CN108219002A - A kind of C peptide based immunogens of recombination and its application - Google Patents
A kind of C peptide based immunogens of recombination and its application Download PDFInfo
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Abstract
The present invention relates to technical field of immunoassay, disclose C peptide based immunogens and its application of a kind of recombination.C peptide based immunogens of the present invention are the fusion proteins formed by C peptides and viral hepatitis type C core protein.The present invention is by selecting carrier protein of the viral hepatitis type C core protein as C peptides, the fusion protein that the two is formed can generate the C peptide-specific antibodies of high-titer, have apparent high-affinity advantage compared to common GST carrier proteins, the auxiliary diagnosis of functional rehabilitation assessment and liver and kidney disease after diabetes diagnosis and parting, the antidiastole of hypoglycemia syndrome, pancreatic islets transplantation can be preferably applied for.
Description
Technical field
The present invention relates to technical field of immunoassay, and in particular to a kind of C peptide based immunogens of recombination and its application.
Background technology
C peptides (C-Peptide) also known as connect peptide, are the secretory products of beta Cell of islet, and there are one common with insulin for it
Precursor -- proinsulin, proinsulin are a very long protein chains, and proinsulin is broken down into three sections under the action of enzyme,
Front and rear two sections couple again, become the insulin being made of A chains and B chains, and intermediate one section independent, and forming one has 31
The polypeptide of amino acid becomes C peptides.
C peptides have following characteristics：(1) in the secretion of the β cells of pancreas islet, Ins is equal with the total gram-molecular weight of C peptides.(2) pancreas islet
Plain half-life period is 4.8min, and it is 17.5min that C peptides, which are 11 minutes proinsulin,.(3) insulin decomposed in liver kidney and C peptides not
Be decomposed is that complete chain is discharged from kidney.(4) C peptides no biological activity, but with very strong species specificity, with anti-insulin
Without cross-immune reaction.Since the concentration of proinsulin is less than 1/10th of C peptides, thus it is general measure C peptides (total C peptides) can generation
Free C peptides in table blood.
C peptides detection stability is strong, is not interfered by exogenous insulin, is better than pancreas in terms of islet beta cell function assessment
Island element, C peptide levels measure, it may be appreciated that the function of diabetic's beta Cell of islet, applied to diabetes diagnosis and parting, and low blood
The antidiastole of sugared syndrome, functional rehabilitation is assessed after pancreatic islets transplantation, the auxiliary diagnosis of liver and kidney disease.
But direct immunization C peptide antigen mouse do not generate anti-C peptide-specific antibodies, add in general carrier protein such as ox
Seralbumin (BSA), hemocyanin (KLH) and gst fusion protein are similarly difficult to obtain the specific antibody of high-affinity
Body.For this special haptens, suitable immunogene is selected to become the key for preparing its excellent antibody.
Invention content
In view of this, the purpose of the present invention is to provide a kind of C peptide based immunogens of recombination, can be produced after making its immune animal
The polyclonal antibody or monoclonal antibody of raw more efficient valency；
Another object of the present invention is to provide a kind of hybridoma obtained using above-mentioned C peptide based immunogens, be made
It can generate the monoclonal antibody of more efficient valency；
Another object of the present invention is to provide a kind of monoclonal antibody obtained using above-mentioned C peptide based immunogens, be made
It has higher potency.
For achieving the above object, the present invention provides following technical solution：
A kind of C peptide based immunogens of recombination are the fusion proteins formed by C peptides and viral hepatitis type C core protein.
The C peptide antibodies of high-affinity (i.e. high-titer) can not be obtained for the general carrier protein of use in the prior art,
The present invention forms new fusion protein, phase using HCV core (viral hepatitis type C core protein) as carrier protein with C peptides
Than the C peptide antibodies that other general carrier proteins can obtain high-titer.
The C peptide based immunogens of recombination of the present invention are further by the C peptides and viral hepatitis type C core by connection peptide
Heart protein form fusion protein by connection, and the peptide therein that connects is commonly used in the art not influence carrier protein and antigen protein
The small peptide of immunogenicity, its sequence is Asp-Gly-Arg in the specific embodiment of the invention.
Preferably, viral hepatitis type C core protein of the present invention selects its the 1st to the 118th amino acid to be formed
Albumen, amino acid sequence such as SEQ ID NO:Shown in 1.The C peptide based immunogens amino acid sequences of recombination of the present invention are
SEQ ID NO:Sequence shown in 1+connection peptide+C peptide sequences.
