CN104418951B - The application of the albumen of people Nectin 2 - Google Patents
The application of the albumen of people Nectin 2 Download PDFInfo
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- CN104418951B CN104418951B CN201310385399.5A CN201310385399A CN104418951B CN 104418951 B CN104418951 B CN 104418951B CN 201310385399 A CN201310385399 A CN 201310385399A CN 104418951 B CN104418951 B CN 104418951B
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- albumen
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- obesity
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70503—Immunoglobulin superfamily
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
Abstract
The present invention relates to the new application of the albumen of people Nectin 2.The albumen of Nectin 2 and its derived protein or its albumen for passing through modification can effectively suppress fat and diabetes, or for the treatment with obesity-related disease, including but not limited to hypertension, hyperlipidemia etc..Nectin 2 can not only reduce blood glucose, also by increasing the energetic supersession level of body, improve the glucose metabolism of body, strengthen the insulin sensitivity of tissue, and strengthen adaptability of the body to cold stimulation by activating body fat acid metabolic, it is that the treatment of diabetes, obesity and relevant disease opens new way.
Description
Technical field
The present invention relates to biomedicine field, specifically, it is related to the application of people's Nectin-2 albumen.
Background technology
Obesity is the result of human body energy metabolism disorder, i.e., remaining when human body energy intake exceedes energy ezpenditure
Under energy be almost completely converted into fat, be stored in whole body white adipose.Obesity is both a kind of independent disease, is II again
Patients with type Ⅰ DM, cardiovascular disease, hypertension, the risk factor of apoplexy and kinds cancer.Obesity worldwide shows outburst
Property popular trend, current China's Overweight people more than 2.7 hundred million, overweight people more than 9.6 thousand ten thousand.Obesity induces many disease bags
Include：Type II diabetes, non-alcohol fatty liver, gall stone, cardiovascular disease and senile dementia, some even the main of cancer are lured
Hair factor.Obesity refer to it is a certain degree of substantially overweight blocked up with fat deposit, due to food intake excessively or organism metabolism changes
Become and cause body fat accumulation excessively to cause excess weight to increase and cause human pathology, a kind of of physiological change shows.With
Normal constitution person compares, fat itself not have too many obvious characteristic, but obesity can bring many serious complication, including
Hypertension, cerebral apoplexy, diabetes, angiocardiopathy, Anomalous lipid metablism, the incidence of tumour and other metabolic diseases substantially increase
Plus, and it is fat with type II diabetes and deposit, show as insulin resistance, Impaired Glucose Tolerance Treated.Obesity has become 21 century shadow
Ring one of Major Risk Factors of people's health, the healthy and quality of life of serious harm people.
However, traditional slimming medicine is mainly used to control appetite and suppresses nutrient absorption, and many fat-reducing on the market
The most side effect of medicine is obvious, or effect is unstable, easily bounce-back.Therefore the rapid obesity controlling of effective method is utilized
Spread it is extremely urgent.
In human body in addition to white adipose, also there is brown fat.Containing abundant mitochondria in brown fat, with straight
The mode of heat of practicing midwifery amount carrys out consumed energy.Result of study shows that activity and the body-mass index of brown fat are in conspicuousness
Negative correlation, and active brown fat can consume substantial amounts of fat and blood glucose, and further prompting brown fat is in energy
Very important effect is played in metabolic process.But still show that brown fat participates in body without any direct evidence so far
Energetic supersession.Current research result is pointed out, and relative to traditional anti-obesity therapy such as control appetite and suppression nutrient absorption, is passed through
The energy ezpenditure of brown fat increase body is likely to turn into fat and diabetes the new method for the treatment of.At present still not by brown
The albumen of fat secretion is used for the relevant report for treating diabetes, obesity and obesity-related disorder.
