CN103865950A - Induced expression and purification method of human-derived insulin-like growth factor binding protein 1 in pichia pastoris, and preparation and application of related antibodies - Google Patents
Induced expression and purification method of human-derived insulin-like growth factor binding protein 1 in pichia pastoris, and preparation and application of related antibodies Download PDFInfo
- Publication number
- CN103865950A CN103865950A CN201210531593.5A CN201210531593A CN103865950A CN 103865950 A CN103865950 A CN 103865950A CN 201210531593 A CN201210531593 A CN 201210531593A CN 103865950 A CN103865950 A CN 103865950A
- Authority
- CN
- China
- Prior art keywords
- igfbp
- antibody
- source
- growth factor
- human
- 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
Images
Landscapes
- Peptides Or Proteins (AREA)
Abstract
The invention relates to an induced expression and purification method of a human-derived insulin-like growth factor binding protein 1 in pichia pastoris. A nucleotide sequence optimized by codons and used for coding the human-derived insulin-like growth factor binding protein 1 is connected with an HIS tag label, then is constructed into a pichia pastoris induced expression vector, next is transferred into pichia pastoris, and is subjected to induced expression; the expression product is subjected to affinity chromatography to obtain a purified human-derived insulin-like growth factor binding protein 1. The invention also relates to related antibodies and an application thereof in preparation of diagnostic kits for premature rupture of fetal membranes. The induced expression and purification method of the human-derived insulin-like growth factor binding protein 1 in pichia pastoris is skillful in design, and thus the human-derived insulin-like growth factor binding protein 1 can be efficiently expressed; and with the human-derived insulin-like growth factor binding protein 1 as an antigen, a high-specific polyclonal antibody and a high-specific monoclonal antibody for recognition of different IGFBP-1 epitopes are obtained, thereby laying the foundation for application of multiple downstream detection kits, and being suitable for large-scale popularization and application.
Description
Technical field
The present invention relates to protein expression, purifying and antibody production techniques field, be particularly related to abduction delivering, purifying and the antibody production techniques field of albumen in pichia yeast, specifically refer to abduction delivering and purification process, associated antibodies preparation and the application of a kind of people source IGFBP-1 in pichia yeast.
Background technology
People source IGFBP-1 (IGFBP1) is one of endocrine hormone important in the human body of finding the eighties in 20th century, it is made up of one group of soluble protein, be divided into six members such as IGFBP1-6, can both and rhIGF-1 (IGF) with high-affinity combination, but different IGFBP is combined rear produced biological action with IGF different.People in 1988 report the full-length cDNA of IGFBP1 for the first time, the main biological significance of IGFBP1 is transportation and regulates the combination of type-1 insulin like growth factor and acceptor, and explaining important in inhibiting (Ferry RJ Jr aspect much physiology, pathological phenomenon and cancer, treating diabetes, Cohen P et al, Insulin-like growth factor binding proteins:new protein, new functions, 1999, Horm Res, 51 (2): 53-67).Due to when pregnant woman conceived 38 weeks (before the expected date of childbirth), there were significant differences for the concentration of IGFBP1 in amniotic fluid and in vaginal secretion (below seeing 2.3), thereby become and detect early one of broken, important indicator of early splitting of fetus amniotic fluid film.
IGFBP1 is totally 234 amino acid, molecular weight is about 25.3-31kD, be positioned human chromosome 7p12-p14, mainly and secretion synthetic by histoorgans such as liver, uterus, decidua, kidney, fetus liver, ovaries, and the histocyte that acts on secretory cell self and vicinity maybe can act on target organ at a distance by blood circulation, major function, except transportation and adjusting IGF-1 and receptors bind, can also be independent of IGF-1 and play a role.
1, the relation of IGFBP1 and disease
The relation of 1.1IGFBP1 and cancer
Cancer is the second largest factor of mankind's death at present, a large amount of evidences show, bad mode of life is as smoking, eat containing higher fatty acid, the food of high sugar and to move very few etc. be the major cause that causes cancer, can cause insulin resistance and hyperglycemia, increase the content of Regular Insulin in blood simultaneously, cause the increase of type-1 insulin like growth factor (IGF1), the synthetic decline of result IGFBP-1 (IGFBP1), stimulate the growth of tumour cell, tumour cell programmed death is suppressed, increase cancered risk, as in lung cancer, prostate cancer, in the evolution of mammary cancer and colorectal carcinoma etc., all there is above situation, the expression regulation of IGFBP1 has participated in the generation of cancer.According to Jing Ma, by comparative analysis, there is dependency (R.James Barnard with the probability of suffering from prostate cancer in the heritable variation of IGF1, IGFBP1 and IGFBP3 in Caucasian, Prevention of Cancer Through Lifestyle Changes.2004, Evid Based Complement Alternat Med.1 (3): 233-239; Fredrick R.Schumacher, Jing Ma, et al, A comprehensive analysis of common IGF1, IGFBP1 and IGFBP3 genetic variation with prospective IGF-1 and IGFBP-3 blood levels and prostate cancer risk among Caucasians, 2010, Hum Mol Genet, 19 (15): 3089-3101).