For the needs of preparation, when carrying out the preparation of C peptide based immunogens of the recombination, amplimer generally comprises screening
Label, such as histidine tag so as to the screening of positive colony below, but screen the C peptides that label is not recombination of the present invention
Immunogene institute is prerequisite.In the specific embodiment of the invention, the C peptide based immunogens amino acid sequence such as SEQ of the recombination
ID NO:Shown in 2.Wherein, 1-6 amino acids sequence screens label for 6 histidines, and 7-124 amino acids sequence is institute
Viral hepatitis type C core protein 1-118 amino acids sequences are stated, 125-127 amino acids sequence is connection peptide sequence,
128-158 amino acids sequence is C peptide sequences.
Mouse is immunized using the C peptide based immunogens of recombination of the present invention and carries out serum titer detection, the results show that simple
Animal generation antibody is hardly immunized because molecular weight is too small in C peptides antigen, though and as C peptides-gst fusion protein antigen energy of control
Generate antibody, but the apparent not C peptide based immunogens as described herein of potency.
It is small after C peptide based immunogens of the present invention are immunized in the experiment of hybridoma cell strain and monoclonal antibody
Mouse by the preparation of hybridoma and the screening of monoclonal antibody, obtains monoclonal antibody potency and can reach 1000K, show anti-C
The monoclonal antibody of peptide has higher potency.Meanwhile to carry out hybridomas thin for the different mouse of continuous immunity of the present invention 50
The preparation of born of the same parents' strain simultaneously filters out 37 plants of monoclonal antibodies, these monoclonal antibodies are provided with the excellent effect of up to 1000K potency,
The reproducibility of C peptide based immunogens high-titer in hybridoma and monoclonal antibody is prepared is shown, can produced
It is applied in industry.
Based on above excellent technique effect, the present invention provides the C peptide based immunogens to prepare anti-C-peptide monoclonal antibody
Application in hybridoma cell strain and/or C peptide antibodies.Wherein, the C peptide antibodies resist for C peptides polyclonal antibody or C peptide monoclonals
Body.
According to above application, the present invention also provides a kind of anti-C-peptide monoclonal antibody hybridoma cell strain, by institute of the present invention
It states after animal is immunized in C peptide based immunogens and prepares.A kind of monoclonal antibody of anti-C peptides is additionally provided simultaneously, by the anti-C peptides list
The secretion of monoclonal hybridomas cell strain obtains.
The high-titer performance of monoclonal antibody based on aforementioned acquisition, the present invention is proposed to be immunized by C peptides of the present invention
Original prepare monoclonal antibody, particularly potency be up to the monoclonal antibody of 1000k prepare detection C peptides, diabetes diagnosis and parting,
After the antidiastole of hypoglycemia syndrome, pancreatic islets transplantation in the product of the auxiliary diagnosis of functional rehabilitation assessment or liver and kidney disease should
With.The form of the product can be any form in checkout and diagnosis field, kit such as based on Elisa principles, immune
Chromatographic test paper, colloid gold test paper etc..Specifically, the present invention provides a kind of C peptides colloidal gold test, key core exists
In using detection line of the monoclonal antibody as Test paper.
In the specific embodiment of the invention, the C peptides colloidal gold test by sample pad, bottom plate, nitrocellulose membrane,
Gold-labelled pad and water absorption pad composition, being drawn on nitrocellulose membrane has T lines (i.e. detection line, by monoclonal antibody of the present invention through packet
Cross after being diluted by buffer solution) and C lines (i.e. nature controlling line, by crossing after the coated buffer solution dilution of sheep anti-mouse igg antibody), specifically
Structure diagram can be found in Fig. 1.
C peptides colloidal gold test of the present invention, with the raising of C peptide concentrations, colour developing on T lines and C lines by
Gradual change is deep, can be used for quantitatively or semi-quantitatively detecting.
By above technical scheme it is found that the present invention is by selecting carrier of the viral hepatitis type C core protein as C peptides
Albumen, the fusion protein that the two is formed can generate the C peptide-specific antibodies of high-titer, have compared to common GST carrier proteins
Standby apparent high-affinity advantage, can be preferably applied for diabetes diagnosis and parting, hypoglycemia syndrome antidiastole,
The auxiliary diagnosis of functional rehabilitation assessment and liver and kidney disease after pancreatic islets transplantation.
