CN106243192A - Vitamin d binding peptide and application thereof - Google Patents
Vitamin d binding peptide and application thereof Download PDFInfo
- Publication number
- CN106243192A CN106243192A CN201610639231.6A CN201610639231A CN106243192A CN 106243192 A CN106243192 A CN 106243192A CN 201610639231 A CN201610639231 A CN 201610639231A CN 106243192 A CN106243192 A CN 106243192A
- Authority
- CN
- China
- Prior art keywords
- vitamin
- binding peptide
- antibody
- binding
- cell
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/82—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving vitamins or their receptors
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Hematology (AREA)
- Genetics & Genomics (AREA)
- Urology & Nephrology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention discloses a kind of vitamin D binding peptide and application thereof, the motif of vitamin D binding peptide is IYSLAASSELPHVYMRKQS.The vitamin D binding peptide of the present invention, can induce the antibody for vitamin D binding protein well, and then vitamin D binding protein is carried out accurate quantification, it is achieved the detection by quantitative of internal vitamin D content as antigen.
Description
Technical field
The present invention relates to a peptide species, particularly to a kind of vitamin D binding peptide and application thereof.
Background technology
Vitamin D is a kind of fatsoluble vitamin being made up of long hydrocarbon chain or condensed ring, is steroid derivatives.It promotees
Enter the formation of normal bone, strengthen small intestinal and the kidney absorption to calcium, phosphorus, ferrum and zinc ion, and can regulate and include skeleton
With heart in the interior health of all muscle, immunoreation, blood glucose and the level of internal insulin.For the mankind, with body
The form of the closely-related most important two kinds of vitamin D of body health status is vitamin D2 and vitamin D3, is called again
Ergocalciferol and cholecalciferol[1].Ergocalciferol and cholecalciferol can absorb from diet and supplement[1][2][3], but very
Few food contains natural calciferol and cholecalciferol.It is true that no matter adult has many sufficient nutrition, they can not
Enough vitamin D can be obtained from diet itself.Therefore, vitamin is under ultraviolet irradiation, percutaneous photochemistry
Effect synthesis.Vitamin D3 is the basal layer at skin, and 7-DHC absorbs the B photon of ultraviolet, generates vitamin
The precursor of D3, the rearrangement being then subjected to a triene structures relevant with variations in temperature forms vitamin D3, lumisterol and speed admittedly
Alcohol (Fig.1.1)[4][5][6].Pointing out Michael in 2004, the synthesis of vitamin D3 is by season, live on-site latitude, kind
Race, age, the calmness of skin pigment and other factor.
Healthy extremely important to people of vitamin D.The effect of vitamin D hormones includes supervising skeleton and muscle
Health, regulates immunoreation, regulates insulin and blood glucose, regulate calcium, phosphorus catabolism.So, vitamin D deficiency is to threaten
One significant problem of human health.The concentration measuring serum 25(OH)VD (25 (OH) D) determines that vitamin D shape
The method of testing that state is best.The horizontal fragmentation of internal 25 (OH) D is as follows[14]:
Vitamin d insufficiency: 21-29 ng/mL (52.5-72.5 mmol/L)
Vitamin D deficiency: < 20 ng/mL (< 50 mmol/L)
Although the diagnosis to vitamin d insufficiency is the most required, but it reflects the health status of human body to a certain extent.
The mensuration of serum parathyroid hormone level potentially contributes to establish the diagnosis of vitamin d insufficiency, such as the patient of vitamin d insufficiency
Often there is the rising of parathyroid hormone level, point out secondary hyperparathyroidism.
Vitamin D deficiency has been demonstrated almost to play in each major disease certain effect.This includes: osteoporosis
Disease and bone amount reduce, the cancer (including breast carcinoma, carcinoma of prostate and colon cancer) of 17 types, heart disease, hypertension, obesity,
Metabolism syndrome, diabetes, autoimmune disease, multiple sclerosis, rheumatic arthritis, osteoarthritis, Bursitis,
Gout, infertile and premenstrual syndrome, Parkinson's disease, depression and seasonal affective disorder, Alzheimer's disease, chronic tired
Labor syndrome, fibromyalgia, chronic pain, periodontal, psoriasis etc..Next be discussed in detail vitamin D deficiency and these
The pathogenetic substantial connection of disease.
In skeletal system, skeleton, intestinal and kidney exist the specific receptor of a lot of vitamin D, it is possible to play it
Bone effectivity, promotes absorbing of calcium.1,25 (OH)2VD3 regulation parathyroid hormone (PTH) and fibroblast
Growth factor-2 3(FGF23) secretion, play an important role to maintaining internal normal bone ore deposit balance and the formation of bone.If grown up
In human body, VD lacks, and will cause osteomalacia, old people's vitamin D deficiency that osteoporosis will occur, and it occurs baby
Time in child's body, arise that rickets.And vitamin D deficiency also can cause muscle weakness, increase fracture and the wind fallen
The serious consequences such as danger.