The relation of 1.2IGFBP1 and type II diabetes and cardiovascular disorder
Clinical observation, type II diabetes people has the probability of larger trouble cardiovascular disorder than non-diabetic people, worse prognosis, the reason that probability increases is except hypertension and hyperlipemia, also relevant with insulin resistance, this is type II diabetes and metabolic syndrome patient's characteristic feature, and these features are all extremely caused due to insulin-like growth factor in body 1 and IGFBP-1, great many of experiments shows, the decline of insulin-like growth factor 1 level increases the risk of suffering from cardiovascular disorder, and IGFBP-1 playing an important role at regulation and control insulin-like growth factor 1 and its acceptor, but relevant Regular Insulin, mechanism of action between rhIGF-1 and IGFBP-1 is not also (the Vivienne A.Ezzat that is perfectly clear, Mark T.Kearney et al, The role of IGF-1 and its binding proteins in the development of type 2 diabetes and cardiovascular disease, 2007, Diabetes Obes Metab, 10 (3): 198-211).
The clinical application of 2IGFBP1 in Obstetric and Gynecologic Department field
2.1 prediction premature labors
IGFBP1 is at pregnant different times, its phosphorylation degree difference, and exist with different isomer.IGFBP1 mainly appears at non-phosphorylating form in serum, amniotic fluid and the decidua tissue of trimester parent, and in the pregnant and late pregnancy period, IGFBP1 mainly appears in maternal serum and decidua tissue with high sulfation form, in amniotic fluid but still take low phosphorylation and dephosphorylation as main.According to clinical observation, before occurring, general premature labor destroys because uterine contraction causes chorion-decidua matrix, chorion-decidua separates, and IGFBP1 in decidua tissue can flow in uterine neck, vagina, still can judge by detecting the phosphorylation degree of IGFBP1 in vagina the generation of premature labor.Detect the susceptibility of IGFBP1 prediction childbirth before 37 weeks according to Zhang Xinling etc., specificity, positive predictive value, negative predictive value is respectively 80.6%, 93.5%, 83.3%, 92.3%, therefore IGFBP1 has higher specific degree as Prediction of Preterm Labor index, be not subject to the interference of extraneous factor, compared with fetal fibronectin (fFN), the positive predictive value of IGFPB1 is higher, wider (the Zhang Xinling of range of application, Li little Mao, Zheng Rongqin etc., the value of IGFBP-1 in uterine neck vaginal secretions in Prediction of Preterm Labor, 2002, practical journal of obstetrics and gynecology, 18(2): 107).
2.2 evaluate cervical ripeness
According to Nuutila etc., high phosphorylation isomer and the cervical ripeness of IGFBP1 in uterine neck vaginal secretions have substantial connection, cervical ripeness and its concentration (the Nuutila M that is proportionate, Hiilesmaa V, Karkkainen T, et al, Phosphorylated isoforms of insulin-like growth factor binding protein-1 in the cervix as a predictor of cervical ripeness, 1999, Obstet Gynecol, 94 (2): 243).Therefore can be used as one of objective indicator of cervix maturation, can judge preferably timing of delivery.
