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Method of determining klotho protein in human blood

A technology for detecting human and protein, applied in the field of immunoassay

Inactive Publication Date: 2004-08-18
PEKING UNIV
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Problems solved by technology

The existence of KL protein in human blood has not been reported

Method used

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Embodiment

[0023] 1. Construction of human Klotho protein expression vector

[0024] 1. Cloning of the C-terminal gene of human Klotho secreted protein

[0025] A pair of primers were designed based on Human Klotho cDNA sequence:

[0026] Sense primer:

[0027] 5'>GTC CCC GGA TCC GTG TCC ATT GCC CTA A<3′

[0028] Anti-sense primer:

[0029] 5′>ACT AGC CTC GAG ATC TCC AGA GCC GAA A<3′

[0030] A 1.5 kb fragment cDNA was amplified from human kidney tissue RNA using primers.

[0031] 2. Construction of expression vector

[0032] The above-mentioned amplified DNA fragment was cloned into pMD18-T vector, and it was verified by sequencing that it belonged to the C-terminal form of Klotho secreted protein (KLSC for short), and then digested with double enzymes (BamHI and HindIII) from the pMD18-T-KLSC plasmid, The 1.2kb fragment was excised and cloned into pET-30a and pET28a vectors to form the KLSC expression vector. Then these vectors were transformed into E. coli BL21 strain to p...

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Abstract

The method is to determine Klotho (KL) protein in human blood via KL protein antibody. The preparation of KL protein antibody includes: amplifying partial cDNA sequence of KL gene from human kidney tissue by RT-PCR technology; cloning the segment cDNA to pET plamid to constitute KL protein segment expressing vector; coverting E.coli cell strain BL21 / DE3 with the expressing vector and IPTE induction to make the converted bacteria to express exogenous protein; collecting and crushing the cultured and induced cell, Ni-NTA combination and separation to obtain purified KL protein segment; injecting the purified KL protein segment to rabbit, collecting rabbit serum and purifying with saturated ammonium sulfate to obtain KL protein antibody. The method of the present invention may be used clinically.

Description

Technical field: [0001] The invention relates to an immunodetection method, in particular to a method for detecting Klotho protein in human blood by using Klotho antibody. Background technique: [0002] Japanese scientists discovered and reported the Klotho (KL) gene for the first time (Kuro-o M., et al. 1997 Nature 390, 45-51). Studies have shown that KL gene is related to some human aging phenotypes and diseases, such as: arteriosclerosis, emphysema, osteoporosis, hypertension, skin atrophy, infertility and short life cycle, etc. (Teruhito Y., et al.2000 Endocrinology 141, 438-445, 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 scientists cloned the human KL gene in 1998. The human KL gene is located on chromosome 13, with a total length of about 50 kb and composed of 5 exons. Due to the diffe...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): G01N33/53G01N33/531G01N33/68
Inventor 顾军肖能明
Owner PEKING UNIV
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