Nectin-2 is a member in immunoglobulin superfamily member Nectin families.Nectin-2 and the family its
Its member Nectin-1, Nectin-3 are similar with Nectin-4 structures, belong to single pass transmembrane albumen, and having 1 outside its film exempts from
Epidemic disease globulin superfamily V spline structures domain and 2 immunoglobulin superfamily C spline structures domains.Research shows that Nectin-2 is various
Wide expression in tissue and cell line, adhesion molecule is played in endothelial cell and epithelial cell, participates in intercellular mutual
Effect and connection.Nectin-2 as cell adhesion molecule, can parent it is of the same race or with xenogenesis interact, i.e., on cell membrane
Nectin-2 extracellular domains can be from the congeneric elements on different cell surfaces or inhomogeneity interaction of molecules.It is right in having been reported
It is more in gene studies such as ApoE, ApoC1 and the Tomm40s relevant with high fat of blood, and it is located at same chromosome with these genes
On, and genetic distance and these genes are nearer and Nectin-2 of the high expression in brown fat is to fat metabolism function
There is not been reported for research.Speculate that Nectin-2 is possible to participate in fatty acid metabolism.
The content of the invention
The purpose of the present invention be from promote energetic supersession brand-new angle there is provided people Nectin-2 albumen in promotion machine
Function in body energy ezpenditure, so as to reach treatment diabetes, the brand-new purposes of fat and relevant disease.
In order to realize the object of the invention, present invention firstly provides a kind of fusion protein, the fusion protein is continuous by three
Immunoglobulin like domain（Immunologlobin-like domain, Ig-like domain）Composition（Fig. 1）, respectively
It is an Ig-like V structures domain and two Ig-like C2-type1 and Ig-like the C2-type2 structures of C-terminal of N-terminal
By two sections of Linker between domain, three domains（Linker1 and Linker2）Connection.Wherein, the Ig-likeV domains of N-terminal,
Middle Ig-like C2-type1 domains and the amino acid sequence of the Ig-like C2-type2 domains of C-terminal are respectively such as
Shown in SEQ ID No.1-3.Every section of Linker is formed by 1-10 amino acid tandem.For example, Linker1 amino acid sequence is
RVIAK, Linker2 amino acid sequence are VRYP.Linker is alternatively other flexible amino acid sequences, such as GGGGS or
GGGGS repeated fragment etc..
The ECD areas of foregoing fusion protein behaviour Nectin-2 albumen（That is extracellular region, Fig. 1）, it is：
1）The albumen being made up of the amino acid sequence shown in SEQ ID No.4；Or
2）Amino acid sequence shown in SEQ ID No.4 is substituted, lacks and/or added one or several amino acid and equal
Function by 1）Derivative albumen；Or
3）The albumen constituted with the amino acid sequence of the homology of amino acid sequence shown in SEQ ID No.4 more than 90%.
The present invention also provides the gene of the above-mentioned fusion protein of coding.
The present invention also provides carrier, engineering bacteria or transgenic cell line containing the gene for encoding above-mentioned fusion protein.
The present invention also provides people's Nectin-2 albumen（Include the ECD areas of people's Nectin-2 albumen）It is used to treat sugar preparing
Application in urine disease, the medicine of fat or obesity-related disease.
The present invention also provides people's Nectin-2 albumen（Include the ECD areas of people's Nectin-2 albumen）It is used to improve energy preparing
Application in the medicine of amount metabolism or promotion heating.
The present invention also provides a kind of pharmaceutical composition for being used to treat diabetes, obesity or obesity-related disease, the medicine
Compositions include the fusion protein or people's Nectin-2 albumen, or their albumen by modification or transformation, optionally include
Pharmaceutically acceptable microcarrier, nano particle and/or auxiliary material etc..
The present invention also provides a kind of pharmaceutical composition for being used to improve energetic supersession or promote heating, described pharmaceutical composition
The fusion protein or people's Nectin-2 albumen including effective dose, or their albumen by modification or transformation, optionally include
Pharmaceutically acceptable microcarrier, nano particle and/or auxiliary material etc..
The heretofore described albumen by modification includes but is not limited to, the fusion protein or people Nectin-2 albumen warp
Acetylation, Pegylation, glycosylation are combined with BSA.
The heretofore described albumen by transformation includes but is not limited to, the fusion protein or people Nectin-2 albumen with
Immunoglobulin Fc segments or the fusion protein for not changing Nectin-2 protein actives constituted with other protein fusions.
Heretofore described obesity-related disease is diabetes or insulin resistance.Preferably, the diabetes be I types or
Type ii diabetes.