2.3 Diagnosis of Premature Rupture
Rupture of membranes is just before giving birth called premature rupture of fetal membrane (Premature rupture of fetal membrane, PROM), the external report of its incidence is about 4.4%-7.6%, and that domestic report is about Song 10%(is beautiful, the application of IGFBP-1 in Diagnosis of Premature Rupture, 2003, shanghai Medicine check magazine, 18(3): 171-172).Premature rupture of fetal membrane is to enclose modal complication of raw phase, can cause serious adverse consequences to pregnant woman, fetus an d neonate.As caused early productive rate to raise, enclosing raw youngster's case fatality rate increases, and intrauterine infection rate and puerperal infection rate raise.The reason of premature rupture of fetal membrane has a lot, mainly to have wound, incompetence of internal orifice of uterus, reproductive tract pathogenic micro-organism ascending infection, mycoplasma infection, amniotic cavity increased pressure, fetal membrane dysplasia etc., therefore, it is particularly important that the early detection of premature rupture of fetal membrane seems, the method that premature rupture of fetal membrane detects clinically at present has microscopy, pH detection paper, early pregnancy β-HCG detection paper and HCG fluorescent polarization immunoassay (FPIA) method detect, but all exist susceptibility not high, process is more loaded down with trivial details, and be especially easily subject to urine, seminal fluid, the impact of blood etc., cause accuracy, sensitivity is all not high.Woyton etc. study discovery, IGFBP-1 when rupture of membranes in amniotic fluid can flow in uterine neck vagina by cut, and height in concentration ratio urine and the blood of IGFBP-1 in amniotic fluid is doubly a lot, as following table shows distribution situation (the Gillian D.Bryant-Greenwood of IGFBP-1 in different body fluid, Eeva-Marja Rutanen, et al, Sequential appearance of relaxin, prolactin and IGFBP-1 during growh and differentiation of the human endometrium, 1993, Mol cell Endocrinol, 95 (1-2): 23-29):
| Sample | The concentration of IGFBP-1 |
| Serum (pregnancy) | 58-600μg/l |
| Urine | Do not detect |
| Seminal fluid | Do not detect |
| Amniotic fluid | 10500-350000μg/l |
So IGFBP-1 can be served as the mark that premature rupture of fetal membrane detects, more existing correlation detection test kit listings at present.But system is complete, and the IGFBP-1 detection method of patent is left to be desired, and present patent application provides from theory to clinical diagnosis, is prepared into the system schema of test kit from antigen-antibody, and convictive data are provided.
The relation of 3.IGFBP-1 and other diseases
In human body other diseases, also there is the variation of different amplitudes in the variation of the expression amount of IGFBP-1, also can be used as Research of predicting markers and the outbreak of corresponding disease is judged, as reports such as Wenjing, IGFBP-1 is likely at insulin sensitivity, in cardiovascular disorder etc. as the evaluation criteria of onset risk, as following table (Wenjing Ruan, Maode Lai, Insulin-likegrowth factor binding protein:a possible marker for the metabolic syndrome, 2010,47:5-14):
About IGFBP-1 as bio signal tag application in the main research of the feasibility of metabolic disorder
In sum, about seeming particularly important for the sensitive detection method of human body IGFBP-1 albumen, and immunology detection is because of its highly sensitive, high specific and simple to operate forming as prefered method, therefore the monoclonal antibody of corresponding anti-IGFBP-1 and the preparation of polyclonal antibody are to determine immunology detection sensitivity and specific key point.The preparation of antibody needs a large amount of highly purified albumen.At present, be to express and purify in bacterium in order to prepare the IGFBP-1 albumen of anti-IGFBP-1 antibody.People source IGFBP-1 has complicated glycosyl and specific space conformation, and bacterial expression system is due to procaryotic limitation, cannot glycosylation IGFBP-1 albumen, and it is potential that also to affect its space conformation normally folding.On this basis, cause that to detect antibody single, the detection sensitivity space that is still improved.Therefore, a kind of abduction delivering and purification process of new people source IGFBP-1 need to be provided, it can high efficient expression people source IGFBP-1, thereby take obtain polyclonal antibody and the monoclonal antibody of the high specific of the different epi-positions of identification IGFBP-1 as antigen, for the application of the multiple detection kit in downstream lays the first stone.The ingenious characteristic of having utilized yeast to have " being eukaryote, Fast-propagation and efficient protein expression " concurrently of the present invention, has overcome this difficult problem.
Summary of the invention
The object of the invention is to have overcome above-mentioned shortcoming of the prior art, abduction delivering and purification process, associated antibodies preparation and the application of a kind of people source IGFBP-1 in pichia yeast is provided, this abduction delivering and purification process design are ingenious, thereby can high efficient expression people source IGFBP-1, thereby take obtain polyclonal antibody and the monoclonal antibody of the high specific of the different epi-positions of identification IGFBP-1 as antigen, for the application of the multiple detection kit in downstream lays the first stone, be suitable for large-scale promotion application.