Description of the drawings
Fig. 1 show C peptides colloidal gold test structure diagram of the present invention；
Fig. 2 show the SDS-PAGE figures of C peptide based immunogens of the present invention；
Fig. 3 show the antiserum titre curve graph of different C peptide based immunogens；Wherein 1 represents C peptide based immunogens of the present invention
Curve, 2 represent C peptide-GST proteantigens curve, 3 represent C peptides curve；
Fig. 4 show the colloidal gold detection strip color developing effect comparison diagram of gradient C peptide concentrations.
Specific embodiment
The invention discloses a kind of C peptide based immunogens of recombination and its application, those skilled in the art can be used for reference in this paper
Hold, be suitably modified technological parameter realization.In particular, it should be pointed out that all similar substitutions and modifications are to those skilled in the art
For be it will be apparent that they are considered as being included in the present invention.C peptide based immunogens of the present invention and its application have passed through
Embodiment is described, and related personnel can significantly not depart from the content of present invention, C peptides described herein exempted from spirit and scope
Epidemic focus and its application are modified or suitably changed with combining, to realize and using the technology of the present invention.
Just a kind of C peptide based immunogens of recombination provided by the present invention and its application are described further below.
Embodiment 1：The preparation of C peptide based immunogens of the present invention
C peptide gene sequences such as SEQ ID NO:Shown in 3, HCV Core (1-118aa) gene order such as SEQ ID NO:4 institutes
Show, it is SEQ ID NO to design HCV Core (1-118aa) primer H1 and H2, H1 gene order:5, H2 gene orders are SEQ ID
NO:Shown in 6, restriction enzyme sites of the GAATTC for EcoR I in H1, CATCACCATCACCATCAC is 6 histidine tags；In H2
AGCTTC is the gene order that the C peptides 5 ' of splicing are held of putting up a bridge, and ACGGCCGTC is connection peptide gene sequence；Design C peptide primer C1 and
C2 is respectively C1SEQ ID NO:7 and C2SEQ ID NO:Shown in 8, GACGGCCGT is to connect peptide gene sequence in C1, in C2
GGATCC is the restriction enzyme site of BamH I, and more than sequence transfers to commercial company to synthesize.
Expand HCV Core (1-118aa) gene (being template with pJFH1 plasmids) and C peptide genes respectively, after use H1
The C peptide based immunogens fusion is expanded with C2, C peptide based immunogens fusion and the carrier PLM1 use that recycling obtains will be purified
I digestion of EcoR I and BamH builds PLM1-Core (1-118)-C peptide expression vectors, plasmid is transferred to e. coli bl21 (DE3)
Competent cell selects positive clone molecule by using the method for bacterium colony PCR.Obtained positive clone molecule is trained in LB culture mediums
It supports, with IPTG induced expressions.Obtained bacterium solution carries out affinity purification with nickel column, is then identified using SDS-PAGE, as a result
See Fig. 2, C peptide based immunogens about 17KD of the present invention.
Embodiment 2：C peptide based immunogens of the present invention are immunized
Appropriate recombinant C peptide based immunogens albumen is mixed into gained emulsion with the dosage of 0.5ml with equivalent Freund's complete adjuvant
It is subcutaneously injected and gives BALB/c mouse (Hunan SJA Laboratory Animal Co. , Ltd, 6 week old are female, 5) abdomen site, the
Primary immunization pneumoretroperitoneum enhancing in 14 days is immune (appropriate antigen is mixed with equivalent incomplete Freund's adjuvant, each 0.5ml), and enhancing is exempted from
After epidemic disease to three needles, adopt tail blood and carry out bioactivity.
At the same time with identical method by recombinant C peptide-GST proteantigens (the far safe limited public affairs of biotinylated biomolecule technology in Hunan
Department's production) and C peptides antigen (Changsha You Bao bio tech ltd) while 5 BALB/c female mices of the same age have been immunized.
Potency reaches the BALB/c mouse of fusion requirement, 3 days before fusion, is carried out eventually with the intraperitoneal injection of doubling dosage antigen
Exempt from.
Embodiment 3：BALB/c mouse serum titer detects
C peptide antigens are diluted with pH9.51 × CB buffer solutions, make its final concentration of 2 μ g/ml, 96 enzymes are added in per hole 0.1ml
Exempt from reaction plate, 4 DEG C overnight.Next day, with the 0.02MpH7.2PBS containing 1%BSA per hole 0.2ml, 37 DEG C 1 hour, for detecting.
