CN1162704C - Method of determining klotho protein in human blood - Google Patents
Method of determining klotho protein in human blood Download PDFInfo
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- CN1162704C CN1162704C CNB021213631A CN02121363A CN1162704C CN 1162704 C CN1162704 C CN 1162704C CN B021213631 A CNB021213631 A CN B021213631A CN 02121363 A CN02121363 A CN 02121363A CN 1162704 C CN1162704 C CN 1162704C
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- albumen
- klotho
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Abstract
The present invention provides a method for detecting Klotho (KL) protein in human blood. KL protein antibodies are used for detecting the KL protein in the human blood. The preparation method for the KL protein antibodies is that an RT-PCR technique is used for amplifying a part of a cDNA sequence of KL genes from RNA of human kidney tissues; the part of the cDNA is cloned to pET plasmid to form an expression vector of the KL protein part; the expression vector is converted to E. Coli bacteria strains BL21/DE3 are converted to bacteria expression foreign protein by the induction of IPTG; cultivated and induced cells are collected and crushed; a Ni-NTA combination and separation method is used for obtaining purified a KL protein part; the obtained purified KL recombination protein part is injected into a rabbit, and blood serum is collected form the rabbit and purified by saturated ammonium sulfate to obtain the KL protein antibodies. Then, the KL protein antibodies are used for detecting the KL protein in the human blood by a Westem method or an ELISA method. The present invention uses the special KL protein part to prepare the antibodies and can specially recognize the KL protein. The prepared antibodies can be used for detecting the KL protein in the human blood and used in clinic and research.
Description
Technical field:
The present invention relates to a kind of immunologic detection method, particularly relate to the method for utilizing Klotho albumen in the Klotho antibody test human blood.
Background technology:
Japan scientist find first and reported Klotho (KL) gene (Kuro-o M., et al.1997 Nature390,45-51).Studies show that the KL gene is relevant with disease with some human old and feeble phenotypes, as: artery sclerosis, pulmonary emphysema, osteoporosis, hypertension, atrophoderma, short (Teruhito Y., etal.2000 Endocrinology 141, the 438-445 of waiting of sterile and life cycle, Den E.A., et al.2002 PNAS 99,856-861, Yoshio O., et al.1998 BBRC 251,15 920-925, Kiyoshi M., et al.2000 BBRC 278,665-670, Noritoshi K., et al.2001 BBRC 280,1015-1020, Yuichiro S., et al.1998 BBRC248,324-329), Japanese scientist had cloned people KL gene in 1998.People KL gene is positioned on No. 13 chromosome, and the about 50kb of total length is made up of 5 extrons.Because the different montage modes of KL and RNA precursor, the KL expression of gene produces two transcripts, the two kinds of patterns of memebrane protein and secretory protein of encoding respectively.The expression of secreted protein be higher than memebrane protein (Yutaka M., et al.1998 BBRC 242,626-630).2000, scientist with adenovirus mediated mode with KL genetic transformation atherosclerotic rat, the rat illness obviously alleviated (Yuichiro S., et al.2000 BBRC 276,767-772).Existing studies show that, the KL gene is relevant with disease anti-ageing and that aging is relevant.Secreting type KL albumen is likely a kind of antidotal hormone in the biosome.
The KL secreted protein can be secreted into the extracellular, however in animal body with human body in, which kind of circulation system the KL secretory protein is present in, and is not clear.In people's blood, exist KL albumen not meet report as yet.
Summary of the invention:
The method that the purpose of this invention is to provide KL albumen in the antibody test blood of human body of a kind of KL of utilization is used for detection clinical and research.
Detection method of the present invention is as follows:
1. prepare the KL protein antibodies
(1) obtains the KL protein fragments
Utilize the RT-PCR technology, from the RNA of people's renal tissue, amplify the Partial cDNA Sequence of KL gene, this segment cDNA is cloned into the pET plasmid, be built into the expression vector of KL protein fragments.
(2) the people's expression and purifying of KL protein fragments of recombinating.
With the expression vector Transformed E .coli bacterial strain BL21/DE3 of above-mentioned structure,, make transformed bacteria express foreign protein by inducing of IPTG.With the cell harvesting that cultivation is induced, fragmentation in conjunction with the method for separating, obtains the KL protein fragments of purifying with Ni-NTA.