To achieve these goals, in a first aspect of the present invention, abduction delivering and the purification process of a kind of people source IGFBP-1 in pichia yeast is provided, be characterized in, after being connected with HIS tag label, the nucleotide sequence of the encoding human source IGFBP-1 of codon optimization is built in pichia spp inducible expression carrier, then proceed to pichia spp and carry out abduction delivering, expression product obtains the people source IGFBP-1 of purifying by affinity chromatography.
Obviously, adopt aforesaid method can express any nucleotide sequence coded people source IGFBP-1.Preferably, described nucleotide sequence is the sequence as shown in SEQ ID NO:1.
Pichia spp inducible expression carrier can be selected pichia spp inducible expression carrier arbitrarily, all can realize the present invention.Preferably, described pichia spp inducible expression carrier is pHBM905a.
Pichia spp also can be selected pichia spp strain arbitrarily, all can realize the present invention.Preferably, described pichia spp is Pichia pastoris GS115.
Described affinity chromatography can adopt any suitable affinity purification column chromatography, and preferably, described affinity chromatography adopts Ni-nickel affinity purification column chromatography.
In a second aspect of the present invention, a kind of antibody is provided, be characterized in, the people source IGFBP-1 that adopts the abduction delivering of above-mentioned people source IGFBP-1 in pichia yeast and purification process to prepare is prepared as antigen-immunized animal.
Described antibody can be any suitable antibody, and preferably, described antibody is polyclonal antibody or monoclonal antibody.
Described polyclonal antibody can adopt any suitable method preparation, more preferably, described polyclonal antibody is specifically adopted with the following method preparation: adopt described people source IGFBP-1 to obtain the rabbit anteserum of the antibody that contains anti-human source IGFBP-1 as antigen immune rabbit, then adopt antibody described in Protein G Sepharose purifying.
Described monoclonal antibody can adopt any suitable method preparation, more preferably, described monoclonal antibody is specifically adopted preparation with the following method: adopt the splenocyte of described people source IGFBP-1 as antigen immune mouse adaptive immune, merge with oncocyte again, screen and obtain the anti-hIGFBP1 cell strain of monoclonal antibody KMM201301 of secretion high-affinity, then adopt the antibody of the anti-hIGFBP1 cell strain of monoclonal antibody KMM201301 secretion of the high-affinity described in Protein G Sepharose purifying.
In a third aspect of the present invention, provide above-mentioned antibody in the application of preparing in diagnosing premature rupture of fetal membrane test kit.
Described diagnosing premature rupture of fetal membrane test kit can be any suitable test kit, and preferably, described diagnosing premature rupture of fetal membrane test kit is latex agglutination diagnosing premature rupture of fetal membrane test kit or colloidal gold method diagnosing premature rupture of fetal membrane test kit.
Beneficial effect of the present invention is:
1, abduction delivering and the purification process of people of the present invention source IGFBP-1 in pichia yeast is to be built in pichia spp inducible expression carrier after the nucleotide sequence of the encoding human source IGFBP-1 of codon optimization is connected with HIS tag label, then proceed to pichia spp and carry out abduction delivering, expression product obtains the people source IGFBP-1 of purifying by affinity chromatography, expression amount can reach about 100-200mg/l; Described human insulin-like growth factor binding protein white-1 is as antigen immune rabbit or mouse, can effectively obtain the polyclonal antibody of high-affinity and the mouse hybridoma cell strain of secrete monoclonal antibody, and test to detect by ELISA and tire, extension rate, from 1:100, is tired and is reached 100 × 2
6above, after Protein G Sepharose resin affinity purification, tire and exceed 10
6; Purified antibody can optimum combination, prepares the sandwich quick diagnosis reagent kit of latex agglutination and Radioactive colloidal gold, for to fetus amniotic fluid film early broken in time, convenient diagnosis, provides foundation for pregnant woman waits that producing seeks medical advice, and is suitable for large-scale promotion application
Accompanying drawing explanation
Fig. 1 is the plasmid map schematic diagram of plasmid pHBM905a.
Fig. 2 is the collection of illustrative plates schematic diagram of pichia spp inducible expression carrier pHBM905aHIGFBP1.
Embodiment
In order more clearly to understand technology contents of the present invention, describe in detail especially exemplified by following examples.