Heretofore described pharmaceutical composition can be slow release formulation.
Heretofore described pharmaceutically acceptable carrier or auxiliary material include but is not limited to, microcarrier, nano particle and/or
Auxiliary material etc..
Fusion protein and/or people's Nectin-2 albumen that the present invention is provided, and their derived protein or their processes
The albumen of modification can effectively suppress obesity or for the treatment with obesity-related disease, blood glucose be reduced, by increasing body
Energetic supersession level, improve body Glucose tolerance test, strengthen tissue insulin sensitivity, and by activate body produce
Heat strengthens adaptability of the body to cold stimulation, so that the treatment for diabetes, fat and obesity-related disease is opened
New way.
Brief description of the drawings
Fig. 1 is Nectin-2 albumen ECD areas of the present invention（That is extracellular region）Structural representation.
Fig. 2 be the embodiment of the present invention 1 in be overexpressed people Nectin-2 albumen and immune globulin respectively in HEK293 cells
The fusion protein of white Fc fragments, immunoglobulin Fc（Control）, the PAGE gel electrophoresis result after protein purification.
Fig. 3 is influence of the Nectin-2 albumen to Mouse Weight in the embodiment of the present invention 2；Wherein, IgG represents obesity mice
Injecting immune globulin Fc reference protein groups, Nectin-2 represents obesity mice injection people Nectin-2 albumen and immunoglobulin
The fusion protein group of Fc fragments.
Fig. 4 is influence of the Nectin-2 albumen to mouse fasting blood-glucose in the embodiment of the present invention 3；Wherein, IgG represents obesity
Mouse injecting immune globulin Fc reference protein groups, Nectin-2 represents obesity mice injection people Nectin-2 albumen and immune ball
The fusion protein group of albumen Fc fragments.
Fig. 5 is influence of the Nectin-2 albumen to mouse energetic supersession in the embodiment of the present invention 4；Wherein, IgG represents obesity
Mouse injecting immune globulin Fc reference protein groups, Nectin-2 represents obesity mice injection people Nectin-2 albumen and immune ball
The fusion protein group of albumen Fc fragments.
Fig. 6 is influence of the Nectin-2 albumen to mouse diet amount in the embodiment of the present invention 4；Wherein, IgG represents fat small
Mouse injecting immune globulin Fc reference protein groups, Nectin-2 represents obesity mice injection people Nectin-2 albumen and immune globulin
The fusion protein group of white Fc fragments.
Fig. 7 is influence of the Nectin-2 albumen to mouse movement amount in the embodiment of the present invention 4；Wherein, IgG represents fat small
Mouse injecting immune globulin Fc reference protein groups, Nectin-2 represents obesity mice injection people Nectin-2 albumen and immune globulin
The fusion protein group of white Fc fragments.
Embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.If not specializing, embodiment
According to conventional laboratory conditions, such as Sambrook equimoleculars Cloning: A Laboratory Manual（Sambrook J&Russell DW,
Molecular cloning:a laboratory manual,2001）, or according to the condition of manufacturer's specification suggestion.
Unless specifically stated otherwise, the C57BL/6J mouse used in following examples are purchased from the magnificent experimental animal of Beijing dimension tonneau
Technology Co., Ltd..Agents useful for same is the reagent for analyzing pure rank, and can be commercially available from regular channel.Insulin is purchased from promise
With Nuo De pharmaceutical Co. Ltds.
The acquisition of the people's Nectin-2 albumen of embodiment 1
According to the people's Nectin-2mRNA sequences announced in GenBank（Accession No.：CR456818.1）, by
The nucleotide sequence of 1-1035 and the gene order of immunoglobulin Fc segments are spliced, and spliced gene order is such as
Shown in SEQ ID No.5.
By artificial synthesized above-mentioned sequence into rear, it is cloned into mammalian expression vector pcDNA3.1, the table built
Instantaneously it is transferred in HEK293 cells up to carrier, realizes the expression of fusion protein, purifies destination protein.SDS- after protein purification
PAGE Gel electrophoresis results are as shown in Figure 2.
