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Recombinant human cystatin C genes, and expression and use thereof

A technology of cystatin and C gene, which is applied in the field of recombinant human cystatin C gene and its expression and application, can solve the problems of poor purity, large batch-to-batch variation, low concentration of cystatin C protein, etc., to solve the bottleneck problem effect

Inactive Publication Date: 2009-04-22
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tracing it to its cause, the anti-cystatin C monoclonal antibody with strong specificity and high sensitivity is required to prepare the cystatin C diagnostic kit, and to obtain the monoclonal antibody, high-purity cystatin C protein must be obtained. Cystatin C protein extracted from placenta, urine and other tissues has low concentration, poor purity, and large batch-to-batch variation, so it cannot be used as an immunogen to prepare monoclonal antibodies or to prepare diagnostic quality controls

Method used

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  • Recombinant human cystatin C genes, and expression and use thereof
  • Recombinant human cystatin C genes, and expression and use thereof
  • Recombinant human cystatin C genes, and expression and use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Preparation of recombinant soluble human cystatin C protein

[0024] (1) Clone the truncated cystatin C gene and construct the expression plasmid

[0025] Primers were designed according to the DNA sequence of the cystatin C gene, the gene was amplified by RT-PCR and ligated with the pMD18-T vector, transformed into Escherichia coli DH5a, single clones were identified by PCR to identify possible positive plasmids, and the plasmids were extracted and identified by double enzyme digestion. After DNA sequencing, the positive plasmid with the correct base and open reading frame was cut out with an appropriate enzyme and connected with the same double-digested PET32a(+) to construct a prokaryotic expression plasmid and transform the expression host strain Rosseta gami II. The positive host bacteria were identified by PCR. The plasmid vectors used in the above experiments were all kept in our laboratory. The expression strain Rosseta gami II was purchased from Merck Company,...

Embodiment 2

[0036] Preparation of Human Cystatin C Diagnostic Reagent

[0037] (1) Preparation of monoclonal antibodies against different epitopes

[0038] Purified recombinant cystatin C protein was prepared according to the monoclonal antibody preparation standard operating procedures to prepare multiple anti-clonal antibodies. A pair of monoclonal antibodies against different sites were screened by site stacking experiments. The specific steps are as follows: take the purified recombinant human cystatin C protein and dilute it to 10ug / ml and pack several plates according to the Elisa standard operating procedures (1-N, N is the number of monoclonal strains to be tested), seal and wash the plates and then add the first strain The monoclonal antibody reaches the saturation concentration of the antigenic site corresponding to the monoclonal antibody (the monoclonal antibody concentration required to saturate the site is completed through the pre-experiment), and incubate at 37°C. In the...

Embodiment 3

[0044] Preparation of Human Cystatin C Diagnostic Quality Control

[0045] Protect buffer (0.02M carbonate buffer, 20% glycerol, 0.8% sucrose, 0.5% BSA, 0.2‰ NaN 3 ) was diluted into five concentrations of 8ug / ml, 4ug / ml, 2ug / ml, 1ug / ml, and 0.5ug / ml, and then freeze-dried to make a human cystatin C diagnostic quality control product.

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Abstract

The invention discloses a recombinant human cystatin C gene and expression and application thereof. A truncated human cystatin C gene has a nucleic acid sequence as shown as in SEQ ID NO:1; the truncated human cystatin C protein has an amino acid sequence as shown in SEQ ID NO:2. The invention uses a genetic engineering technique to express and purify a great deal of stable recombinant soluble cystatin C protein and obtains a pair of monoclonal antibodies using the protein to prepare stable cystatin C diagnostic reagents and quality control products thereof, thereby solving bottleneck problems of independent research and development of a cystatin testing reagent box.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant human cystatin C gene and its expression and application. Background technique [0002] Cystatin C (Cystatin C) is a member of the cysteine ​​protease inhibitor superfamily 2. Its molecular weight is 13kD, which is a low molecular weight non-glycosylated protein composed of 122 amino acids (M Abrahamson, et al. Biochemical Journal, 1990.), contains two pairs of disulfide bonds, and can be constant in all nucleated cells , continuous transcription and expression, can be regarded as House keeping genes (Huang Junfu et al., Medical Clinical Biochemistry and Laboratory Science Volume). Recent studies have found that cystatin C is involved in many physiological and pathological processes in the body, including the invasive growth and metastasis of tumors, the occurrence and development of inflammation, and tissue fibrosis (Zhang Yaoquan, Yuan Fahuan, etc., 2003,...

Claims

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Application Information

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IPC IPC(8): C12N15/12C12N15/70C12N1/21C07K14/47C07K16/18G01N33/577G01N33/531
Inventor 喻堃王保宁周文娟吴丹陈丹
Owner SICHUAN MACCURA BIOTECH CO LTD
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