Structure and the expression of embodiment 1pHBM905aHIGFBP1 expression vector
1, the optimization of the nucleic acid codon of human insulin-like growth factor binding protein white-1 and synthetic
In Genebank, search the protein sequence of human insulin-like growth factor binding protein white-1, optimize nucleic acid codon synthetic white-1 nucleotide sequence (being the sequence shown in SEQ ID NO:1) of human insulin-like growth factor binding protein that obtains of complete sequence by website http://helix.nih.gov according to pichia yeast codon-bias.
And design synthetic primer, and adding respectively nucleic acid restriction endonuclease site and several protection base at 5 ' end and the 3 ' end of sequence, primer is P1:5 '-ATA
cTCGAGaTGCACCATCATCATCATCATGCCCCATGGCAATGTG-' 3 (sequence shown in SEO IDNO:2), P2:5 '-ATA
tATTGTACGTTGAAGTAAATCTGGCAGTTGGG-' 3 (sequence shown in SEQ ID NO:3).
Setting-out part is respectively XhoI and NotI restriction endonuclease sites, and tilted letter is HIS sequence label, adopts single stage method synthetic.
Adopt P1 and P2 to carry out pcr amplification to above-mentioned sequence, 50ul PCR reaction system is as follows:
Reaction conditions:
98 ℃ of sex change, lO second; Anneal 58 ℃, 15 seconds; Extend 72 ℃, 40 seconds; 28 circulations, 72 ℃ are extended 1 minute.
2, the structure of pHBM905aHIGFBP1 expression vector
The expression vector that the present embodiment adopts is pHBM905a, is carrier pPIC9K transformation is obtained, and its plasmid map is shown in Fig. 1.After specifically transforming as follows, obtain plasmid pHBM905a:
A, first use BglII digested plasmid pPIC9K, remove Amp
rwith pBR322 fragment, then form plasmid pPIC9K 1 from connecting, then design primer, two ends all add SalI restriction endonuclease site, this fragment increases;
The fragment expanding in b, 1 and plasmid pPIC9K 1 are used respectively the effect of SalI restriction endonuclease, then are connected to form plasmid pPIC9K 2;
C, by the Kan in plasmid pPIC9K 1
rgene fragment is cut away and is connected in multiple clone site, forms plasmid pHBM905a.
After the fragment recovery purifying that above-mentioned PCR is obtained, cut with XhoI and NotI enzyme, pHBM905a also adopts XhoI and NotI enzyme to cut simultaneously, and then two endonuclease bamhis connect, thereby His-IGFBP-1 nucleotide sequence is built into pHBM905a, finally obtain pHBM905aHIGFBP1 expression vector, see Fig. 2.
3, the abduction delivering of human insulin-like growth factor binding protein white-1
The pHBM905aHIGFBP1 expression vector SalI enzyme building is cut plasmid linearization, has also removed the resistant gene AmpR on carrier simultaneously, then transforms pichia yeast GS115 by electricity, selects positive colony, carries out abduction delivering screening; Adopt following method to identify: (1) SDS-PAGE electrophoresis detection; (2) Dot Blot; (3) Western blotting; (4) order-checking, and filter out the correct recombinant bacterium (about 100-200mg/l) of evaluation sequence that expression amount is higher.
4, the purifying of human insulin-like growth factor binding protein white-1
Because the human insulin-like growth factor binding protein that pichia yeast is expressed white-1, with HIS label, mainly adopts nickel post affinity purification, after purifying, purity reaches more than 90%.
5, pichia yeast is expressed human insulin-like growth factor binding protein is white-1(IGFBP-1) and the preparation of polyclonal antibody
1) preparation of antibody
A. the negative control while extracting non-immune normal rabbits blood separation of serum 1ml as bioactivity;
B. the IGFBP-1 after purifying is mixed with Freund's complete adjuvant, with muscle and the injection of intracutaneous multiple location, antigen concentration is 0.5mg/ml respectively, every injection 1ml antigen;
C. after surrounding, strengthen weekly immunity once again, the same initial immunity of injection system and injection volume, just immunological adjuvant has changed Freund's incomplete adjuvant into, strengthens altogether immunity three times;
D. strengthen three rear neck artery bloodletting of immunity, collect serum;
2) antiserum(antisera) purifying
A. the serum of collecting filters with 0.45 μ m filter, then adds 1 × PBS(PBS pH8.0 of 10% volume);
B. by the serum of handling well by the speed of 0.5ml/min be added to pre-treatment Protein A affinity column;
C. use pH3.0 0.1M glycine solution, by the speed wash-out of 2.0ml/min, collect elutriant;
D. finally in elutriant, add the neutralization of 20%1M Tris damping fluid;
E. in the elutriant of collecting at the EP pipe with 1.0ml, every pipe is got 5 μ l, makes SDS-PAGE electrophoresis detection;