(3) acquisition of KL protein antibodies.
With the KL recombinant protein fragment injection rabbit of the purifying of above-mentioned acquisition, collect serum on one's body from rabbit, behind the saturated ammonium sulfate purifying, obtain the antibody of anti-KL albumen.
2. utilize the KL albumen in the KL protein antibodies detection human blood that makes.
Can adopt following two kinds of methods to detect:
(1) with the KL albumen in the Western method detection human blood.
With 10 times of human serum dilutions, carry out electrophoresis after, change on the film, be incubated cultivation with above-mentioned antibody and film, carry out the signal demonstration then.Utilize this method can detect that molecular weight is the KL albumen of 62.2kD in the human blood.
(2) with the KL albumen in the ELISA method detection human blood
After 10 times of human serum dilutions, be embedded in 96 orifice plates, be incubated cultivation with above-mentioned antibody, carry out input then.Utilize this method can detect KL albumen in the human blood.
Advantage of the present invention and good effect:
1. the present invention utilizes the antibody of KL protein fragments preparation specifically, can discern KL albumen specifically.
2. the present invention utilizes the antibody of preparation to detect KL albumen in people's blood, is used for clinical and research.
Embodiment:
One, the structure of people Klotho protein expression vector
1. the clone of people Klotho secreted protein C terminal gene
With Human Klotho cDNA sequences Design a pair of primer:
Sense?primer：
5′＞GTC?CCC?
GGA?TCC?GTG?TCC?ATT?GCC?CTA?A＜3′
Anti-sense?primer：
5′＞ACT?AGC?
CTC?GAG?ATC?TCC?AGA?GCC?GAA?A＜3′
Utilize primer from people's nephridial tissue RNA, to amplify the 1.5kb segment cDNA.
2. the structure of expression vector
The dna fragmentation of above-mentioned amplification is cloned on the pMD18-T vector, belong to the terminal form of Klotho secreted protein C (being called for short KLSC) through sequence verification, then from pMD18-T-KLSC plasmid double digestion (BamHI and HindIII), downcut the 1.2kb segment, and endonuclease bamhi is cloned on pET-30a and the pET28a carrier. formed the expression vector of KLSC.With these carrier Transformed E .coli BL21 bacterial strain, prepare the expression system of KLSC again.
Two, prokaryotic expression, purifying and the Polyclonal Antibody Preparation of 6 * His reorganization KLSC albumen
1. prokaryotic expression, the affinitive layer purification of 6 * His reorganization KLSC albumen
Above-mentioned expression bacterium 37 ℃ of cultivations, is worked as OD
600Adding IPTG (final concentration is 1mM) at=0.8 o'clock induced respectively 2,4,6 hours.The thalline of getting behind the inducing culture is centrifugal, and the thalline of collecting is resuspended in Binding Buffer (NaH
2PO
450mM, NaCl 300mM, imidazoles 10mM, pH8.0) ultrasonic, fragmentation.
2. the purifying of 6 * His reorganization KLSC albumen
With the thalline of above-mentioned the ultrasonication at room temperature centrifugal 20-30 of 10000g minute, get supernatant and Ni-NTA gluing and close 2 hours (30 ℃ of 200rpm), used post to separate, wash post with Buffer MCAC-2 again, stepwise elution (MCAC-20,40,60 then, 80,100,200; ), Fractional Collections detects with 12%SDS-PAGE, and silver dyes evaluation.
MCAC-0:Tris-HCl 20mmol/L, NaCl 0.5mol/L, glycerine 10% (v/v), PMSF1mmol/L, urea 8mol/L; MCAC-2,20,40,60,80,100,200 promptly add 0.02,0.2,0.4,0.6,0.8,1 respectively, the 2mol/L imidazoles in MCAC-0.
Three, the preparation of 6 * His reorganization KLSC protein polyclone antibody
Preparation material: the big grey rabbit of livid purple indigo plant, Buddhist formula Freund's complete adjuvant and Buddhist formula Freund.
