 seconds at 94 C., annealing for 30 seconds at 55 C., and extension for 1 minute at 72 C.; extension is finally carried out for 5 minutes at 72 C. The PCR product is identified with 1% agarose gel electrophoresis (AGE) and recovered with a TaKaRa gel recovery kit.
[0060] The obtained mutant gene is subjected to double-enzyme digestion with Nde I and Hind III and then cloned to a prokaryotic expression vector pET21b to construct an expression plasmid pET-EX-L21K-mhIgG1Fc (shown in part B of
Example 4 Cloning and Expression of a Long-Acting Fusion Protein EX-hIgG1Fc of Wild Exendin-4
[0061] The coding gene (SEQ ID NO: 14) of a long-acting fusion protein EX-hIgG1Fc of wild Exendin-4 is synthesized and cloned by Nanjing Jinsirui Biotechnology Co., Ltd. The gene is subjected to double-enzyme digestion with Nde I and Hind III and then sub-cloned to a prokaryotic expression vector pET21b to construct an expression plasmid pET-EX-hIhG1Fc (
[0062] The long-acting fusion protein EX-hIgG1Fc of Wild Exendin-4 is expressed with reference to the protein expression method in Example 2. After expression, broken cell solution is centrifuged at 4 C. (12000 rpm, 20 min), and supernatant is taken for 12% SDS-PAGE electrophoretic analysis. A result is that there is no target protein expression band at the molecular weight of about 30 KD, indicating that the fusion protein cannot be expressed in a soluble form.
Example 5 Separation and Purification of Fusion Proteins EX-mhIgG1Fc and EX-L21K-mhIgG1Fc
[0063] 1. Collection of bacterial sludge and breaking of cell walls: fermentation broth is centrifuged at 42 C. (10000 rpm, 10 min) to collect bacterial sludge, wet bacterial sludge is weighed and resuspended with PBS buffer in a ratio of 1:15 (g/ml), and the bacterial sludge is washed twice to three times. The bacterial sludge is resuspended in a ratio of 10% (w/v) with broken bacterial buffer (PBS buffer (pH 7.4) containing 1 mM PMSF and 1 mM EDTA), cells are broken three times with an ATS homogenizer (AH100B, ATS Engineering Inc., Canada), and the pressure of the homogenizer in the process of broking is maintained to be 800900 bar. The broken cells are centrifuged at 4 C. (12000 rpm, 20 min) to collect supernatant of broken wall solution.
[0064] 2. Protein A affinity chromatography: a HiTrap rProtein A FF affinity chromatographic prepacked column (a product from GE company) is sufficiently balanced with balance buffer PBS (pH7.4). The supernatant of broken wall solution is subjected to suction filtration via a 0.22 m water phase filter membrane, and then loaded at a flow velocity of 0.5 ml/min. After loading is completed, the chromatographic column is rinsed with the balance buffer to remove hybrid proteins that are not bound. Then, eluting is carried out with elution buffer (0.1M citric acid, pH is adjusted to 4.0 with NaOH), and 450 l of neutralization buffer (which is formed by mixing 1M Tris hydrochloride buffer (pH is adjusted to 9.0) and glycerinum in a ratio of 1:2) is added into each ml of collected solution.
[0065] 3. Ammonium sulfate precipitation: after filtered by the 0.22 m membrane, ice-cold saturated ammonium sulfate solution is dropped into the above protein-like sample solution placed on ice bath at a flow velocity of 1 ml/min until a concentration is 60%, and a target protein is precipitated. Slow stirring is carried out with a magnetic stirrer in the whole process. Centrifugation is carried out at 4 C. (10000 rpm, 20 min) to collect precipitate, the collected precipitate is dissolved with a proper amount of PBS buffer (pH7.4) to obtain concentrated target protein solution.
[0066] 4. Gel chromatography: a Superdex 200 Increase 10/300 GL gel filtration prepacked column (a product from GE Company) is sufficiently balanced with PBS buffer (pH7.4). 500 l of concentrated target protein solution precipitated by ammonium sulfate is taken and centrifuged at 40 C. (10000 rpm, 10 min) to collect supernatant, the supernatant is loaded to the balanced chromatographic column at a flow velocity of 0.4 ml/min. Target protein eluting solution is collected and eluted, and filtered and sterilized with a 0.22 m filter membrane on an ultra-clean platform. Subsequently, the obtained substance is sub-packed, and stored at 70 C.