F. shampoo adopts dialysis membrane dialysis, the PBS damping fluid that dialyzate is 0.01M;
G. adopt ELISA method to detect antibody titer.
6, pichia yeast is expressed human insulin-like growth factor binding protein is white-1(IGFBP-1) and the preparation of monoclonal antibody
1) antigen injection
Antigen (IGFBP-1) and Freund's complete adjuvant are fully mixed by 1:1, and the mixed solution of multiple spot subcutaneous injection 100 μ g antigens is to Bal b/c mouse; After approximately three weeks, again antigen and Freund's incomplete adjuvant are fully mixed by 1:1, the same mode of multi-point injection that adopts is by subcutaneous the mixture injection mouse of same antigen and Freund's incomplete adjuvant, after one week, adopt indirect ELISA method to detect antibody titer, finally choose conduct that antibody titer the is high object of immunity again, at tail vein and spleen injections of antigens 300 μ g/ only.
2) cytogamy
Tail vein supplementary immunization is after three days, after immune mouse eyeball blood sampling (preparing serum as positive control), slaughters, and aseptic its spleen of getting, prepares splenocyte, the SP2/0 cell of spleen cell and logarithmic phase merged with 50%PEG4000; And after non-immune BAL b/c eyeball of mouse blood sampling (preparing serum as negative control), get peritoneal macrophage as trophocyte.Cell after fusion suspends with HAT substratum, plants in 96 orifice plates, puts 37 ℃, 5%CO
2cultivate in incubator, after 5 days, use HAT substratum instead, routine observation, in the time that hybridoma rises by 1/10 left and right to hole floorage, starts to detect screening.
7, the specificity purifying of antibody
1) preparation of IGFBP-1 affinity column
From yeast, purify and be applied to IGFBP-1 prepared by antibody, by the specificity purifying with this strain antibody by use.IGFBP-1 is arrived to protein A chromatography column with halfcystine disulfide-bonded.
2) being further purified of antibody
This strain antibody is crossed to post, with pH3.00.1M glycine solution wash-out, collect elutriant, it is neutral adjusting pH value.
Finally in elutriant, add the neutralization of 20%1M Tris damping fluid.
8, antibody is applied to the preparation of diagnostic kit
1) latex agglutination test kit
From the polyclone of high titre and monoclonal antibody, filter out a strain antibody the strongest with facing Prenatal puerperas (before 37 weeks) amniotic fluid IGFBP-1 albumen (antigen) binding specificity.Adopt the antibody consistent with antigen concentration to mix with different concns latex proportioning, and with antigen in conjunction with observing latex agglutination degree, by photoabsorption detection by quantitative latex agglutination intensity, filter out the proportioning of antibody and latex.With this proportioning, change the concentration of antigen, each experiment repeats to average and standard deviation for five times, draws the best ratio of antigen, antibody, latex.Be applied to the preparation of latex agglutination test kit.
2) Radioactive colloidal gold double fastener heart test kit
From the polyclone of high titre and monoclonal antibody, filter out two strain antibodies the strongest with facing Prenatal puerperas (before 37 weeks) amniotic fluid IGFBP-1 albumen (antigen) binding specificity.One strain antibody A is combined with antigen; Another strain antibody B first mixes with Radioactive colloidal gold, and then with the former further combined with; Thereby form the agglomeration of antibody A-Ag-Ab B-Radioactive colloidal gold, and manifest the peculiar redness of high density Radioactive colloidal gold.Adopt antibody A and the antibody B consistent with antigen concentration, mix with different concns Radioactive colloidal gold proportioning, and with antigen in conjunction with observation, with photoabsorption detection by quantitative colored intensity, filter out the proportioning of antibody and latex.With this proportioning, change the concentration of antigen, each experiment repeats to average and standard deviation for five times, draws the best ratio of antigen, antibody A, antibody B, Radioactive colloidal gold.Be applied to the preparation of Radioactive colloidal gold double fastener heart test kit.
As can be seen from above, because yeast can carry out glycosylation modified to expressing protein, and yeast is eukaryote, therefore the human insulin-like growth factor binding protein white-1 that adopts method of the present invention to express, compared with traditional protokaryon bacterial expression, there is different glycosylation modified and space conformations, and expression amount is high, can reaches about 100mg/l; Through ELISA checking, polyclonal antibody and monoclonal antibody avidity and specificity are better, dilution 10
6still can obtain good result.