In terms of immune system, 1,25 (OH)2VD3 can suppress the generation of lymphopoiesis and immunoglobulin, prolongs
The process of slow bone-marrow-derived lymphocyte activation plasmablast, has benefit to autoimmune disease, moreover it is possible to induction peptide expression, kills
Sick and wounded pathogenic microorganism[15].VD level can increase the risk of autoimmune disease when reducing, such as type 1 diabetes, multiple sclerosis
And clone disease[16].Vitamin D deficiency can also increase the risk of viral infection, including HIV (human immunodeficiency virus) (HIV) and popularity
Flu[17][18][19], also it is a tuberculous risk factor[20].Supplement 1,25 (OH)2VD3 is to these diseases and inflammation
Property arthritis, inflammatory bowel and pulmonary tuberculosis etc. there is prevention or therapeutical effect, it is also possible to reduce and systemic lupus erythematosus (sle) occur
Risk.
In terms of tumor, there are bioactive 1,25 (OH)2VD3 has antiproliferative and promotes differentiation most cells
Effect, has potential anti-tumor activity.Particularly 1,25 (OH)2VD3 stimulates sub-p21 and p27 of Cyclin-dependent kinase and thin
The expression of intercellular adhesion molecule CAM 120/80, suppresses the transcriptional activity of beta-catenin simultaneously.Vitamin D can also resist antibacterial
Infect with virus.The vitamin D nutrition situation of internal abundance can prevent a series of cancer, such as bladder cancer, carcinoma of prostate, first
Shape adenocarcinoma, lymphoma, gastric cancer etc.[21].But research finds, taking Vitamin D supplements does not has notable shadow to cancered risk
Ring[22].And some researchs have shown that, the mortality rate of vitamin D3 reduction cancer, but the character quilt of the data about this respect
Pay close attention to[23]。
Activated vitamin D regulation hormone secretion, the intracellular calcium that there is VDR and regulation intracellular calcium concentration of the β of pancreas combines
Albumen-D28K, 1,25 (OH)2VD and vitamin D receptor (VDR) combine scalable insulin secretion.Vitamin D deficiency, it is possible to
Increase the risk that type 2 diabetes mellitus occurs.Serum 25(OH) VD can be as immunomodulator for renal transplantation, serum 25(OH)
D has the function of protection transplanted kidney in renal transplantation[24]。
In terms of cardiovascular disease, vitamin D deficiency crowd's coronary heart disease, heart failure and the prevalence of peripheral vascular disease
Dramatically increase[25].Vitamin D deficiency easily causes the infection of respiratory tract, and there is dependency with asthma.In essence
God's disease aspect, the shortage of vitamin D may increase depression, schizophrenia, cognitive disorder and autism-spectrum obstacle etc.
The risk of mental illness；. also it is the risk factor of chronic kidney disease generation.Gestation period VD lacks and preeclampsia, insulin
Resist relevant with gestational diabetes.
Vitamin D nutritional status in vivo, the generation development with the most multiple systems, multiple disease has close
Relation, is referred to as " rising star of disease prevention "[26].But long-term or heavy dose of use vitamin D, can make serum 25(OH)
The concentration of VD raises and hypercalcemia, and clinical signs goes out poisoning symptom.Researcher is had to think, serum 25(OH) VD > 100ng/
Ml claims poisoning by vitamin D.Therefore, vitamin D plays important role in human body, and in detection bodies, the level of vitamin D is also
Become a kind of trend.
Vitamin D is to have inertia biologically, and it just must can be activated through twice hydroxylating in vivo and have life
Thing activity.Vitamin D enters body-internal-circulation system, and the carrier protein in blood i.e. vitamin D binding protein is combined, and then transports
It is passed to the site of target tissue and storage, mainly fat and muscle[7].When vitamin D is transported to liver, it is through C25 site
The hydroxylation of CYP27A1 enzyme changes into 25(OH)VD, is also called calcifediol (25OHD).This is that vitamin D is at body
Interior first time metabolism, 6 six cytochrosome P-450 isoforms (CYP27A1, CYP2R1, CYP2C11 CYP3A4, CYP2D25
And CYP2J3) all take part in the hydroxylated activity of vitamin D3 25[8].25(OH)VD transports out liver and vitamin D
Associated proteins combines, and is then transported to kidney under the most hydroxylated effect of CYP27B1 enzyme in C1 α site, is finally converted to
There is bioactive calcitriol, i.e. 1 α, 25(OH)2D.As internal 1 α, 25(OH)2During D content abundance, it is at CYP24 enzyme
24,25 (OH) are changed under effect2D(Fig.1.2).Calcitriol is the activated form in kidney, and also can be with vitamin D
Associated proteins combines, and is transported to the target tissue that vitamin D receptor is expressed, mainly has skeleton, small intestinal and parathyroid gland, then
Its effect is played with the form of genome or non genome.The concentration of 25(OH)VD is the highest, the most stable, and the half-life is
Long (more than two weeks), is vitamin D prevailing traffic form in vivo.Therefore, serum 25 (OH) D level is to evaluate internal dimension
The maximally effective index of raw element D-state[9]。
List of references:
[1] Holick M F . High prevalence of vitamin D inadequacy and implications
for health [J]. Mayo Clin. Proc. 2006,81(3): 353–373.
