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Method for preparing recombinant cystatin C

A technology of cystatin and fusion protein, applied in the field of preparation of recombinant cystatin C, which can solve the problems of limited source, low yield and high cost

Pending Publication Date: 2022-04-29
DAAN GENE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past, Cys C was mainly extracted from patient urine, not only the source was limited, but also the cost was high and the yield was extremely low

Method used

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  • Method for preparing recombinant cystatin C
  • Method for preparing recombinant cystatin C
  • Method for preparing recombinant cystatin C

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] 1) Taking the human Cys C gene provided by NCBI as a reference, combined with the test design requirements of the present invention, the amino acid sequence shown in SEQ ID NO: 1 was determined without a tagged vector, after optimization of the synonymous codon preference of Escherichia coli ( SEQ ID NO: 2), the connection vector is pET-28a(+) (C-terminal fusion expression (His)6 tag in the vector), synthesized by Nanjing GenScript Biotechnology Co., Ltd.

[0089] The amino acid sequence of the tagged fusion Cys C protein is shown in SEQ ID NO: 3. In the fusion Cys C protein, the first 26 amino acids at the N-terminal of SEQ ID NO: 1 are excised to better promote the soluble expression of Cys C protein. After optimization of synonymous codon preference in Escherichia coli (SEQ ID NO:4), the connection vector was pET-32a(+) (the C-terminal fusion expression (His)6 tag in the vector), provided by Nanjing GenScript Biotechnology Co., Ltd. Ltd Synthetics.

[0090] 2) The r...

Embodiment 2

[0098] Example 2 Purification of a large amount of expression products

[0099] Cultivate 1.5L of bacterial liquid in a shake flask, and the wet weight of bacterial cells collected by centrifugation is 26g. Weigh about 4g of bacteria, add 35ml LysisBuffer and resuspend on ice. After sonication, centrifuge at 20,000 rpm at 4°C for 30 min, take the supernatant, and filter it with a needle filter of 0.22 μm to obtain the filtered bacterial liquid.

[0100] After filtering, pass through Ni-column affinity chromatography, and the protein eluted with 50mM Tris-HCl, 50mM NaCl, 200mM imidazole pH7.0 is the target protein, and 7ml of protein is obtained by elution with a concentration of 2.13mg / ml. The electrophoresis diagram is as follows image 3 shown.

[0101] The calculated target protein expression content is 97mg / L, and the purity can reach 95%.

Embodiment 3

[0102] Embodiment 3 protein stability test

[0103] The obtained target protein was divided into 2mL EP tubes, 1mL / tube, and sealed with parafilm.

[0104] There are 3 tubes in each batch, one of which is placed at 4°C as a control, and the remaining 4 tubes are placed at 37°C for an accelerated one-week test.

[0105] Samples were taken for identification on 3 and 7 days respectively, and the stability of the protein was tested by UNcle (Unchained Lab, United States, UNcle multifunctional protein stability analysis system).

[0106] Figure 4 The results showed that the stability of the recombinant fusion Cys C protein was better after being placed at 37°C for 3 and 7 days, and there was no significant difference with that at 4°C.

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Abstract

The invention discloses a method for preparing recombinant cystatin C. Specifically, gene engineering escherichia coli is adopted for recombinant fusion expression of Cys C protein, a specific tag sequence is fused at the N terminal of the expressed Cys C recombinant protein, so that soluble high-efficiency expression in an escherichia coli system is realized, and the protein is high in stability and has excellent immunological activity.

Description

technical field [0001] The invention belongs to the field of biotechnology. More specifically, it relates to methods for preparing recombinant cystatin C. Background technique [0002] Cystatin C (Cystatin C or Cys C for short), also known as cysteine ​​protease inhibitor C, is a cysteine ​​protease inhibitor. Cystatin C is an ideal endogenous marker to reflect changes in glomerular filtration rate (GFR). At present, cystatin C is recognized as an indicator of renal function at home and abroad. [0003] The traditional method of extracting natural cystatin from placenta, urine and other tissues has problems such as low efficiency, low purity, high purification cost, and unknown potential risks. At the same time, the purification steps are complicated, and it is very difficult to obtain high-purity protein. The polyantiserum prepared by immunizing with this natural protein also has serious crossover with other components in human serum, which brings great difficulties to pr...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N15/62C12N15/70C12N1/21C12P21/02C12R1/19
CPCC07K14/8139C12N15/70C12P21/02C07K2319/21C12N2800/22
Inventor 蒋析文黄黉颜青青况修丽肖兰花
Owner DAAN GENE CO LTD
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