CN103550790B - Periostin antibody and application thereof in preparation of medicine - Google Patents
Periostin antibody and application thereof in preparation of medicine Download PDFInfo
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Abstract
The invention relates to the field of medicine preparation, in particular to application of Periostin genes and Periostin antibodies in medicine preparation. The Periostin antibody prepared by the invention can be used for neutralizing Periostin in obese mice and remarkably reducing the liver/body weight ratio and the content of liver and serum TG, so that the Periostin antibody has the effects of remarkably improving the development of NAFLD and reducing the content of TG in serum.
Description
Technical field
The present invention relates to field of medicine preparation, particularly periostin gene and the application of periostin antibody in medicine preparation.
Background technology
Non-alcoholic fatty liver disease (NAFLD) has become an important public health problem.In in the past 10 years, the prevalence of western countries NAFLD rises to 20-30%, and the sickness rate of obese people NAFLD is even more than 50%.In recent years, along with the change of people life style and dietary structure, the prevalence of China some areas adult NAFLD also more than 15%, and gradually becomes younger, the health of serious harm people.Except the health caused except NAFLD itself and psychological problems, its adjoint multiple relevant disease is as type 2 diabetes mellitus, cardiovascular and cerebrovascular disease, hepatic fibrosis, liver cirrhosis even hepatocarcinoma etc., if Diagnosis and Treat not in time, this disease meeting serious threat human physical and mental health and quality of life, also considerably increase the financial burden of family and society.
The essence of NAFLD is the excessive accumulation of triglyceride in liver (TG).In hepatocyte, triglyceride mainly comes from hepatocellular picked-up and the biosynthesis of self.On the other hand, in liver, the outlet of triglyceride is mainly beta oxidation and is released into blood with synthesis VLDL.The exception of above-mentioned arbitrary link all can cause the excessive accumulation of triglyceride in liver.
Hyperlipemia refers to that human body blood lipid level is too high, directly can cause the disease of some serious harm healths, as atherosclerosis, coronary heart disease, pancreatitis etc.Hyperlipemia is a kind of common disease, and in the middle of middle-aged and elderly people, sickness rate is high, can be divided into constitutional and Secondary cases two class.Constitutional is relevant with heredity with congenital, and the multiple metabolic disorder disease (diabetes, hypertension, myxedema, hypothyroidism, obesity, hepatorenal disease, adrenal cortex function are hyperfunction) of being born in of Secondary cases, or with other factor age, sex, season, drink, smoking, diet, physical exertion, psychentonia, emotional activity etc. be relevant.Under normal conditions, most of patients non-evident sympton and abnormal sign, many people raise owing to just finding that there is blood plasma lipoprotein level when other reasons carries out blood biochemical inspection, if so can diagnose out hyperlipemia in time, patient can be allowed better to be treated.
Periostin (Periostin) is a kind of extracellular matrix protein by osteoblast or fibroblasts to secrete, belongs to bundle protein (fasciclin) family member.Show according to current research, Periostin is up-regulated under multiple pathologic condition, comprises the Bone tumour etc. of colon cancer, breast carcinoma, but the effect of Periostin in NAFLD and hyperlipemia not yet has relevant report.
Summary of the invention
The object of this invention is to provide the application of periostin (Periostin) gene in preparation screening non-alcoholic fatty liver disease (NAFLD) reagent and hyperlipemia reagent, by the clinical practice of this diagnostic reagent, NAFLD patient can be diagnosed out in time, to treat in time, improve the cure rate of patient.
Another object of the present invention is to provide a kind of Periostin antibody, and this Periostin antibody tool is significantly improved NAFLD progress and reduce the effect of Triglycerides in Serum (TG) content.
Another object of the present invention is to provide the application of above-mentioned Periostin antibody in preparation treatment NAFLD medicine.
Another object of the present invention is to provide the application of above-mentioned Periostin antibody in preparation treatment hyperlipidemia.