[2] Calvo M S, Whiting S J, Barton C N, Whiting, Barton. Vitamin D
intake: a global perspective of current status[J]. Nutr. 2005,135 (2): 310–
316.
[3] Norman A W. From vitamin D to hormone D: fundamentals of the vitamin
D endocrine system essential for good health. Am. J. Clin. Nutr. 2008,88 (2):
491S–499S.
[4] Holick M F , McLaughlin J A , Clark M B , Doppelt S H . Factors that
influence the cutaneous pHotosynthesis of previtamin D3 . Science.1981,211 :
590-593.
[5] Holick M F , MacLaughlin J A , Clark M B , Holick S A , Potts JT Jr ,
Anderson R R , Blank I H , Parrish J A , Elias P . pHotosynthesis of
previtamin D3 in human and the pHysiologic consequences. Science.1980, 210 :
203-205.
[6] Holick M F , Richtand N M , McNeill S C , Holick S A , Frommer J E ,
Henley J W , Potts J T . Isolation and identification of previtamin D3 from
the skin of exposed to ultraviolet irradiation . Biochemistry.1979,18 : 1003-
1008.
[7] Heaney R P , Horst R L , Cullen M , Armas L A . Vitamin D3
distribution and status in the body . J AmColl Nutr.2009,28 : 252 – 253.
[8] Ghoreschi K., U. Mrowietz and M. Röcken (2003) A molecule solves
psoriasis? Systemic therapies for psoriasis inducing interleukin 4 and Th2
responses. J. Mol. Med. 81, 471-480.
[9] Holick M F. The use and interpretation of assays for vitamin D and
its metabolites [J].J Nutr, 1990, 120(Suppl11),21:464-1469.
[10] Verboven C, Rabijns A, De Maeyer M, Van Baelen H, Bouillon R, De
Ranter C. A structural basis for the unique binding features of the human
vitamin D-binding protein[J]. Nat. Struct. Biol. 2002, 9 (2): 131–136.
[11] Mikkelsen M, Jacobsen P, Henningsen K. Possible localization of Gc-
System on chromosome 4. Loss of long arm 4 material associated with father-
child incompatibility within the Gc-System[J]. Hum Hered, 1977, 27 (2): 105–
107.
[12] D. D. Bikle, P. K. Siiteri, and E. Ryzen. Serum protein binding of
1,25-dihydroxyvitamin D: a reevaluation by direct measurement of free
metabolite levels[J]. The Journal of ClinicalEndocrinology & Metabolism,
1985, 61(5): 969–975.
[13] R. F. Chun, B.E.Peercy, E. S. Orwoll, C.M.Nielson, J. S.Adams, and
M. Hewison. Vitamin D and DBP: the free hormone hypothesis revisited[J]. The Journal of Steroid Biochemistry andMolecular Biology, 2013.
[14] Hollis B W, Wagner C L. Normal serum vitamin D levels[M]. N Engl J Med. Feb 3 2005;35,2(5):515-6; author reply 515-516.
[15] Zhou Xueying. Progress of Vitmin D Research. preclinical medicine and clinic, 1998,18:415 ~ 418.
[16] SMOLDERS J.DAMOISEAUX J, MEN HEERE P, et a1.Vitamin D as an immune
Modulator in multiple sclerosisz a review [-J] .J Neuroimmunol, 2008,194 (1 2):
7-17.
[17] Beard J A, Bearden A, Striker R. Vitamin D and the anti-viral state
[J]. Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology ,2011, 50 (3): 194–200.
[18] Spector S A (Feb 2011). Vitamin D and HIV: letting the sun shine in
[R]. Topics in antiviral medicine ;2011,19 (1): 6–10.
[19] Cannell J J, Vieth R, Umhau J C, Holick M F, Grant W B, Madronich S,
Garland C F, Giovannucci E. Epidemic influenza and vitamin D. Epidemiology and Infection ;2006, 134 (6): 1129–1140.
