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Recombined human cystatin-C protein with natural activity and preparation method thereof

A cystatin and protein technology, which is applied in the field of recombinant human cystatin C protein and its preparation, can solve the problems of large batch-to-batch variation, low cystatin C protein concentration and high purification cost

Active Publication Date: 2013-04-03
深圳前海菲鹏基因科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The reason is that the preparation of cystatin C diagnostic kit requires highly specific and sensitive cystatin C monoclonal antibody or polyantiserum, and high-purity cystatin C must be obtained in order to obtain the monoclonal antibody or polyantiserum. Cystatin C protein, the traditional 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 or polyclonal antibodies, even if it is used as an immunogen to prepare Diagnostic products used in methods such as immunoturbidimetry also have serious crossover problems. To extract high-purity immunogens, the purification cost is quite high

Method used

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  • Recombined human cystatin-C protein with natural activity and preparation method thereof
  • Recombined human cystatin-C protein with natural activity and preparation method thereof
  • Recombined human cystatin-C protein with natural activity and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1 Preparation of recombinant human cystatin C protein

[0039] (1) Artificially synthesize human cystatin C gene and construct expression plasmid

[0040] According to the reported cystatin C gene sequence, overlapping PCR primers were designed to artificially synthesize the human cystatin C gene, the sequence of which is set forth in SEQ ID NO:1. The gene was connected with the pMD18-T vector, transformed into Escherichia coli, a single clone was picked, the correct positive plasmid was identified by PCR, and the plasmid was extracted and identified by enzyme digestion. The plasmids with correct PCR identification and enzyme digestion identification were sent to Invagen Company for sequencing, and the results were completely consistent with the designed sequence. The plasmid with correct sequencing was digested with the enzymes corresponding to the enzyme cutting sites designed at both ends of the plasmid, and the excised target sequence was connected into the...

Embodiment 2

[0056] Example 2 The mass ratio of trypsin is 50:1 to digest recombinant human cystatin C protein

[0057] The undigested recombinant human cystatin C protein prepared in Example 1 was added with recombinant trypsin at a mass ratio of recombinant cystatin C:trypsin of 50:1, and digested in a water bath at 37° C. for 5 min. After digesting the sample, adjust the pH to 9.5 with 0.5M NaOH, and place it at 4°C for 1 hour to completely inactivate trypsin. After digestion, the protein was dialyzed with 20mM PBS overnight, bound on a nickel agarose gel FF column for 1 hour, the excised part with his tag and residual recombinant trypsin were removed by reverse affinity, and the flow-through protein sample after binding was collected. It is the final purified recombinant human cystatin C protein, and the SDS-PAGE electrophoresis analysis shows that the protein purity is over 95%. Activity was evaluated according to step (5) in Example 1.

Embodiment 3

[0058] Example 3 The mass ratio of trypsin is 1000:1 to digest recombinant human cystatin C protein

[0059] The undigested recombinant human cystatin C protein prepared in Example 1 was added with recombinant trypsin at a mass ratio of recombinant cystatin C:trypsin of 1000:1, and digested in a water bath at 37° C. for 30 min. After digesting the sample, adjust the pH to 9.5 with 0.5M NaOH, and place it at 4°C for 1 hour to completely inactivate trypsin. After digestion, the protein was dialyzed with 20mM PBS overnight, bound on a nickel agarose gel FF column for 1 hour, the excised part with his tag and residual recombinant trypsin were removed by reverse affinity, and the flow-through protein sample after binding was collected. It is the final purified recombinant human cystatin C protein, and the SDS-PAGE electrophoresis analysis shows that the protein purity is over 95%. Activity was evaluated according to step (5) in Example 1.

[0060] Table 2: Comparison of enzyme cl...

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Abstract

The invention discloses a recombined human cystatin-C protein with natural activity and a preparation method thereof. According to the preparation method, lots of stable recombined human cystatin-C proteins are expressed and purified by employing a gene engineering technique, and the recombined human cystatin-C proteins are cut by utilizing the protease, so that the recombined human cystatin-C protein has high natural activity. The recombined human cystatin-C protein can be used for immune preparation of monoclonal antibodies and multi-antibody blood serum, and excellent raw materials are provided for preparing stable cystatin-C detection agents and quality control products thereof.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a recombinant human cystatin C protein with natural activity and a preparation method thereof. Background technique [0002] Cystatin C (Cystatin C, CysC), also known as cystatin C, has a molecular weight of 13kD and contains 120 amino acid residues. It is a non-glycosylated oval small molecular protein, and its precursor The molecule contains a signal peptide of 26 amino acids. Cystatin C is widely present in various body fluids such as blood, cerebrospinal fluid, saliva, semen, and urine, with the highest content in cerebrospinal fluid and the lowest in urine, and all nucleated cells can stably produce cystatin C. Under physiological conditions, an important function of cystatin C is to inhibit the activity of endogenous cysteine ​​proteases. It plays an important role in the conversion of intracellular proteins, the degradation of collagen, and the separation of prot...

Claims

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Application Information

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IPC IPC(8): C12N15/70C07K14/81C07K16/38G01N33/68C12R1/19
Inventor 崔鹏李泓彦何志强胡鹏杨耿周范凌云
Owner 深圳前海菲鹏基因科技有限公司
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