Mouse tail blood is taken, serum diluting multiple is followed successively by：AB row -2000, CD row -20000, EF rows -200000, GH row -2000000,
1-5 is classified as 5 mice serums that C peptide antigens are immunized, and 6-10 is classified as 5 mice serums that C peptide based immunogens of the present invention are immunized,
11-15 is classified as 5 mice serums that recombinant C peptide-GST proteantigens are immunized, and the 16th is classified as blank control, diluted serum
0.1ml/ holes in detection plate, 37 DEG C 1 hour, the sheep of 10000 times of diluted horseradish peroxidases labels is added in after washing six times
Anti- mouse IgG (Sinocare Biosensing Co., Ltd) after 37 DEG C is ibid washed for 1 hour, 100 μ l is added in per hole and contain 0.02% (M/
V) 3.3 ' .5.5 ' -- tetramethyl benzidine, 0.003% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C 10 points
Clock adds in 1M sulfuric acid solutions, per 50 μ l of hole, surveys 450nm absorption values.As a result such as Tables 1 and 2, using dilution as horizontal axis, OD450
Measured average value is longitudinal axis drawing 3.
Table 1
| 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | |
| A | 0.178 | 0.159 | 0.274 | 0.277 | 0.161 | 2.687 | 3.199 | 2.55 |
| B | 0.197 | 0.144 | 0.233 | 0.234 | 0.158 | 2.73 | 3.042 | 2.517 |
| C | 0.12 | 0.133 | 0.125 | 0.133 | 0.159 | 0.755 | 1.828 | 0.659 |
| D | 0.13 | 0.116 | 0.147 | 0.131 | 0.157 | 0.634 | 1.756 | 0.723 |
| E | 0.146 | 0.133 | 0.095 | 0.112 | 0.159 | 0.143 | 0.34 | 0.17 |
| F | 0.133 | 0.135 | 0.092 | 0.11 | 0.156 | 0.133 | 0.315 | 0.135 |
| G | 0.135 | 0.128 | 0.097 | 0.107 | 0.16 | 0.096 | 0.104 | 0.088 |
| H | 0.118 | 0.125 | 0.115 | 0.109 | 0.16 | 0.095 | 0.104 | 0.09 |
Table 2
| 9 | 10 | 11 | 12 | 13 | 14 | 15 | 16 | |
| A | 3.179 | 2.755 | 1.879 | 1.774 | 1.629 | 1.739 | 1.672 | 0.159 |
| B | 3.092 | 2.859 | 1.507 | 1.442 | 1.32 | 1.391 | 1.462 | 0.156 |
| C | 2.774 | 0.82 | 0.341 | 0.386 | 0.299 | 0.369 | 0.308 | 0.157 |
| D | 2.743 | 0.871 | 0.335 | 0.339 | 0.244 | 0.295 | 0.327 | 0.156 |
| E | 0.682 | 0.123 | 0.124 | 0.114 | 0.109 | 0.112 | 0.114 | 0.159 |
| F | 0.646 | 0.134 | 0.115 | 0.125 | 0.11 | 0.108 | 0.106 | 0.155 |
| G | 0.153 | 0.09 | 0.089 | 0.085 | 0.089 | 0.084 | 0.088 | 0.16 |
| H | 0.138 | 0.093 | 0.084 | 0.079 | 0.082 | 0.087 | 0.081 | 0.158 |
Fig. 3 is the antiserum titre curve graph detected according to ELISA method, which reflects the overall trend of serum titer,
It is more intuitive.It may determine that from table 1-2 and Fig. 3, simple C peptides antigen is anti-because the too small hardly immune animal of molecular weight generates
Body, C peptide-GST recombinant antigens can generate antibody, but potency is not as good as C peptide based immunogens of the present invention height, C peptide based immunogens serum of the present invention
Potency can reach fusion requirement, meet the experiment immunogene requirement of follow-up hybridoma cell strain and monoclonal antibody.
Embodiment 4：The foundation of hybridoma cell strain and the preparation of anti-C peptides protein monoclonal antibody
(1) preparation of feeder cells
Feeder cells are made with Kunming mouse peritoneal macrophage.1 day before fusion, Kunming mouse draws neck to put to death, the leaching of 75% alcohol
5-10min is steeped, in super-clean bench, abdominal cut skin and peritonaeum under sterile working inject HAT cell culture fluids with aseptic straw
5-10ml is rinsed repeatedly, recycles flushing liquor, and adjustment cell concentration is 1 × 105A/ml adds in 96 porocyte culture plates, 100 μ
L/ holes, 37 DEG C, 5%CO2Overnight incubation.