[20] Nnoaham K E, Clarke A. Low serum vitamin D levels and tuberculosis:
a systematic review and meta-analysis[J]. International Journal of Epidemiology ; 2008, 37 (1): 113–119.
[21] the Peripheral Blood In Patients With Breast Cancer 25(OH)VD such as Zhuan Zhigang, Yu Jianmin, Jiang Beiqi measure and clinical meaning
J ］, Chinese journals of practical medicine, 2010,26(2): 244 ~ 245.
[22] Bolland M J, Grey A, Gamble G D, Reid I R. The effect of vitamin D
supplementation on skeletal, vascular, or cancer outcomes: a trial sequential
meta-analysis[J]. Lancet Diabetes Endocrinol (Meta-analysis); 2014, 85(13):
202-209.
[23] Bjelakovic G, Gluud L L, Nikolova D, Whitfield K, Wetterslev J,
Simonetti R G, Bjelakovic M, Gluud C (January 10, 2014). Vitamin D
supplementation for prevention of mortality in adults[J]. The Cochrane database of systematic reviews; 2014.
[24] Teng Donghai, Liu Zhigang, flat 1 α of Lu one, what 25-dihydroxyvitamin D3 and the like was applied in renal transplantation grinds
Study carefully present situation and prospect " Sichuan medical science " 11 phases of volume 24 in 2003.
[25] KIM D H, SABOUR S, SAGAR U N, et a1.Prevalence of hypovitaminosis D in
cardiovascular diseases(from the National Health and Nutrition Examination
Survey 2001 to 2004) [J] .Am J Cardiol, 2008,102 (11): 1,540 1544.
[26] Autier P Gandini S. Vitamin D supplementation and total mortality ；a
Meta-analysis o randomized controlled trials [J]. Arch Intern Med, 2007,167
(16)；1730-1732..
Summary of the invention
It is an object of the invention to provide a kind of vitamin D binding peptide and application thereof.
The technical solution used in the present invention is:
A kind of vitamin D binding peptide, its motif is IYSLAASSELPHVYMRKQS(SEQ ID NO:1).
Further, one end of motif connects the aminoacid having not more than 3.
Further, its sequence is IYSLAASSELPHVYMRKQS.
The application in preparation vitamin D binding protein antigen of the said vitamin D binding peptide.
A kind of vitamin D binding protein antibody, is had the carrier induced animal body of said vitamin D binding peptide to obtain by coupling
Arrive.
The preparation method of a kind of vitamin D binding protein antibody, including by above-mentioned vitamin D binding peptide and increase immunity
The carrier conjugation of originality, afterwards stimulating animal body, then prepare and filter out monoclonal antibody, finally gives vitamin D and combines egg
Bai Kangti.
As the further improvement of said method, increasing immunogenic carrier is purified protein derivative (PPD) PPD.
A kind of vitamin D immue quantitative detection reagent box, described test kit is ELISA kit, and its vitamin D used combines
Protein antibodies is had the carrier induced animal body of said vitamin D binding peptide to obtain by coupling.
The invention has the beneficial effects as follows:
The vitamin D binding peptide of the present invention, can induce the antibody for vitamin D binding protein well as antigen,
And then vitamin D binding protein is carried out accurate quantification, it is achieved the detection by quantitative of internal vitamin D content.
Accompanying drawing explanation
Fig. 1 is the selection result of the mouse immune power of VD2；
Fig. 2 is the hybridoma the selection result of VD2.
Detailed description of the invention
Below in conjunction with experiment, further illustrate technical scheme.
The preparation of antigen:
1) the peptide sequence IYSLAASSELPHVYMRKQS(taking synthetic is labeled as VD2) it is dissolved in 2ml 0.02 M at 4 DEG C
Hydrochloric acid in, be then then added to the ether of 40ml extracts, stand after fully shaking up, be layered after take the layering of bottommost,
The polypeptide i.e. dissolved；
2) outfit of PPD: the PPD weighing 10mg is dissolved in the phosphate buffer (PBS) of 1ml, adds 10mg's
Malsac(EPSILON-N-MALEIMIDOCAPROIC ACID-(2-NITRO-4-SULFO)-pHENYL ESTER SODIUM
SALT, Ε-N-maleimidohexanoic acid (2-N-4-S) phenyl ester sodium salt) mix 30 minutes.Then draw mixed liquor to join in advance
With in the cylinder of washed twice of 0.1M PBS (5-10ml), after a period of time, collect the layering of gray with EP pipe, be dissolving
After PPD.Polypeptide after dissolving and PPD again together with join 0.5M PBS(0.5ml) in, mistake in the cold air of liquid nitrogen rapidly
Once, then shake up overnight in room temperature.Within second day, it is diluted to 5ml with PBS again, standby in subpackage EP pipe (1ml/ pipe), remaining
-80 DEG C can be stored into；Obtain being coupled to, by polypeptide, the antigen that tuberculin is made.