The present invention is by chip gene expression profile technology, compare the differential expression of two groups of C57BL/6 mouse liver genes of high fat and low fat diet nursing, therefrom find: in the obesity mice liver of high fat diet induction, the expression of Periostin gene is significantly raised (p<0.05).
We are by Real-timePCR and ELISA experiment further, confirm the unconventionality expression of Periostin in obesity mice liver.In addition, in the ob/ob obesity mice that db/db obesity mice and the leptin receptor of leptin receptor shortage lack, also obtain identical result, that is:, during obesity, the expression of liver Periostin is significantly raised.
Further, we find that the expression of the Periostin of fatty liver patient significantly raises, and present obvious positive correlation with liver TG content.As can be seen here, the high expressed of Periostin is a characteristic change of NAFLD.
There is developing effect for inquiring into Periostin at NAFLD, testing by adenovirus mediated process LAN system, specificity process LAN Periostin in C57BL/6 mouse liver.Compared with organizing mice with process LAN green fluorescent protein (GFP), after Periostin process LAN, liver TG content increases, liver/weight ratio increases, and Triglycerides in Serum (TG) especially low density lipoprotein, LDL-TG (VLDL-TG) content raises.
Yihong-the brazilwood extract dyeing (H & E) of liver organization paraffin section and oil red dyeing (OilRedO) also confirm: after Periostin process LAN, hepatocyte presents ballooning degeneration.In addition, in the people source hepatoma H22 cells of In vitro culture and mouse primary hepatocytes (MPH), add the Periostin albumen of purification, also find: Periostin can promote the deposition of intracellular triglyceride.
We prepared Periostin antibody in and obesity mice body in Periostin.Give db/db mouse tail vein injection antibody after two weeks (5mg/kg), same matched group (injection IgG) is compared, Periostin neutralizing antibody does not affect the body weight of obesity mice, but can significantly reduce liver/weight ratio, reduce liver and serum TG content, increasing serum beta-hydroxybutyric acid level, increases the expression of triglyceride and fatty acid beta oxidation related gene.
The aminoacid sequence of the Periostin albumen of mice of the present invention is as shown in SEQIDNo.1;
1mvpllplyallllflcdinpanansyydkvlahsrirgrdqgpnvcalqqilgtkkkyfs
61scknwyqgaicgkkttvlyeccpgymrmegmkgcpavmpidhvygtlgivgatttqhysd
121vsklreeiegkgsytyfapsneawenldsdirrglennvnvellnalhshmvnkrmltkd
181lkhgmvipsmynnlglfinhypngvvtvncarvihgnqiatngvvhvidrvltqigtsiq
241dfleaeddlssfraaaitsdlleslgrdghftlfaptneafeklprgvlerimgdkvase
301almkyhilntlqcseaitggavfetmegntieigcegdsisingikmvnkkdivtkngvi
361hlidevlipdsakqvielagkqqttftdlvaqlglasslkpdgeytllapvnnafsddtl
421smdqrllklilqnhilkvkvglsdlyngqiletiggkqlrvfvyrtaicienscmvrgsk
481qgrngaihifreiiqpaekslhdklrqdkrfsiflslleaadlkdlltqpgdwtlfaptn
541dafkgmtseerelligdknalqniilyhltpgvyigkgfepgvtnilkttqgskiylkgv
601netllvnelkskesdimttngvihvvdkllypadipvgndqllellnklikyiqikfvrg
661stfkeipmtvyrpamtkiqiegdpdfrlikegetvtevihgepvikkytkiidgvpveit
721ekqtreeriitgpeikytristgggetgetlqkflqkevskvtkfieggdghlfedeeik
781rllqgdtpakkipankrvqgprrrsregrsq
Then the 761st wherein is chosen to be antigen fragment to 790 amino acids fragments (wkvtkfieggdghlfedeeikrllqgdtpak), step obtains Periostin antibody then to carry out prepared by antigen production, animal immune, fusion and monomer Selection, ascites etc.