Method: get Buddhist formula Freund's complete adjuvant and 1mg/mL 30a-KLSC antigen equal-volume mixing and emulsifying first week, every rabbit injection 2mL emulsification antigen: injection for the second time is with Buddhist formula Freund and 1mg/mL 30a-KLSC antigen equal-volume mixing and emulsifying after 10 days, every rabbit injection 1mL emulsification antigen, injection in per 10 days later on once, (date is respectively March 11 to inject 4 times altogether, March 21, April 1, April 11), 7 days (being April 18) serum that takes a morsel carries out the ELISA detection after the last injections of antigens, serum is got in bloodletting then, with saturated ammonium sulphate method IgG purification.
Four, utilize rabbit anti-KLSC to detect the existence and the form of klotho albumen in the human serum
1. detect klotho albumen in the human serum with the Western method
With 10 times of human serum dilutions, the KLSC antibody dilution is 10
-4, the positive contrast of antigen 28a-KLSC, arabidopsis leaf total protein, people HUVEC cell and mouse RAW total protein of cell are contrast.The klotho albumen that the quantity that all has tested serum does not wait exists, and molecular weight is 62.2kD.
2. with the KL albumen in the ELISA method detection human blood
After 10 times of human serum dilutions, be embedded in 96 orifice plates, (dilutability is 10 with above-mentioned antibody
-4) be incubated cultivation, carry out input then.Utilize this method can detect KL albumen in the human blood.
Claims (4)
1, a kind of method that detects the Klotho albumen in the human blood is characterized in that it being to utilize the Klotho protein antibodies to detect Klotho albumen in the human blood.
2. the method for the Klotho albumen in the detection human blood as claimed in claim 1 is characterized in that the preparation method of Klotho protein antibodies is as follows:
(1) obtains the KL protein fragments: utilize the RT-PCR technology, from the RNA of people's renal tissue, amplify the Partial cDNA Sequence of KL gene, this segment cDNA is cloned into the pET plasmid, be built into the expression vector of KL protein fragments;
(2) the people's expression and purifying of KL protein fragments of recombinating:,, make transformed bacteria express foreign protein by inducing of IPTG with the expression vector Transformed E .coli bacterial strain BL21/DE3 of above-mentioned structure; With the cell harvesting that cultivation is induced, fragmentation in conjunction with the method for separating, obtains the KL protein fragments of purifying with Ni-NTA;
(3) acquisition of KL protein antibodies: with the KL recombinant protein fragment injection rabbit of the purifying of above-mentioned acquisition, collect serum on one's body, behind the saturated ammonium sulfate purifying, obtain the antibody of anti-KL albumen from rabbit.
3. the method for the Klotho albumen in the detection human blood as claimed in claim 1 or 2 is characterized in that it being the KL albumen that adopts in the Western method detection human blood.
4. the method for the Klotho albumen in the detection human blood as claimed in claim 1 or 2 is characterized in that it being the KL albumen of using in the ELISA method detection human blood.
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| Application Number | Priority Date | Filing Date | Title |
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| CNB021213631A CN1162704C (en) | 2002-06-17 | 2002-06-17 | Method of determining klotho protein in human blood |
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| CNB021213631A CN1162704C (en) | 2002-06-17 | 2002-06-17 | Method of determining klotho protein in human blood |
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| CN1162704C true CN1162704C (en) | 2004-08-18 |
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Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100381577C (en) * | 2004-04-08 | 2008-04-16 | 华东师范大学 | Screening method of klothe gene promotor activator |
| UA109888C2 (en) * | 2009-12-07 | 2015-10-26 | ANTIBODY OR ANTIBODILITY ANTIBODY OR ITS BINDING TO THE β-CLOTE, FGF RECEPTORS AND THEIR COMPLEXES | |
| CN102507925B (en) * | 2011-11-30 | 2013-10-30 | 尉军 | Improved detection technology for antibodies in human blood |
| CN109030835B (en) * | 2018-09-06 | 2021-06-22 | 江苏省人民医院（南京医科大学第一附属医院） | Method for analyzing effect of Rab8 on Klotho expression regulation in non-small cell lung cancer |
| TW202323277A (en) * | 2021-09-23 | 2023-06-16 | 大陸商江蘇恆瑞醫藥股份有限公司 | Anti-klb antibodies and uses |
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