The PPD configured is mixed homogeneously with Freund adjuvant (Freund adjuvant), obtains VD2 immunizing agent.
Immune mouse: first step elder generation bacillus calmette-guerin vaccine afterbody subcutaneous injection immune mouse, makes mice produce immunoreation, further
Produce the antibody carrier protein as next step polypeptide immune.After one month, start the immunity of polypeptide for the first time, first group of mice
The VD2 immunizing agent that intramuscular injection 0.1ml is equipped with；The normal saline of control group mice intramuscular injection equivalent, injects first
The pure polypeptide of 20ug to be included at least in immunizing dose.After one month, all mices repeat booster immunization of injection.Warp
After crossing immunity in 20 days, cut mouse tail and take blood, measure the mice immunity programm to the immunizing agent of injection.Afterbody takes 2-3 and bleeds dilute
Release in the PBS of 0.5ml, be then centrifuged for taking its supernatant and store.With store supernatant as sample, carry out with elisa technique
Detection, sees immunne response situation in Mice Body.Then the mice that immunoreation is strong is carried out a booster immunization again.After 28 days
The mice that a front immunne response is strong is only injected the last booster immunization of synthetic peptide.During to experimental group booster immunization, comparison
Group all injects the normal saline of same dosage.Again after 3 days, kill the strong mice of immunoreation and take its spleen, then with without blood
Clear DMEM rinses several times, adds frozen stock solution (90%FBS+10%DMSO), and liquid nitrogen cryopreservation is standby.
The screening of mouse tail blood
The afterbody blood taking mice is sample, with being coated liquid (0.2M bicarbonate solution, pH9.6) as diluent, this polypeptide
It is installed to required volume, the concentration dilution of polypeptide to 10ug/ml, add the dilution containing polypeptide of 100ul in each hole
Liquid, seals with diaphragm seal, under moist environment room temperature, places 2 days.After hatching 2 days, first wash 2 times with cleaning mixture, wash
The formula washing liquid is: 0.05M Tris-HCl pH7.5,0.15M NaCl, 0.05%Tween-20.Wash away and unnecessary be coated liquid,
The confining liquid (5%BSA, PBS, 0.2% Tween-20) adding 100 ul closes one hour.After one hour, wash 2 times,
Add the blood plasma that 100 ul take, incubated at room temperature 2 hours from mouse tail.Wash away the antibody in uncombined serum the most again,
Addition Tris is conjugated buffer (1%BSA, 25Mm Tris-HCl, 0.15M NaCl) and presses the enzyme mark of 1:1000 dilution proportion
Two anti-(anti-mouse IgG), incubated at room temperature, after 1 hour, washes away the most specific binding antibody and enzyme labelled antibody, often
Hole adds the tetramethyl benzidine peroxidase substrate of 50ul, and after the color of our enough needs occurs, every hole adds
The stop buffer (1M sulfuric acid) of 100ul.Within adding stop buffer 30 minutes, read each hole at 450nm by microplate reader
Under absorbance.