Beneficial effect of the present invention is:
1, the Periostin during the Periostin antibody that prepared by the present invention can be used for and in obesity mice body, remarkable reduction liver/weight ratio, liver and serum TG content, so, Periostin antibody tool is significantly improved NAFLD development and reduce the effect of TG content in serum, can be applicable to preparation treatment NAFLD and hyperlipidemia.
2, by experiment, find in the obesity mice liver of high fat diet induction, the expression of Periostin gene is significantly raised, the expression of the Periostin of fatty liver patient significantly raises, and present obvious positive correlation with liver TG content, serum TG content, illustrate that Periostin gene can be used for preparation screening NAFLD medicine.
3, the present invention provides more experimental data and scientific basis, for Diagnosis and Treat NAFLD and hyperlipemia are laid a good foundation early clinically for more reasonably applying Periostin and Periostin antibody clinically.
Accompanying drawing explanation
Fig. 1 is that the Periostin in the mouse liver that high fat is fed and low fat is fed expresses comparison diagram.
Fig. 2 be leptin receptor lack db/db obesity mice and leptin lack ob/ob obesity mice liver in Periostin express comparison diagram.
Fig. 3 is the expression comparison diagram of the Periostin of normal person and fatty liver patient, and presents obvious positive correlation with liver tg content.
Fig. 4 is by adenovirus mediated process LAN system, specificity process LAN Periostin in C57BL/6 mouse liver, process LAN green fluorescent protein comparison diagram in control group mice liver, wherein, A is liver tg content balance figure, B is that liver/weight ratio increases comparison diagram, and C is serum triglycerides comparison diagram.
Fig. 5 is by adenovirus mediated process LAN system, specificity process LAN Periostin in C57BL/6 mouse liver, process LAN green fluorescent protein in control group mice liver, Yihong-brazilwood extract dyeing of the liver organization paraffin section of two groups of mices and oil red staining versus figure.
Fig. 6 is the Periostin albumen adding purification in the people source hepatoma H22 cells cultivated and mouse primary primary hepatic cell, demonstrates the deposition that Periostin can promote intracellular triglyceride.
Fig. 7 be preparation Periostin antibody in and the comparison diagram of obesity mice, wherein, Fig. 7 A is the protein immunoblotting Experimental comparison figure of three kinds of mouse genotypes primary hepatic cells; 7B is that the db/db obesity mice that leptin receptor lacks gave Periostin antibody treatment after 2 weeks, compares, blood-serum P eriostin content balance figure with matched group IgG group.
Fig. 8 is the db/db obesity mice tail vein injection Periostin antibody and the comparison diagram of injection IgG after two weeks that give leptin receptor shortage, wherein, Fig. 8 A is liver/weight ratio comparison diagram, Fig. 8 B is liver tg content balance figure, Fig. 8 C is serum triglycerides content balance figure, Fig. 8 D is the horizontal comparison diagram of serum beta-hydroxybutyric acid.
Fig. 9 gives the db/db obesity mice tail vein injection Periostin antibody that leptin receptor lacks and the expression comparison diagram injecting the fatty acid beta oxidation related gene of IgG after two weeks.
Detailed description of the invention
Below in conjunction with embodiment, the invention will be further described:
Mice: C57BJ/6 wild type male mice (8-12 age in week), purchased from Shanghai Slac Experimental Animal Co., Ltd..Db/db, ob/ob mice (8-12 age in week), purchased from Nanjing University model organism center.Mouse feeder is at Shanghai Endocrine-Metabolic Diseases Inst.'s cleaning grade Animal House, and raising temperature is 21 ± 1 DEG C, humidity 55 ± 10%, and light application time is 8:00AM-8:00PM.
Normal diet is purchased from Shanghai Slac Experimental Animal Co., Ltd., fatty containing 10Kcal%, 70Kcal% carbohydrate, 20Kcal% protein.
High lipid food purchased from American ResearchDiet company (D12492), 60Kcal% fat, 20Kcal% carbohydrate, 20Kcal% protein.