(2) preparation of immune spleen cell
3-5 days after mouse final immunization, spleen is aseptically taken out, cell suspension is made.
(3) preparation of myeloma cell
Murine myeloma cell Sp2/0 takes two big bottles, cell suspension is made, and centrifugation is abandoned supernatant, broken up.
(4) cell fusion and HAT selection hybridomas
In the sterile plastic cement centrifuge tubes of 50mL, immune mouse spleen cell will be added in above-mentioned myeloma cell, 1500rpm from
The heart abandons supernatant, fully breaks up, and is slowly added to 1ml fusion agents (Sinocare Biosensing Co., Ltd), quiet 1 minute after fusion
1min is put, the basic culture solution for being slowly added to 50ml terminates cell fusion, and 1200rpm is centrifuged 8 minutes.Supernatant is abandoned, 100ml's
HAT liquid mediums are gently suspended, and divide equally in 96 well culture plates for having feeder cells in 10 pieces, 100 μ l/ holes, 37 DEG C, 5%CO2 trainings
It supports.Culture changed HT culture solutions to the 7th day.
(5) detection of antibody
C peptide antigens are diluted with 1 × CB of pH9.5 buffer solutions, make its final concentration of 2 μ g/ml, 96 enzymes are added in per hole 0.1ml
Exempt from reaction plate, 4 DEG C overnight.It is next day, 37 DEG C 1 small with the PBS of the 0.02MpH7.2 containing 1%BSA per hole 0.2ml capping plates
When, it pats dry, detection is spare.Recombination fusion after the 10-14 days, take cell conditioned medium 0.1ml/ holes in detection plate, 37 DEG C 1 hour,
Sheep anti-mouse igg (the three limited public affairs of promise bio-sensing share of 10000 times of diluted horseradish peroxidases labels are added in after washing six times
Department), after 37 DEG C are ibid washed for 1 hour, 100 μ l are added in per hole and contain 0.02% (M/V) 3.3 ' .5.5 ' -- tetramethyl benzidine,
0.003% (V/V) hydrogen peroxide, pH5.0 citrate phosphate buffers, 37 DEG C 10 minutes, add in 1M sulfuric acid solutions, per 50 μ l of hole,
Survey 450nm absorption values.Blank value >=2.0 are subtracted as positive cell hole with measured value.
(6) limiting dilution and positive colony screening
Monoclonal is screened with limiting dilution assay, two kinds of albumen of monoclonal C peptides albumen and Core (1-118aa) albumen are resisted
Body surface position is screened, only selection C peptide protein positives value >=2.0, and the monoclonal hole that Core (1-118aa) albumen is no positive.
(7) preparation of monoclonal antibody
6-8 weeks BALB/c male mice, every mouse peritoneal inject 0.5ml Freund's incomplete adjuvants, pneumoretroperitoneum injection in 7-10 days
500000 hybridomas.Inoculating cell can generate ascites after 9-15 days, the health status of close observation animal and sign of ascites as,
Before mouse is frequency domain dead, when ascites is as more as possible, neck is drawn to put to death mouse, is taken out ascites with dropper, collect ascites, centrifugation
Supernatant is taken, -20 DEG C of refrigerators is put in and saves backup.Ascites adds in isometric saturated ammonium sulfate after being diluted with 3 times of volume PBS, uses PBS
After redissolution, 4 DEG C of dialysed overnights, after protein A affinity chromatography column (being purchased from Niu Long Bioisystech Co., Ltd).When in OD280nm
Under adsorptive value when reaching baseline, then eluted with the eluent (pH3.0) of 0.1M and recycle the antibody.The solution recycled is used
0.1MTRIS (pH9) is neutralized, 4 DEG C of dialysed overnights after neutralization.
(8) monoclonal antibody antibody titration
It being detected with indirect elisa method, measures the potency of antibody, antibody dilutes 100 times, 1000 times, 10000 times, and 100000
Times, 1000000 times, such as table 3.
Table 3
As shown in Table 2, antibody titer can reach 1000K, illustrate that anti-C-peptide monoclonal antibody has higher effect
Valency.