Cell merges
Recover the myeloma cell (SP2) of mice of a cryopreservation tube, with complete culture solution (500mlDMEM, the 50ml tire cattle of DMED
Serum, 10ml penicillin/streptomycin) cultivate, because SP2 cell is suspension cell, so each little culture bottle adds
The culture fluid of 10ml, gives the vivosphere that cell is enough.SP2 cell at least started to cultivate before merging for 1 week, and every day observes
The growth conditions of cell, after the time of general one week, takes some Cell saps and carries out cell counting, it is ensured that have enough living
SP2 cell is used for merging, 2x10 to be had7Individual cell.SP2 cell, room is collected with the sterile centrifugation tube of 50ml after counting
Under temperature, the centrifugal force of 1000rpm 10 minutes, draw old culture fluid, exhaust as far as possible.It is subsequently adding 20ml preheating
Without the DMEM of serum, blow and beat up and down several times, be centrifuged 15 minutes under 193xg centrifugal force, be repeated 3 times this step fully to wash away
Stick to the serum on cell, exhaust supernatant.While processing SP2, process the spleen cell of mice together, without blood
Clear DMEM culture fluid it is cut into small pieces the spleen being used for merging and resolves into single spleen cell, collecting spleen cell, with
The method same with SP2 processes spleen cell.With the resuspended spleen cell of DMEM culture fluid without serum, then spleen cell
Transferring in the centrifuge tube of collection SP2 cell, after being sufficiently mixed, centrifugal (193 × g) 15 minutes, are repeated 2 times, and fully absorb
Clear liquid avoids diluting the concentration of next step PEG, affects the fusion of two kinds of cells.Before cell merges, it is necessary to without serum
DMED culture fluid, HAT select culture fluid and the aseptic PEG being equipped with to be placed on 37OPreheat in the water-bath of C.The formula of PEG
It is in the DMEM culture fluid of the serum-free that the PEG of 10mg is dissolved into 10ml, because the viscosity of PEG is relatively big, is difficult to molten at low temperatures
Solve, so dissolving should be placed in water-bath, carry out filtering holding aseptic with filter after being completely dissolved.In 1 minute slowly
PEG preheated for dropping 1.5ml in cleaned spleen cell and SP2 cell centrifugation pipe, rock lightly 2 minutes,
Ensureing the abundant fusion of cell, this step will be 37OThe warm water of C is carried out.After 2 minutes, add the serum-free DMEM training of 1ml
Nutrient solution, drips complete in one minute, be then sequentially added into the DMEM culture fluid of 2ml, 5ml, 6ml, 6ml serum-free, progressively dilute
The concentration of PEG, completed the most respectively in one minute, left at room temperature 2 minutes, was then transferred in incubator stand 5 minutes,
The centrifugal force of 100xg 10 minutes, removes supernatant and guarantees to completely remove intracellular PEG, it is to avoid the PEG poison to cell
Property injury.Then the HAT selective medium adding 20ml preheating is the most intracellular to merge, transfers to one lightly after piping and druming
Individual big culture bottle (75cm2In), it is repeated once.Culture bottle is placed in incubator cultivation 2 hours, after 2 hours,
The cell merged is transferred in 96 orifice plates, and each hole adds the HAT culture fluid of 200ul, carries out cultivating (37 in being then put into incubator
℃, 5% CO2).
Technology screening hybridoma
HAT culture fluid is the selective medium of a kind of mammaliancellculture, and it contains hypoxanthine (H), amino is talked endlessly
Purine (A) and thymus pyrimidine (T).Aminopterin-induced syndrome is a kind of initial route of synthesis effectively suppressing RNA and DNA, thus can hinder
The propagation of myeloma cell's (SP2 cell).The amino that the main path of the myeloma cell's synthetic DNA not merged is cultured in base
Pterin blocks, the most for want of hypoxanthine-guanine-phosphoribosyltransferase (HGPRT), it is impossible to utilize time Huang in culture medium
Purine completes the building-up process of DNA and dead.And the splenocyte not merged because of can not in vitro long-term surviving and dead.Only melt
Close successful hybridoma and have the HPRT enzyme obtained in spleen cell, under the catalytic action of HPRT enzyme, can be with choosing
Select the hypoxanthine added in culture medium and thymus pyrimidine as raw material by middle route of synthesis synthesis RNA and DNA, make fusion
Cell can survive and infinite multiplication.
Could survive through the screening of HAT culture fluid, only hybridoma, after about 14 days, which detection is to determine
The generation antibody that a little clone body are interesting.Not all splenocyte forming hybridoma is B cell, therefore, can mix
There is hybridoma can not produce antibody；Also having some hybridomies is that B cell fusion forms, and has the spy of output antibody
Property, but have the inertia producing antibody.It is important that a step be to filter out the clone of the cell that can produce monoclonal antibody
Body.Substantial amounts of clone may need rapid screening and cultivate the satisfactory clone filtered out further.
The success or not merged is the specificity produced the hybridoma under optimum condition by indirect ELISA technology
The screening of antibody determines.
Being coated 96 hole elisa plates with the polypeptide of 10ug/ml, add 100ul in every hole is coated liquid, overnight at room temperature.The
Two days, wash away unnecessary be coated liquid after, add the confining liquid of 100ul, room temperature is closed one hour.After absorbing confining liquid,
Every hole add the cultivation of 100ul the culture supernatants of cell, then seal, incubated at room temperature is overnight.Within second day, use cleaning mixture
Washing away the antibody in unconjugated culture fluid, in absorbent paper, bang dries, and is subsequently added the two of 50ul and resists (with mouse tail sieve
Antibody used by choosing is the same), hatch 1 hour.Then wash away the most specific binding enzyme labelled antibody, dry 96 orifice plates, then add
Entering the tmb substrate of 50ul, lucifuge, the moment notes observing color change, occurs that color change and color are deep at first, hybridization is described
The ability that oncocyte produces antibody is strong.