To sum up, abduction delivering of the present invention and purification process design are ingenious, thereby can high efficient expression people source IGFBP-1, thereby take obtain polyclonal antibody and the monoclonal antibody of the high specific of the different epi-positions of identification IGFBP-1 as antigen, for the application of the multiple detection kit in downstream lays the first stone, be suitable for large-scale promotion application.
In this specification sheets, the present invention is described with reference to its specific embodiment.But, still can make various modifications and conversion obviously and not deviate from the spirit and scope of the present invention.Therefore, specification sheets and accompanying drawing are regarded in an illustrative, rather than a restrictive.
Claims (11)
1. people source IGFBP-1 abduction delivering and the purification process in pichia yeast, it is characterized in that, after being connected with HIS tag label, the nucleotide sequence of the encoding human source IGFBP-1 of codon optimization is built in pichia spp inducible expression carrier, then proceed to pichia spp and carry out abduction delivering, expression product obtains the people source IGFBP-1 of purifying by affinity chromatography.
2. abduction delivering and the purification process of people according to claim 1 source IGFBP-1 in pichia yeast, is characterized in that, described nucleotide sequence is the sequence as shown in SEQ ID NO:1.
3. abduction delivering and the purification process of people according to claim 1 source IGFBP-1 in pichia yeast, is characterized in that, described pichia spp inducible expression carrier is pHBM905a.
4. abduction delivering and the purification process of people according to claim 1 source IGFBP-1 in pichia yeast, is characterized in that, described pichia spp is Pichia pastoris GS115.
5. abduction delivering and the purification process of people according to claim 1 source IGFBP-1 in pichia yeast, is characterized in that, described affinity chromatography adopts Ni-nickel affinity purification column chromatography.
6. an antibody, it is characterized in that, the people source IGFBP-1 that adopts the abduction delivering of people according to claim 1 source IGFBP-1 in pichia yeast and purification process to prepare is prepared as antigen-immunized animal.
7. antibody according to claim 6, is characterized in that, described antibody is polyclonal antibody or monoclonal antibody.
8. antibody according to claim 7, it is characterized in that, described polyclonal antibody is specifically adopted with the following method preparation: adopt described people source IGFBP-1 to obtain the rabbit anteserum of the antibody that contains anti-human source IGFBP-1 as antigen immune rabbit, then adopt antibody described in Protein G Sepharose purifying.
9. antibody according to claim 7, it is characterized in that, described monoclonal antibody is specifically adopted preparation with the following method: adopt the splenocyte of described people source IGFBP-1 as antigen immune mouse adaptive immune, merge with oncocyte again, screen and obtain the anti-hIGFBP1 cell strain of monoclonal antibody of secretion high-affinity, then adopt the antibody of the anti-hIGFBP1 cell strain of monoclonal antibody secretion of the high-affinity described in Protein G Sepharose purifying.
According to the arbitrary described antibody of claim 6-9 in the application of preparing in diagnosing premature rupture of fetal membrane test kit.