(9) C peptides-Core (1-118aa) is tested as the reproducibility of immunogene
By process as described above, 50 BALB/c mouses are immunized with C peptide based immunogens of the present invention, are sieved by clone
Choosing, prepares 39 plants of monoclonal antibodies, wherein 37 plants of potency are up to 1000K, potency is as shown in following table 4-7 altogether.
Table 4
Table 5
Table 6
Table 7
The potency that table 4-7 can be seen that antibody almost can reach 1000k, be system using recombinant immune original work of the present invention
The immunogene of standby C-peptide monoclonal antibody, can prepare a batch well has the C peptide monoclonal antibodies of high-titer, shows the C peptides and exempts from
The reproducibility of epidemic focus high-titer in hybridoma and monoclonal antibody is prepared, can industrially be applied.
Embodiment 5：The preparation of colloidal gold fast detecting test paper
1. the preparation of nitrocellulose filter
It is coated with the preparation of buffer solution：0.02MPH7.5Tris buffer solutions containing 3% methanol, 2% sucrose are coating buffer solution
Put 4 DEG C of spare, terms of validity one week.
The preparation of nitrocellulose filter：With coating buffer solution by potency of the present invention the anti-C peptides of 1000K monoclonal antibody
Antibody is diluted to 0.5~2mg/ml, is marked as T lines, as detection line, T lines are close to gold-labelled pad end, away from gold-labelled pad end about 7mm；With
Sheep anti-mouse igg antibody (Arist) is diluted to 0.2-0.5mg/ml by coating buffer solution, is marked as C lines, as nature controlling line, C lines lean on
Nearly water absorption pad.Two linear distance 3-5mm, uniformly.37 DEG C of drying, encapsulation are spare.
2. the preparation of colloidal gold, golden labeled monoclonal antibody
(1) preparation of solution
1. the preparation of gold chloride：With ultrapure water dissolution gold chloride, 1% solution is made into, puts 4 DEG C of spare, terms of validity 3 months.
2. the preparation of trisodium citrate：With ultrapure water dissolution sodium citrate, 1% solution is made into, puts 4 DEG C of spare, terms of validity 3
My god.
3. the preparation of 0.2M potassium carbonate：It is prepared with ultra-pure water, puts 4 DEG C of spare, term of validity half a year.1000ml0.2M potassium carbonate
Solution formula：27.6g potassium carbonate, ultra-pure water are settled to 1000ml.
4. the configuration of 10%BSA：It is prepared with ultra-pure water, puts 4 DEG C of spare, terms of validity one month.100ml10%BSA is formulated：
10gBSA powder, ultra-pure water are settled to 100ml.
5. the preparation of the golden re-suspension liquid of label：0.5% casein (Casein), 0.1% Sodium azide (NaN3), 0.2% tween-
20,3% sucrose, 0.01MpH7.2PBS solution, 0.22 μ membrane filtration mistakes put 4 DEG C of spare, terms of validity 6 months.
(2) preparation of colloid gold particle：1% gold chloride is diluted to 0.04% with ultra-pure water, puts in electric jacket and boils, press
5ml1% trisodium citrates are added in per 100ml, continue to boil, until liquid stops heating in shiny red.The colloid prepared
Golden appearance should it is pure, bright, without precipitation and floating material, the term of validity two weeks.
(3) preparation of colloid gold label monoclonal antibody：The pH value of colloidal gold is adjusted to 7-8 with 0.2M potassium carbonate, by 5~
10 μ g antibody/ml colloidal golds add in the monoclonal antibody of the anti-C peptides of the present invention, mixing, are added with stirring 10%BSA to final concentration of
0.1%.10000rpm, 4 DEG C of centrifugation 10min, abandon supernatant, will the precipitation golden re-suspension liquid resuspension of suitable label, put 4 DEG C it is spare,
Term of validity two weeks.
3. the preparation of gold-labelled pad
Gold pad per 0.6cmx10cm is uniformly coated with 5%~20% golden labelled antibody of 300ul, is dried in 37 DEG C,
It encapsulates, is saved backup in dry proof cabinet.
4. the preparation of test strips sample pad
(1) preparation of sample pad treatment fluid：
0.2% Tween-20,1%BSA, 0.05%NaN3,0.01MpH7.2PBS solution puts 4 DEG C of spare, terms of validity two
A month.
(2) preparation of sample pad：
Sample pad is cut into 2.8cmx30cm and is soaked in 5min in sample pad treatment fluid, 37 DEG C of drying encapsulate, dry anti-
It is saved backup in damp case.