[0067] Samples collected in various steps are purified and subjected to 12% SDS-PAGE electrophoretic analysis. Results are seen in
Example 6 Molecular Weight Detection and Purity Analysis of Fusion Proteins EX-mhIgG1Fc and EX-L21K-mhIgG1Fc
[0068] Analysis is carried out on a high performance liquid chromatography (HPLC) system (LC-2010A HT, SHIMADZU Corp., Japan) utilizing Size Exclusion Chromatography (SEC), wherein, a chromatographic column is Shodex PROTEIN KW-802.5 (SHOW A DENKO K.K., Japan), a mobile phase is 0.2M phosphate buffer (pH7.4) and contains 0.1M Na.sub.2SO.sub.4 (which is added according to specification requirement of a chromatographic column), a flow velocity is 0.7 ml/min, and a detection wavelength is 280 nm.
[0069] Single analysis and mixed analysis are carried out on four standard proteins purchased from Shanghai Yuanye Biotechnology Company. Results show that retention times of four standard proteins BSA (MW=67 kDa), chicken ovalbumin (MW=43 kDa), chymotrypsinogen (MW=25 kDa) and lysozyme (MW=14.4 kDa) are respectively 11.152 min, 11.938 min, 13.284 min and 14.31 min (shown in part A of
lgMW=0.2965Ve+4.1308.
[0070] The retention time of the long-acting fusion protein EX-mhIgG1Fc of wild Exendin-4 is 11.532 min, as shown in
[0071] The retention time of the long-acting fusion protein EX-L21K-mhIgG1Fc of the high-activity Exendin-4 mutant is 11.534 min, as shown in
[0072] Accordingly, the purified long-acting fusion protein EX-mhIgG1Fc of wild Exendin-4 and the long-acting fusion protein EX-L21K-mhIgG1Fc of the high-activity Exendin-4 mutant are both present in a soluble dimer form.
[0073] In addition, purity analysis is carried out on SEC-HPLC analysis results of the above two proteins by adopting a peak area comparison method. A result shows that the purity of the purified EX-mhIgG1Fc sample is 98.296% (shown in part B of
Example 7 In-Vivo Hypoglycemic Test for Type II Diabetic Model Mouse C57BL/KsJ-db/db
[0074] 50 six-week-old type II diabetic mice C57BL/KsJ-db/db (namely, S.Cg-Dock7.sup.m+/+Lepr.sup.db/JNju mouse) and 10 control mice C57BLKS/JNju are purchased from Nanjing Biomedical Research Institute of Nanjing University (certificate number: 201602819, and license number: SCXK (Su) 2015-0001). The mouse are fed in a SPF-grade animal house with a humidity of 40-60% at room temperature of 25 C. for 12 h each under light and dark conditions, and an experiment is carried out after adaptive feeding for one week. Blood glucose concentration is measured using a Roche Accu-Chek Performa glucometer and blood glucose test paper. Experiment grouping and administration modes are seen in Table 1.
TABLE-US-00005 TABLE 1 Experimental Animal Grouping And Administrations Adminis- Adminis- tration tration Group Mice n Drug dosage mode 1 C57BLKS/JNju 8 NS ip. 2 BKS.Cg-Dock7.sup.m+/+ 8 NS ip. Lepr.sup.db/JNju 3 BKS.Cg-Dock7.sup.m+/+ 8 Ex-4 10 nmol/kg ip. Lepr.sup.db/JNju 4 BKS.Cg-Dock7.sup.m+/+ 8 EX- 10 nmol/kg ip. Lepr.sup.db/JNju mhIgG1Fc 5 BKS.Cg-Dock7.sup.m+/+ 8 EX-L21K- 5 nmol/kg ip. Lepr.sup.db/JNju mhIgG1Fc 6 BKS.Cg-Dock7.sup.m+/+ 8 EX-L21K- 10 nmol/kg ip. Lepr.sup.db/JNju mhIgG1Fc 7 BKS.Cg-Ddck7.sup.m+/+ 8 EX-L21K- 20 nmol/kg ip. Lepr.sup.db/JNju mhIgG1Fc Note: NS is normal saline; Ex-4 is positive drag acetic Exendin-4, a product from Shanghai Gil Co., Ltd (CATALOG number: 052143; batch number: P160102-CQ052143; molecular formula: C.sub.184H.sub.282N.sub.50O.sub.60S.sub.1; purity is 99.41%; molecular weight: 4186.66 Da).