11. application according to claim 10, is characterized in that, described diagnosing premature rupture of fetal membrane test kit is latex agglutination diagnosing premature rupture of fetal membrane test kit or colloidal gold method diagnosing premature rupture of fetal membrane test kit.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210531593.5A CN103865950A (en) | 2012-12-11 | 2012-12-11 | Induced expression and purification method of human-derived insulin-like growth factor binding protein 1 in pichia pastoris, and preparation and application of related antibodies |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210531593.5A CN103865950A (en) | 2012-12-11 | 2012-12-11 | Induced expression and purification method of human-derived insulin-like growth factor binding protein 1 in pichia pastoris, and preparation and application of related antibodies |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103865950A true CN103865950A (en) | 2014-06-18 |
Family
ID=50904958
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201210531593.5A Pending CN103865950A (en) | 2012-12-11 | 2012-12-11 | Induced expression and purification method of human-derived insulin-like growth factor binding protein 1 in pichia pastoris, and preparation and application of related antibodies |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103865950A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838732A (en) * | 2016-05-16 | 2016-08-10 | 上海蓝庄生物医药科技有限公司 | Sika deer insulin-like growth factor eukaryotic expression purification method |
| CN106636175A (en) * | 2016-09-30 | 2017-05-10 | 湖北科技学院 | Induced expression method of cell injury repair protein in pichia pastoris and purification method and application of cell injury repair protein |
| CN109142565A (en) * | 2018-07-27 | 2019-01-04 | 重庆早柒天生物科技股份有限公司 | The screening technique of premature rupture of fetal membranes pregnant woman's vaginal fluid differential protein based on iTRAQ technology |
| CN109320601A (en) * | 2018-10-18 | 2019-02-12 | 天津林达生物科技有限公司 | Recombinate IGF-1 albumen and high efficient expression and its purposes in terms of promoting cell Proliferation |
-
2012
- 2012-12-11 CN CN201210531593.5A patent/CN103865950A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105838732A (en) * | 2016-05-16 | 2016-08-10 | 上海蓝庄生物医药科技有限公司 | Sika deer insulin-like growth factor eukaryotic expression purification method |
| CN106636175A (en) * | 2016-09-30 | 2017-05-10 | 湖北科技学院 | Induced expression method of cell injury repair protein in pichia pastoris and purification method and application of cell injury repair protein |
| CN109142565A (en) * | 2018-07-27 | 2019-01-04 | 重庆早柒天生物科技股份有限公司 | The screening technique of premature rupture of fetal membranes pregnant woman's vaginal fluid differential protein based on iTRAQ technology |
| CN109320601A (en) * | 2018-10-18 | 2019-02-12 | 天津林达生物科技有限公司 | Recombinate IGF-1 albumen and high efficient expression and its purposes in terms of promoting cell Proliferation |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP5727938B2 (en) | How to test the high risk of developing pre-eclampsia | |
| JP5461998B2 (en) | Biomarkers for reproductive medicine and biology | |
| CN105934671B (en) | For assessing the method and reagent of gestational diabetes mellitus | |
| WO2022099465A1 (en) | Hybridoma cell strain 9c1, plgf-1 monoclonal antibody, preparation method therefor and application thereof | |
| CN103865950A (en) | Induced expression and purification method of human-derived insulin-like growth factor binding protein 1 in pichia pastoris, and preparation and application of related antibodies | |
| CN102887943A (en) | B cell epitope peptide segment of amino-terminal pro-brain natriuretic peptide and applications thereof | |
| CN106831976A (en) | Milk cow PAG1 polypeptides, its polyclonal antibody and application | |
| CN111996173B (en) | Hybridoma cell strain, preparation method and application thereof, monoclonal antibody and application thereof | |
| Senna et al. | Study of plasma adrenomedullin level in normal pregnancy and preclampsia | |
| CN103463629B (en) | A kind of preparation method of premature ovarian failure non-human mammal model and application thereof | |
| CN102659929B (en) | Transient receptor potential channel-6 (TRPC6) antigen polypeptide and anti-TRPC6 monoclonal antibody | |
| CN112946299B (en) | Application of product of quantitative FTL in preparation of preeclampsia diagnosis tool | |
| CN112358546B (en) | Hybridoma cell strain 9C1, PLGF-1 monoclonal antibody and application thereof | |
| US20070072245A1 (en) | Method and means for the determination of defined states or modifications in the mucus of the uterus or in the epithelium of other organs | |
| CN104945506A (en) | Immunohistochemical reagent for mammary cancer diagnosis and prognosis judgment | |
| CN104945496A (en) | Polypeptide and application thereof in preparing and purifying EHD2-specific antibody | |
| CN114213534B (en) | Anti-human PlGF (platelet-derived growth factor) murine monoclonal antibody and application thereof | |
| CN110079506B (en) | Monoclonal antibody of human serum fibrin ficolin-2 and application thereof | |
| CN108774287A (en) | A kind of VEGF-B monoclonal antibodies and kit | |
| US7943325B2 (en) | Diagnosis of fertility conditions using a serine protease | |
| EP4276109A1 (en) | Biomarker for determining fertility, and determining method using same | |
| CN101968482A (en) | Method for morphologically detecting angiotensin II type 1 receptor autoantibody | |
| WO2013099376A1 (en) | Mature ovum marker for use in in vitro fertilization, and use thereof | |
| CN106589123A (en) | Method for preparing anti-hyperglycosylation human chorionic gonadotropin antibody | |
| JP2022023020A (en) | Method for predicting probability of contracting obstetrical emergency including hypertensive disorders of pregnancy and hellp syndrome by measuring blood uromodulin concentration |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140618 |