5. the assembling of test strips
Nitrocellulose filter, gold-labelled pad, absorption pad, sample pad by Fig. 1 are stacked gradually, are cut into the examination of 3.0mm wide
Item, 20 parcels add in drier 1 and wrap, Vacuum Package.Room temperature preservation, the term of validity 1 year.
Embodiment 6：Colloidal gold fast detecting test paper detects C peptide antigens
What it is with the preparation of embodiment 5 is strip, and detection C peptide antigen concentrations are respectively：0,0.5ng/ml, 1ng/ml, 3ng/ml,
5ng/ml, 10ng/ml, 16ng/ml, 20ng/ml detected value interpretation data such as table 8, corresponding strip colour developing situation such as Fig. 4.
Table 8
| Quality-control product concentration (ng/ml) | 0 | 0.5 | 1.0 | 3.0 | 5.0 | 10.0 | 16.0 | 20.0 |
| Corresponding colour atla value | B | C9 | C8 | C7 | C6 | C4 | C2 | C1 |
Can be obtained according to 8 data of table and Fig. 4 color developing effects, the anti-detection charge product of monoclonal of the anti-C peptides of the present invention by
For low value to high level, colour developing is also more and more apparent, will can be used for quantitatively or semi-quantitatively detecting.
The above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, various improvements and modifications may be made without departing from the principle of the present invention, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Sinocare Biosensing Co., Ltd
<120>A kind of C peptide based immunogens of recombination and its application
<130> MP1725641
<160> 8
<170> SIPOSequenceListing 1.0
<210> 1
<211> 118
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 1
Met Ser Thr Asp Pro Lys Pro Gln Arg Lys Thr Lys Arg Asp Thr Asn
1 5 10 15
Cys Arg Pro Glu Asp Val Lys Phe Pro Gly Gly Gly Gln Ile Val Gly
20 25 30
Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro Arg Leu Gly Val Arg Thr
35 40 45
Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro Arg Gly Arg Arg Gln Arg
50 55 60
Phe Pro Lys Asp Arg Arg Ser Thr Gly Lys Ala Trp Gly Lys Pro Gly
65 70 75 80
Arg Pro Trp Pro Leu Tyr Gly Asn Glu Gly Leu Gly Trp Ala Gly Trp
85 90 95
Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser Trp Gly Pro Thr Asp Pro
100 105 110
Arg His Arg Ser Arg Asn
115
<210> 2
<211> 158
<212> PRT
<213>Artificial sequence (Artificial Sequence)
<400> 2
His His His His His His Met Ser Thr Asp Pro Lys Pro Gln Arg Lys
1 5 10 15
Thr Lys Arg Asp Thr Asn Cys Arg Pro Glu Asp Val Lys Phe Pro Gly
20 25 30
Gly Gly Gln Ile Val Gly Gly Val Tyr Leu Leu Pro Arg Arg Gly Pro
35 40 45
Arg Leu Gly Val Arg Thr Thr Arg Lys Thr Ser Glu Arg Ser Gln Pro
50 55 60
Arg Gly Arg Arg Gln Arg Phe Pro Lys Asp Arg Arg Ser Thr Gly Lys
65 70 75 80
Ala Trp Gly Lys Pro Gly Arg Pro Trp Pro Leu Tyr Gly Asn Glu Gly
85 90 95
Leu Gly Trp Ala Gly Trp Leu Leu Ser Pro Arg Gly Ser Arg Pro Ser
100 105 110
Trp Gly Pro Thr Asp Pro Arg His Arg Ser Arg Asn Asp Gly Arg Glu
115 120 125
Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly
130 135 140
Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln
145 150 155
<210> 3
<211> 93
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
gaagctgaag acttgcaagt tggtcaagtt gaattgggtg gtggtccagg cgcgggtagc 60
ttgcaaccat tggcgttgga aggttctttg caa 93
<210> 4
<211> 278
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
atgagcacaa atcctaaacc tcaaagaaaa accaaaagaa acaccaaccg tcgcccagaa 60
gacgttaagt tcccgggcgg cggccagatc gttggcggag tatacttgtt gccgcgcagg 120
ggccccaggt tgggtgtgcg cacgacaagg aaaacttcgg agcggtccca gccacgtggg 180
agacgccagc ccatccccaa agatcggcgc tccactggca aggcctgggg aaaaccaggt 240
cgcccctggc ccctatatgg gaatgaggga ctcggctg 278
<210> 5
<211> 81
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
cgcgcgaatt caggaggaat ttaaaatgag aggatcgcat caccatcacc atcacatgag 60
cacaaatcct aaacctcaaa g 81
<210> 6
<211> 32
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
agcttcacgg ccgtccacgt tgcgcgacct at 32
<210> 7
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gacggccgtg aagctgaaga cttgc 25
<210> 8
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
ggatccttgc aaagaacctt c 21
Claims (10)
1. the C peptide based immunogens of a kind of recombination, which is characterized in that melted by what C peptides and viral hepatitis type C core protein were formed
Hop protein.