Cell: people source hepatoma H22 cells, purchased from Chinese Academy of Sciences's Shanghai school of life and health sciences cell bank, adopts DMEM culture fluid to cultivate, adds 10% hyclone and 100U/ml penicillin and 100g/ml streptomycin in training liquid.Cell every 2 goes down to posterity once.
Embodiment 1
C57BL/6 mice is divided into two groups, one group is that high fat is fed 12 weeks, another group is that low fat is fed 12 weeks, by chip gene expression profile technology, compare the differential expression of two groups of mouse liver genes of high fat group and low fat nursing group, therefrom find: in the obesity mice liver of high fat diet induction, express and significantly raise (p<0.05).Tested by Real-timePCR and ELISA, detect db/db obesity mice that Periostin gene lacks at the obesity mice of high fat nursing group, leptin receptor, expression in ob/ob obesity mice that leptin lacks and fatty liver patient, result shows: the unconventionality expression of Periostin gene in the obesity mice liver of high fat nursing group, specifically as shown in Figure 1; The ob/ob obesity mice that the db/db obesity mice lacked for leptin receptor and leptin lack, the expression of two groups of obesity mice liver Periostin is significantly raised, specifically as shown in Figure 2; In fatty liver patient, the expression of Periostin significantly raises, and presents obvious positive correlation with liver TG content, specifically as shown in Figure 3.
Experiment is by adenovirus mediated process LAN system, specificity process LAN Periostin in C57BL/6 mouse liver, simultaneously, test as a comparison with specificity process LAN green fluorescent protein (GFP) in C57BL/6 mouse liver and detect, testing result is: compared with organizing mice with process LAN green fluorescent protein (GFP), after Periostin process LAN, the increase of mouse liver TG content, liver/weight ratio increase, in serum, TG content increases, especially in serum, low density lipoprotein, LDL-TG (VLDL-TG) content raises, and specifically sees shown in Fig. 4 A-4C.Simultaneously, specificity process LAN Periostin mouse liver tissue and specificity process LAN GFP mouse liver tissue are carried out Yihong-brazilwood extract dyeing (H & E) and oil red dyeing (OilRedO) experiment of paraffin section, experiment confirms: after Periostin process LAN, hepatocyte presents ballooning degeneration, as shown in Figure 5.
Add the Periostin albumen of purification in the people source hepatoma H22 cells cultivated in vitro and mouse primary hepatocytes (MPH), specifically as shown in Figure 6, find: Periostin can promote the deposition of intracellular triglyceride.
By above-mentioned experiment, describe Periosin and express with the generation of NAFLD, hyperlipemia, develop to have and close contact relation.
Embodiment 2
Periostin antibody manufacturing process.
1), the aminoacid sequence (as shown in SEQIDNo.1) of mice Periostin albumen is:
1mvpllplyallllflcdinpanansyydkvlahsrirgrdqgpnvcalqqilgtkkkyfs
61scknwyqgaicgkkttvlyeccpgymrmegmkgcpavmpidhvygtlgivgatttqhysd
121vsklreeiegkgsytyfapsneawenldsdirrglennvnvellnalhshmvnkrmltkd
181lkhgmvipsmynnlglfinhypngvvtvncarvihgnqiatngvvhvidrvltqigtsiq
241dfleaeddlssfraaaitsdlleslgrdghftlfaptneafeklprgvlerimgdkvase
301almkyhilntlqcseaitggavfetmegntieigcegdsisingikmvnkkdivtkngvi
361hlidevlipdsakqvielagkqqttftdlvaqlglasslkpdgeytllapvnnafsddtl
421smdqrllklilqnhilkvkvglsdlyngqiletiggkqlrvfvyrtaicienscmvrgsk
481qgrngaihifreiiqpaekslhdklrqdkrfsiflslleaadlkdlltqpgdwtlfaptn
541dafkgmtseerelligdknalqniilyhltpgvyigkgfepgvtnilkttqgskiylkgv
601netllvnelkskesdimttngvihvvdkllypadipvgndqllellnklikyiqikfvrg
661stfkeipmtvyrpamtkiqiegdpdfrlikegetvtevihgepvikkytkiidgvpveit
721ekqtreeriitgpeikytristgggetgetlqkflqkevskvtkfieggdghlfedeeik
781rllqgdtpakkipankrvqgprrrsregrsq
2), the choosing of antigen fragment:
The 761st wherein to 790 amino acids fragments (wkvtkfieggdghlfedeeikrllqgdtpak) are chosen to be antigen fragment.