The detailed process of cell transfecting, cell culture and protein purification is as follows：First by HEK293 cell suspension cultures in
In 293FreeStyle culture mediums（GIBCO）, in 5%CO2Under the conditions of cultivate, carbon dioxide shaking table（INFORS）Rotating speed be
125-135rpm.Final concentration of the 1 × 10 of cell during transfection expression6Individual cell/mL, transfection reagent is 293Fectin
(Invitrogen).48h and 96h after transfection, adds the 293FreeStyle culture mediums of original transfection cumulative volume 30%.After transfection
6-7 days, nutrient solution supernatant was collected by centrifugation in 2500rpm, and NaCl to final concentration of 3M is added thereto, and then adding Tris makes its end
Concentration is 50mM, and regulation pH value to 8.5, then 6000rpm centrifuge 10min fully after dissolving, finally by supernatant with 0.22 μm
Membrane filtration, then with 5mL Protein A (ToyoScreen AF-rProtein A-650F, TOSOH) in AKTA
Purified on Purifier (GE) protein purification system.First use equilibrium liquid（50mM Tris/HCl, 3.0M NaCl, pH8.5）
Balance after 5 column volumes of pillar, pillar is washed with equilibrium liquid for loading and continuation until UV absorption is to baseline, then uses eluent
（25mM citric acids, 4.5mM sodium citrates, pH3.0）Elution, efflux is collected when UV absorption is more than 5mAU.Then add
5%v/v neutralizers（2M Tris-HCl, pH9.0）Neutralized, PAGE gel electrophoresis is carried out to the destination protein of purifying.
Application of the people Nectin-2 albumen of embodiment 2 in obesity is suppressed
In order to verify the anti-obesic action of the Nectin-2 albumen prepared in embodiment 1, build in the present embodiment as follows
High lipid food inducing obesity group mouse model：
The C57BL/6J male mices of 6 week old are taken, 60Kcal% high lipid food is fed（Research diet,
D12451）, body weight is recorded weekly.8 weeks are induced after high fat diet inducing obesity group is randomly divided into control group after its obesity（IgG,
10）With experimental group group（Nectin-2,10）；Then by experimental group, Nectin-2 is used（Prepared in embodiment 1）Processing, with
The dose subcutaneous injection in 5mg/kg body weight/week；And by the same dose of IgG of control group（Immunoglobulin Fc is used as control egg
In vain）Processing, is administered, weekly administration in hypodermic mode, and first 2 weeks each test group of animals of detection are administered and averagely ingest daily
Amount, and in the record body weight weekly of administration.After successive administration 14 weeks, the neck that breaks puts to death mouse, takes out liver, epididymal adipose tissues and subcutaneous
Fat, weighs.
From figure 3, it can be seen that Nectin-2 processing can effectively suppress fat, specially 50.85 ± 0.5g of control group,
46.85 ± 1.1g of experimental group（p<0.05）.
Application of the people Nectin-2 albumen of embodiment 3 in reduction fasting blood-glucose
Existing result of study shows that obesity is usually associated with serious insulin resistance.In the present embodiment, it is further right
The mouse of the 9th week is administered in embodiment 2, tests below has been carried out：
The mouse overnight fasting after 9 weeks will be administered in embodiment 2 and survey fasting blood glucose level.
As a result as shown in figure 4, the control group of high fat diet induction（IgG）Fasting blood-glucose（5.84mmol）It is significantly higher than
The experimental group fasting blood-glucose of Nectin-2 processing（5.2mmol, p<0.05）.
Application of the people Nectin-2 albumen of embodiment 4 in energy ezpenditure is promoted
Mouse after being administered the 10th week in Example 2, its oxygen demand is detected with the LabMaster of TSE companies.From Fig. 5
As can be seen that Nectin-2 albumen increases the oxygen demand of body（p<0.05）, illustrate that Nectin-2 albumen can improve body
Integral energy is metabolized.But Nectin-2 is to feed intake（Fig. 6）And amount of exercise（Fig. 7）Have no significant effect.
As can be seen here, Nectin-2 albumen can increase the energetic supersession level of body.
Although above the present invention is described in detail with a general description of the specific embodiments,
On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements, belong to the scope of protection of present invention without departing from theon the basis of the spirit of the present invention.