Once confirming required hybridoma, these cells must be cloned several times, with guarantee to obtain one stable, with
The clone cell of class, and guarantee that it is strictly monoclonal antibody.So, thin for hybridoma strong for the antibody-producing ability filtered out
Born of the same parents transfer to 24 holes, 6 holes, little culture bottle from 96 orifice plates successively, and culture fluid selects culture fluid conversion from HAT the most successively
Become HT culture fluid, complete culture solution.
The satisfactory clone cell obtained is carried out cell cultivation, collects the supernatant of Cell sap, i.e. obtain without
The monoclonal antibody of purification.Use affinity purification that monoclonal antibody is purified subsequently.
The selection result of the mouse immune power of vitamin D binding protein synthesis polypeptide VD2 immunity is as shown in Figure 1.From figure
In it can be seen that immunoreactive strong and weak different to synthetic peptide of each mice, each mice of same group is to same polypeptide
Immunoreation power also differ.Upper figure is the result with 96 orifice plate detections, and the sample added when figure below is detection is in 96 holes
The control site of plate, each color represents each mice.Using 96 orifice plates as solid phase, 20 holes of C1-C10 and D1-D10
It is coated VD2；C11 is as comparison the most fixing upper VD2.According to principle, this step experiment be to survey by the indirect method of ELISA
The situation that mice internal antibody produces, color illustrates that the concentration of the antibody in sample is the highest the most deeply.In picture from the graph permissible
Finding out, the color in experimental group hole is all deep than the color of control wells, illustrates that mice has immunoreation to being expelled to internal polypeptide.With
Contrast between experimental group difference mice, the color in hole corresponding to No. 8 of VD2 group and No. 9 mices relatively deeply, i.e. No. 8 and No. 9 mices pair
The immunne response of VD2 is stronger.Therefore, in the immunity later stage, No. 8 and No. 9 mices of VD2 group are killed.
The screening of polypeptide hybridoma
Fig. 2 is the first time result to VD2 filtering hybridoma.96 orifice plates are to be coated with VD2 polypeptide, specific to merge
The screening of VD2 hybridoma.Fig. 2 is that VD2 filtering hybridoma is added the color shown by stopped reaction liquid, and figure below is
With the absorbance under 450nm wavelength of the hole corresponding to 96 orifice plates.H12 is control wells, and addition is do not cultivated cell new
Fresh HAT culture fluid.From picture, only C6, E6, E7 these three hole color is relatively deep, and corresponding absorbance is the highest, is required
The hybridoma wanted.
<110>Guangzhou General Hospital Guangzhou Military Command
<120>vitamin D binding peptide and application thereof
<130> VD2
<160> 1
<170> PatentIn version 3.5
<210> 1
<211> 19
<212> PRT
<213>artificial polypeptide
<400> 1
Ile Tyr Ser Leu Ala Ala Ser Ser Glu Leu Pro His Val Tyr Met Arg
1 5 10 15
Lys Gln Ser
Claims (8)
1. a vitamin D binding peptide, its motif is IYSLAASSELPHVYMRKQS.
Vitamin D binding peptide the most according to claim 1, it is characterised in that: one end of motif connects not more than 3
Aminoacid.
Vitamin D binding peptide the most according to claim 1, it is characterised in that: its sequence is
IYSLAASSELPHVYMRKQS。
4. vitamin D binding peptide application in preparation vitamin D binding protein antigen, wherein, vitamin D binding peptide such as right
Require described in 1～3 any one.
5. a vitamin D binding protein antibody, it is characterised in that: had the right described in requirement 1～3 any one to tie up life by coupling
The carrier induced animal body of element D binding peptide obtains.
6. a preparation method for vitamin D binding protein antibody, including raw by the dimension described in claims 1 to 3 any one
Element D binding peptide and increase immunogenic carrier conjugation, afterwards stimulating animal body, then prepare and filter out monoclonal antibody,
Obtain vitamin D binding protein antibody eventually.