3), antigen is produced:
Transformed by genetic fragment synthesis, expression vector establishment, positive clone identification, positive colony plasmid extraction and expression bacterium, a large amount of induced protein expresses generation, Ni post affinity purification step obtains proteantigen for immunity.
4), animal immune:
By step 3) in the antigen mixed immunity Mus that obtains be age the female BAl BIc/c healthy mice 3 of 8 ~ 12 weeks.Take mice tachysynthesis mode, the mixing of the spleen of 3 mices is merged.
5), fusion and monoclonal antibody screening:
The splenocyte of mice best for immunoreation and myeloma cell (SP2/0) are merged, cell after fusion is through dilution, be placed in 96 well culture plates and cultivate, cultivate and carry out ELISA detection in 10-14 days, the cell selected in the high hole of OD value carries out limiting dilution assay sub-clone, until positive boring ratio rate is 100%, obtain building the successful cell strain of strain and carrying out amplification culture, cell number is by (1-2) x10
6/ pipe carries out frozen.
6), ascites preparation:
By step 5) in cell strain adopt mice celiac inoculation to prepare ascites, within 10-14 days, collect ascites, adopt ProteinG column purification ascites, obtain Protein antibody.
Knock out type mouse primary hepatocytes at wild type mouse primary hepatocytes, Periostin, Periostin knocks out in the type mouse primary hepatocytes transfection Perisotin adenovirus hepatocyte of 36 hours, tested by protein immunoblotting, verify the specificity of the Protein antibody obtained, specifically as shown in Figure 7 A; The db/db obesity mice tail vein injection that leptin receptor lacks gave Periostin antibody treatment after 2 weeks, compared with matched group, and blood-serum P eriostin level declines, and sees shown in Fig. 7 B.
Embodiment 3
To give in db/db mouse tail vein injection embodiment 2 the Periostin antibody two weeks of preparation, injection volume is 5mg/kg, give another db/db mouse tail vein injection IgG two weeks, injection volume is that 5mg/kg compares as a control group, and testing result is as follows: the liver/weight ratio of Periostin antibody injection mice group significantly reduces; TG content in liver and serum also obviously reduces; Serum b-hydroxybutyric acid level also raises greatly, specifically as D shown in FIGS. 8 A-8.The expression of fatty acid beta oxidation related gene simultaneously too increases, as shown in Figure 9.
By above-mentioned experiment, illustrate that Periostin antibody tool is significantly improved the effect of NAFLD development, is simultaneously also improved effect for hyperlipemia, can be applicable to the medicine preparing treatment NAFLD and hyperlipemia.
Claims (3)
1. a periostin antibody, is characterized in that: the antigen fragment of this antibody is for aminoacid sequence is for the 761st shown in SEQIDNo.1 is to the amino acid fragment of 790.
2. the application of periostin antibody as claimed in claim 1 in preparation treatment non-alcoholic fatty liver disease medicine.
3. the application of periostin antibody as claimed in claim 1 in preparation treatment hyperlipemia medicine.
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| Expression of the extracellular matrix protein periostin in liver tumours and bile duct carcinomas;Marc-Oliver Riener et al.;《Histopathology》;20101231;第56卷;第600–606页 * |
| Irisin is inversely associated with intrahepatic triglyceride contents in obese adults;Hui-Jie Zhang et al.;《Journal of Hepatology》;20130509;第59卷;第557–562页 * |
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