Bibliography：
1、Gao X,et al.Prevalence and trend of hepatitis C virus infection
among blood donors in Chinese mainland:a systematic review and meta-
analysis.BMC Infect Dis2011;11:88.
2nd, Samanta D, Ramagopal UA, Rubinstein R, et al.Structure of Nectin-
2reveals determinants of homophilic and heterophilic interactions that
Control cell-cell adhesion [J] .Proc Natl Acad Sci U S A, 2012,109（37）：14836-
14840.
3rd, Lopez M, Aoubala M, Jordier F, et al.The human poliovirus receptor
related2protein is a new hematopoietic/endothelial homophilic adhesion
Molecule [J] .Blood, 1998,92（12）：4602-4611.
4th, Takahashi K, Nakanishi H, Miyahara M, et al.Nectin/PRR:an
immunoglobulin-like cell adhesion molecule recruited to cadherin-based
Adherens junctions through interaction with Afadin, a PDZ domain-containing
Protein [J] .J Cell Biol, 1999,145（3）：539-549.
Claims (6)
1. a kind of fusion protein, it is characterised in that be made up of three continuous immunoglobulin like domain, be N-terminal respectively
One Ig-like V structures domain and the two Ig-like C2-type 1 and the domains of Ig-like C2-type 2 of C-terminal, three
By two sections of Linker connections between domain, every section of Linker is formed by 1-10 amino acid tandem；
Wherein, the Ig-like V structures domain of N-terminal, two Ig-like C2-type 1 of C-terminal and the knots of Ig-like C2-type 2
The amino acid sequence in structure domain is respectively as shown in SEQ ID No.1-3；
The amino acid sequence of the fusion protein is as shown in SEQ ID No.4.
2. encode the gene of fusion protein described in claim 1.
3. engineering bacteria or transgenic cell line containing gene described in claim 2.
4. people Nectin-2 albumen encodes the medicine that the gene of the albumen is used to improve energetic supersession or promote to generate heat in preparation
In application.
5. a kind of be used to treat diabetes, the pharmaceutical composition of fat and relevant disease, it is characterised in that the drug regimen
Thing includes the fusion protein described in claim 1, or it is by modification or improved albumen；Optionally include pharmaceutically acceptable
Microcarrier, nano particle and/or auxiliary material；
Described to refer to by improved albumen, the albumen merges the fusion protein constituted with immunoglobulin Fc segments；
The albumen by modification is finger protein by including acetylation, Pegylation, glycosylation or being combined with BSA.
6. pharmaceutical composition according to claim 5, it is characterised in that it is slow release formulation.
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| PCT/CN2014/084265 WO2015027820A1 (en) | 2013-08-29 | 2014-08-13 | Use of human nectin-2 protein |
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| US20030044893A1 (en) * | 2000-10-05 | 2003-03-06 | Baum Peter R. | Nectin polypeptides, polynucleotides, methods of making and use thereof |
| CN1880455A (en) * | 2005-06-17 | 2006-12-20 | 中国医学科学院基础医学研究所 | Human tumor suppressor gene NECL2 and apoptosis of leukemia cells induced by its interactive protein |
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| WO1999063063A1 (en) * | 1998-06-03 | 1999-12-09 | Northwestern University | Cellular proteins which mediate herpesvirus entry |
| US20100150924A1 (en) * | 2007-05-22 | 2010-06-17 | Elior Peles | Regulation of myelination by nectin-like (necl) molecules |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030044893A1 (en) * | 2000-10-05 | 2003-03-06 | Baum Peter R. | Nectin polypeptides, polynucleotides, methods of making and use thereof |
| CN1880455A (en) * | 2005-06-17 | 2006-12-20 | 中国医学科学院基础医学研究所 | Human tumor suppressor gene NECL2 and apoptosis of leukemia cells induced by its interactive protein |
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| Title |
|---|
| HSV vector-mediated transduction and GDNF secretion from adipose cells;J Fradette et al.;《Gene Therapy》;20041007;第12卷(第1期);第48-58 * |
| 细胞黏附力和黏附分子对癌侵袭性影响的研究展望;许洪卫;《世界华人消化杂志》;20050915;第13卷(第17期);第2057-2060页 * |
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