[0075] 1. Cute Hypoglycemic Test
[0076] Blood glucose levels of each group of mouse are detected before administration. As shown in Table 2 and
[0077] After mouse are administrated, blood glucose levels after administration for 1, 2, 4, 8, 12, 24, 36, 48, 60, 72, 96 and 120 h are continuously monitored. Results are as shown in Table 2 and
TABLE-US-00006 TABLE 2 Hypoglycemic Activity of Ex-4/FC and EX-L21K on BKS.Cg-Dock7.sup.m +/+ Lepr.sup.db/JNju Mouse Blood glucose (mM) EX- EX-L21K- EX-L21K- EX-L21K- Administration Ex-4 mhIgG1Fc mhIgG1Fc mhIgG1Fc mhIgG1Fc time (h) CON MOD (10 nmol/kg) (10 nmol/kg) (5 nmol/kg) (10 nmol/kg) (20 nmol/kg) 0 8.58 24.16 24.53 23.311.05.sup.#### 23.04 22.41 24.99 0.25 0.81.sup.#### 1.20.sup.#### 1.23.sup.#### 1.51.sup.#### 1.43.sup.#### 1 7.79 22.23 10.63 19.18 15.18 15.2 14.98 0.33 0.90 1.07**** 1.27.sup. 1.58***.sup. 1.98***.sup. 1.65***.sup. 2 7.40 21.88 10.79 16.6 11.28 14.3 12.46 0.21 1.28 1.70*** 1.40**.sup. 1.49**** 1.58**** 1.36**** 4 8.45 18.96 10.71 10.08 9.16 8.48 9.54 0.30 1.64 1.65**** 1.49**** 1.42**** 0.89**** 1.20**** 8 8.79 21.81 15.31 11.83 9.91 8.01 8.73 0.34 1.55 1.79**** 1.24**** 1.84****.sup. 0.78****.sup. 1.08****.sup. 12 8.76 23.58 24.43 17.58 13.06 10.13 53 0.23 0.74 1.44 1.41**.sup. 1.51****.sup. 1.03****.sup. 0.75****.sup. 24 8.38 21.36 20.48 16.28 13.58 8.5 7.49 0.17 1.10 0.88 1.53* 1.80****.sup. 0.96****.sup. 0.66****.sup. 36 8.99 23.59 26.59 19.24 17.34 12.9 11.95 0.43 0.75 1.11 1.49*.sup. 1.53**.sup. 1.96****.sup. 1.16****.sup. 48 8.65 22.38 24.76 19.99 16.46 12.96 12.2 0.37 1.48 1.06 1.64.sup. 1.48**.sup. 1.06****.sup. 0.63****.sup. 60 8.91 26.86 27.19 25.01 22.39 21.36 21.5 0.29 0.99 0.95 1.06 1.52*.sup. 0.64**.sup. 0.90**.sup. 72 8.95 24.64 25.34 21.36 18.85 17.4 14.1 0.38 1.26 1.01 1.23 1.59**.sup. 1.21***.sup. 1.06****.sup. 96 8.21 22.98 23.68 21.35 18.71 17.33 14.46 0.24 1.06 0.75 0.98 1.59.sup. 0.95**.sup. 1.54****.sup. 120 9.08 25.08 24.83 23.75 23.35 22.96 22.76 0.27 0.85 0.68 1.12 0.87 0.65 1.03 Note: compared with normal group, .sup.#### represents P < 0.0001; compared with model group, *, **, *** and **** respectively represents P < 0.05, P > 0.01, P < 0.001 and P < 0.0001; compared with Ex-4, .sup., .sup., .sup. and .sup. respectively represents P < 0.05, P < 0.01, P < 0.001 and P < 0.0001; (n = 8, means SEM).
[0078] Hypoglycemic effects of Ex-4, EX-mhIgG1Fc and EX-L21K-mhIgG1Fc (5-20 nmol/kg) are further analyzed and estimated using a method of area under the curve (AUC). As shown in
[0079] In summary, three drugs Ex-4, EX-mhIgG1Fc and EX-L21K-mhIgG1Fc (5-20 nmol/kg) all have hypoglycemic effects, among them, Ex-4 fast works and exhibits a significant hypoglycemic effect after administration for 1 h. EX-mhIgG1Fc relatively slowly works and exhibits the hypoglycemic effect of after administration for 2 h, but its hypoglycemic effect lasts for a long time and can be extended to 48 h after administration. EX-L21K-mhIgG1Fc exhibits the hypoglycemic effect within 1 h after administration, and its hypoglycemic effect lasts for a longer time and can be extended to 96 h after administration.
[0080] 2. Intraperitoneal Glucose Tolerance Test (IPGTT)
[0081] Various groups of animals are fasted overnight for 18 h before test. Firstly, normal saline, Ex-4, EX-mhIgG1Fc and EX-L21K-mhIgG1Fc are respectively intraperitoneally injected. After administration for 2 h, 1.5 g/kg glucose is intraperitoneally injected to each mouse, blood is taken via caudal vein, and blood glucose concentrations in 120, 0, 15, 30, 45, 60, 90, 120 and 180 min are recorded. During the test, animals are normally dieted and supplied with water.
[0082] IPGTT test results are seen in
[0083] Area under the curve is further applied for analysis. Results are as shown in