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Recombinant human cystatin C coding gene and expression method

An expression method and coding gene technology, which are applied in the field of recombinant human cystatin C coding gene and expression, can solve the problems of hindering popularization and application, limited antibody sources, low expression efficiency, etc., and meet the limitations of preparation conditions and selection of vector types. less, the construction process is simple, and the expression efficiency is high.

Inactive Publication Date: 2012-09-19
GUANGZHOU GENERAL HOSPITAL OF GUANGZHOU MILITARY COMMAND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003]Because the source of Cys C and its antibody is limited, the existing kits for detecting Cys C are expensive, which hinders its promotion and application in China
At present, the Cys C coding gene is usually obtained by PCR technology, cloned and then recombinantly expressed, but the expression efficiency is extremely low, which is difficult to meet the needs of the market

Method used

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  • Recombinant human cystatin C coding gene and expression method
  • Recombinant human cystatin C coding gene and expression method
  • Recombinant human cystatin C coding gene and expression method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The expression method of recombinant human cystatin C protein comprises the following steps:

[0035] 1) Add NdeI restriction site (catatg) to the 5' end of the target gene (nucleotide sequence is SEQ ID NO: 1), and EcoRI restriction site (gaattc) to the 3' end to synthesize the entire sequence to obtain Synthetic gene sequences;

[0036] 2) The synthetic gene sequence was cloned into the pMD18-T vector, and sequenced to confirm the successful construction of the recombinant T vector;

[0037] 3) The target gene sequenced correctly was excised from the recombinant T vector with NdeI and EcoRI endonucleases, and purified to obtain the target gene. The prokaryotic expression vector pET-22b(+) was subjected to the same enzyme digestion treatment, and the large fragment gene was purified. Then connect with the target gene obtained after double enzyme digestion, transfer into competent cell DH5α, extract the plasmid, and obtain the recombinant expression plasmid pET-CysC; ...

Embodiment 2

[0042] The expression method of recombinant human cystatin C protein comprises the following steps:

[0043]1) Transfer the recombinant expression plasmid pET-Cys C obtained in step 3) of Example 1 into the competent expression host strain E.coli BL21(DE3), and spread it on LB solid medium (containing 100 mg / L ampicillin) above, cultured at 37°C for 16 hours, and the positive clones were screened out as engineering bacteria;

[0044] 2) Inoculate the engineered bacteria into LB culture solution (containing 100 mg / L ampicillin), culture at 37°C, and wait until the OD of the bacteria solution 600nm At 0.6, add IPTG with a final concentration of 0.1 mmol / L, incubate at 37°C for 10 h, and centrifuge at 4°C to obtain bacterial cells;

[0045] 3) Same as Step 6 of Example 1).

Embodiment 3

[0047] The expression method of recombinant human cystatin C protein comprises the following steps:

[0048] 1) Transfer the recombinant expression plasmid pET-Cys C obtained in step 3) of Example 1 into the competent expression host strain E.coli BL21(DE3), and spread it on LB solid medium (containing 100 mg / L ampicillin) above, cultured at 37°C for 24 hours, and the positive clones were screened out as engineering bacteria;

[0049] 2) Inoculate the engineered bacteria into LB culture solution (containing 100 mg / L ampicillin), culture at 37°C, and wait until the OD of the bacteria solution 600nm At 1.0, add IPTG with a final concentration of 10 mmol / L, incubate at 37°C for 2 h, and centrifuge at 4°C to obtain bacterial cells;

[0050] 3) Same as Step 6 of Example 1).

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Abstract

The invention discloses a recombinant human cystatin C coding gene and an expression method. The nucleotide sequence of the coding gene is shown in SEQ ID NO:1, and the expression method of recombinant human cystatin C protein comprises the following steps that the recombinant human cystatin C coding gene is inserted into a polyclone locus of a prokaryotic expression carrier, a recombinant expression plasmid is constructed, the recombinant expression plasmid is converted to an expression host bacterium escherichia coli, and positive clone bacteria are screened out to obtain engineering bacteria; and the engineering bacteria are fermented, induced, separated and purified to obtain the recombinant human cystatin C protein. The recombinant human cystatin C coding gene provided by the invention can express the human cystatin C protein, the operation is convenient, the purity of the human cystatin C protein expressed by the coding gene reaches more than 90%, the expression efficiency is high, and no higher expression efficiency exists currently.

Description

technical field [0001] The invention belongs to the field of biomedical engineering, in particular to a recombinant human cystatin C coding gene and an expression method. Background technique [0002] Cystatin C (cystatin C, Cys C) is a cysteine ​​protease inhibitor protein. In 1983, Anastasi et al. first isolated and purified cysteine ​​protease inhibitor (cysteine ​​proteinase inhibitor, CPI) from egg white. Named as cystatin C, also known as γ-trace protein and γ-postglobulin, it is widely present in nucleated cells and body fluids of various tissues, and is a low molecular weight, basic non-glycosylated protein with molecular weight It is 13.3KD, composed of 122 amino acid residues, and can be produced by all nucleated cells in the body with a constant production rate. Circulating cystatin C is cleared only by glomerular filtration, is an endogenous marker of changes in glomerular filtration rate, and is reabsorbed in the proximal convoluted tubule, but is completely el...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/15C12N15/70
Inventor 张宏斌
Owner GUANGZHOU GENERAL HOSPITAL OF GUANGZHOU MILITARY COMMAND
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