Preparation method the most according to claim 6, it is characterised in that: increasing immunogenic carrier is purified protein derivative (PPD)
PPD。
8. a vitamin D immue quantitative detection reagent box, described test kit is ELISA kit, it is characterised in that: its dimension used
Raw element D associated proteins antibody is had the right the carrier induced animal of vitamin D binding peptide described in requirement 1～3 any one by coupling
Body obtains.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610639231.6A CN106243192A (en) | 2016-08-04 | 2016-08-04 | Vitamin d binding peptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610639231.6A CN106243192A (en) | 2016-08-04 | 2016-08-04 | Vitamin d binding peptide and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106243192A true CN106243192A (en) | 2016-12-21 |
Family
ID=58078975
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610639231.6A Pending CN106243192A (en) | 2016-08-04 | 2016-08-04 | Vitamin d binding peptide and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106243192A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111351945A (en) * | 2020-03-18 | 2020-06-30 | 东南大学 | Application of vitamin D binding protein as marker in diagnosis of mental disease depression |
| CN112773889A (en) * | 2020-12-15 | 2021-05-11 | 重庆医科大学附属永川医院 | VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539779A (en) * | 2010-12-27 | 2012-07-04 | 中国科学院上海生命科学研究院 | Application of Vitamin D binding protein serving as marker of diabetes |
| CN102628872A (en) * | 2012-03-31 | 2012-08-08 | 广州菲康生物技术有限公司 | Reagent box for detecting 25 hydroxyl vitamin D and manufacturing method thereof |
| CN103163306A (en) * | 2013-02-05 | 2013-06-19 | 北京九强生物技术股份有限公司 | 25 hydroxyl vitamin D detection kit and preparation method thereof |
-
2016
- 2016-08-04 CN CN201610639231.6A patent/CN106243192A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102539779A (en) * | 2010-12-27 | 2012-07-04 | 中国科学院上海生命科学研究院 | Application of Vitamin D binding protein serving as marker of diabetes |
| CN102628872A (en) * | 2012-03-31 | 2012-08-08 | 广州菲康生物技术有限公司 | Reagent box for detecting 25 hydroxyl vitamin D and manufacturing method thereof |
| CN103163306A (en) * | 2013-02-05 | 2013-06-19 | 北京九强生物技术股份有限公司 | 25 hydroxyl vitamin D detection kit and preparation method thereof |
Non-Patent Citations (2)
| Title |
|---|
| 姜冰洁: "一种新型维生素D结合蛋白单克隆抗体的制备", 《广州中医药大学 硕士学位论文》 * |
| 宋帅等: "抗原表位研究方法进展", 《动物医学进展》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111351945A (en) * | 2020-03-18 | 2020-06-30 | 东南大学 | Application of vitamin D binding protein as marker in diagnosis of mental disease depression |
| CN112773889A (en) * | 2020-12-15 | 2021-05-11 | 重庆医科大学附属永川医院 | VDBP/VSIG 4-based immune negative regulation multivalent vaccine and preparation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101415403B (en) | Solid oral form of a medicinal preparation and a method for the production thereof | |
| US5643736A (en) | Monoclonal antibodies for human osteogenic cell surface antigens | |
| EP3146967B1 (en) | Use of an isoquinoline alkaloid derivative for preparing drug capable of promoting ampk activity | |
| JPH05501400A (en) | Polypeptide analogs of apolipoprotein E, diagnostic systems and uses of the analogs | |
| WO1996024848A9 (en) | Monoclonal antibodies for human osteogenic cell surface antigens | |
| POWERS et al. | Characterization of monoclonal antibody 3G5 and utilization of this antibody to immobilize pancreatic islet cell gangliosides in a solid phase radioassay | |
| CN106243192A (en) | Vitamin d binding peptide and application thereof | |
| CN106220710A (en) | A kind of vitamin D binding peptide and application thereof | |
| Sonoda et al. | Targeting hepatic oxidative stress rescues bone loss in liver fibrosis | |
| US9707222B2 (en) | Isoquinoline alkaloid derivative for activating AMP-dependent protein kinase | |
| JP5535638B2 (en) | Method for reducing phosphate absorption | |
| Goldstein | The Gordon Wilson lecture. The history of the development of thymosin: chemistry, biology and clinical applications. | |
| CN101671655B (en) | Monoclonal antibody hybridoma cell of HIV P24 and application | |
| Crowther et al. | Immunological and insulin secretory studies on isolated porcine islets of Langerhans | |
| JP5701504B2 (en) | Method for reducing phosphate absorption | |
| CN112480167B (en) | Isocarbophos hapten, artificial antigen and antibody as well as preparation method and application thereof | |
| KR101289023B1 (en) | A Method for culturing endocrine cells Using Concave Microwell | |
| US20120128706A1 (en) | Immune adjuvant comprising atp | |
| CN105085669A (en) | Vitamin B5 conjugate and preparation method and application thereof | |
| CN105859878A (en) | Preparation method of anti-HPV infection antibody | |
| CN106366174A (en) | Application of velvet antler polypeptide to reducing adhesive force of adipose derived stem cells | |
| JPH01168299A (en) | Monoclonal antibody and hybridoma producing the same | |
| CN110201152B (en) | G3bp2 polypeptide vaccine and application thereof | |
| CN112552409B (en) | Monoclonal antibody for resisting SPTSSA and application thereof | |
| US20150342908A1 (en) | Secoaporphine alkaloid derivative for activating amp-dependent protein kinase |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20161221 |
|
| RJ01 | Rejection of invention patent application after publication |