2. C peptide based immunogens according to claim 1, which is characterized in that the C peptides and viral hepatitis type C core protein by
Connect peptide connection.
3. C peptide based immunogens according to claim 2, which is characterized in that the connection peptide is Asp-Gly-Arg.
4. C peptide based immunogens according to claim 1 or claim 2, which is characterized in that the viral hepatitis type C core protein is it
1st to the 118th amino acids formed albumen.
5. C peptide based immunogens according to claim 4, which is characterized in that the viral hepatitis type C core protein the 1st to
118 amino acids formed amino acid sequences such as SEQ ID NO:Shown in 1.
6. C peptide based immunogens described in claim 1-5 any one are preparing anti-C-peptide monoclonal antibody hybridoma cell strain and/or C
Application in peptide antibody.
A kind of 7. anti-C-peptide monoclonal antibody hybridoma cell strain, which is characterized in that the C peptides as described in claim 1-5 any one
It is prepared after immunogen immune animal.
8. a kind of monoclonal antibody of anti-C peptides, which is characterized in that anti-C-peptide monoclonal antibody hybridoma is thin as described in claim 7
Born of the same parents' strain secretion obtains.
9. monoclonal antibody described in claim 8 is preparing detection C peptides, diabetes diagnosis and parting, the mirror of hypoglycemia syndrome
Not Zhen Duan, the application after pancreatic islets transplantation in the product of the auxiliary diagnosis of functional rehabilitation assessment or liver and kidney disease.
10. a kind of C peptides colloidal gold test, which is characterized in that tried using monoclonal antibody described in claim 8 as detection
The detection line of paper.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109705221A (en) * | 2018-12-27 | 2019-05-03 | 美康生物科技股份有限公司 | C peptide based immunogens and its monoclonal antibody pair and the antibody are to the application in C peptide magnetic microparticle chemiluminescence immunoreagent |
| CN117362432A (en) * | 2023-10-16 | 2024-01-09 | 郑州伊美诺生物技术有限公司 | C peptide recombinant rabbit monoclonal antibody, preparation method and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004014936A2 (en) * | 2002-08-02 | 2004-02-19 | Commissariat A L'energie Atomique | Mixture of peptides from c and ns3 proteins of the hepatitis c virus and applications thereof |
| CN102286107A (en) * | 2011-07-13 | 2011-12-21 | 天津迈迪瑞康生物医药科技有限公司 | Method for efficiently expressing recombinant hepatitis C virus multi-epitope antigen and application thereof |
| CN105601750A (en) * | 2016-01-22 | 2016-05-25 | 宁波美康生物科技股份有限公司 | Genetic recombinant human C-peptide fused protein and preparation method and application thereof |
-
2018
- 2018-01-22 CN CN201810058354.XA patent/CN108219002B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004014936A2 (en) * | 2002-08-02 | 2004-02-19 | Commissariat A L'energie Atomique | Mixture of peptides from c and ns3 proteins of the hepatitis c virus and applications thereof |
| CN102286107A (en) * | 2011-07-13 | 2011-12-21 | 天津迈迪瑞康生物医药科技有限公司 | Method for efficiently expressing recombinant hepatitis C virus multi-epitope antigen and application thereof |
| CN105601750A (en) * | 2016-01-22 | 2016-05-25 | 宁波美康生物科技股份有限公司 | Genetic recombinant human C-peptide fused protein and preparation method and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109705221A (en) * | 2018-12-27 | 2019-05-03 | 美康生物科技股份有限公司 | C peptide based immunogens and its monoclonal antibody pair and the antibody are to the application in C peptide magnetic microparticle chemiluminescence immunoreagent |
| CN117362432A (en) * | 2023-10-16 | 2024-01-09 | 郑州伊美诺生物技术有限公司 | C peptide recombinant rabbit monoclonal antibody, preparation method and application thereof |
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