Recombinant cystatin C protein and application of recombinant cystatin C protein to detection kit

A cystatin and reagent kit technology, applied in the field of medical immunology in vitro diagnosis, can solve the problems of hindering widespread use, cumbersome operation, high cost, etc., and achieve the effect of low cost, large amount of expression, and high accuracy

Pending Publication Date: 2019-07-05
ZONHON BIOPHARMA INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The disadvantages of the first two methods, such as high cost, cumbersome operation, and long cycle, have hindered their widespread clinical use. With the popularization of automatic biochemical analyzers, biochemical turbidimetry has short reaction time, good precision, and is easy to use. With advantages such as automation, it has become the mainstream detection method in clinical practice.

Method used

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  • Recombinant cystatin C protein and application of recombinant cystatin C protein to detection kit
  • Recombinant cystatin C protein and application of recombinant cystatin C protein to detection kit
  • Recombinant cystatin C protein and application of recombinant cystatin C protein to detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1 Preparation of rhCysC protein

[0040] 1. Construction of expression vector of rhCysC gene

[0041] According to the cDNA sequence of hCysC published by NCBI (GenBank accession number: NM_000099.3), the inventor obtained the rhCysC gene of the present invention after codon optimization of the gene, as shown in SEQ ID No:2.

[0042] The optimized rhCysC whole gene was introduced into the NcoI restriction site sequence at the 5' end, and the BamHI restriction site sequence was introduced at the 3' end, and the whole gene was synthesized, and the synthetic gene fragment was constructed into the pUC57 plasmid (provided by Nanjing Provided by GenScript Technology Co., Ltd.), a long-term storage plasmid was obtained, which was designated as pUC57-rhCysC plasmid. The pUC57-rhCysC plasmid was used as a template for PCR amplification, and the primer sequences used were as follows:

[0043] Upstream primers:

[0044] M13F: CGC CAG GGT TTT CCC AGT CAC GAC

[0045] ...

Embodiment 2

[0078] Example 2 Preparation of rabbit polyclonal antibodies

[0079] 1. Three days before immunization, blood was collected from the ear vein as a negative control (500ul per mouse, left at 37°C for 1 hour, centrifuged at 10,000rpm for 15 minutes, absorbed serum and stored at -20°C);

[0080] 2. After 3 days, mix rhCysC (1 mg / rabbit) with complete Freund's adjuvant in an equal volume to form a "water-in-oil" form, and inject it subcutaneously at multiple points on both sides of the rabbit's spine;

[0081] 3. The second immunization after 2 weeks: mix rhCysC (1 mg / rabbit) with incomplete Freund's adjuvant in an equal volume to form a "water-in-oil" form, and inject subcutaneously on both sides of the spine (injection sites should be distinguished For the first time); one week after the immunization, blood was collected from the ear vein (the blood processing method is the same as step 1);

[0082] 4. The third immunization after 2 weeks: mix rhCysC (1 mg / rabbit) with an equa...

Embodiment 3

[0089] Example 3 Preparation and use of the detection kit

[0090] The detection kit of the present invention is a liquid double reagent, which includes reagent R1, reagent R2 and calibrator, see below for details:

[0091] 1. Reagent R1: Add stabilizer (for example: NaCl), coagulant (for example: PEG6000), protective agent (for example: BSA), preservative (for example: Proclin 300), chelating agent (for example: EDTA), stirred and mixed, that is the R1 reagent.

[0092] 2. R2 reagent: Dilute the rabbit anti-human cystatin C polyclonal antibody to 2 mg / mL with MES buffer to prepare antibody diluent; use MES buffer to dilute latex microspheres (polystyrene latex, purchased from Japan JSR company, particle size 100-400nm) is diluted to 1% (mass concentration), then add 0.5%-2% (mass concentration) of EDC, stir and react at 30°C for 30min, remove unreacted by centrifugation after the reaction EDC, then add rabbit anti-human cystatin C polyclonal antibody dilution, stir and reac...

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Abstract

The invention relates to a recombinant cystatin C protein (rhCysC) and the application of the recombinant cystatin C protein to a detection kit for detecting serum cystatin C (CysC) and belongs to thefield of in vitro diagnosis of medical immunology. According to the recombinant cystatin C protein (rhCysC) and the application of the recombinant cystatin C protein of the invention, the gene sequence of coded rhCysC, which is obtained through specially carrying out codon optimization on an escherichia coli expression system, is provided, wherein the sequence is as shown as SEQ ID NO: 2. With the sequence transferred, the expression efficiency of heterologous genes in a host bacterium can be remarkably improved. The recombinant expression method of the rhCysC has the characteristics of shortperiod, large expression quantity and low cost; the rhCysC prepared by the method can be used as an effective immunogen for the preparation of rabbit polyclonal antibodies. With the polyclonal antibodies applied to the preparation of a latex-enhanced immunoturbidimetric detection kit, the content of CysC in serum can be accurately detected. Compared with a commercially available product, the recombinant cystatin C protein (rhCysC) has better repeatability and higher accuracy, and shows a good clinical application prospect.

Description

technical field [0001] The invention belongs to the field of in vitro diagnosis of medical immunity, in particular to a recombinant cystatin C protein and its application in a detection kit for detecting the content of cystatin C (CysC) in serum. Background technique [0002] Cystatin C is a positively charged basic secretory protein with a molecular weight of 13KD, widely present in various body fluids, such as semen, saliva, urine, etc., without tissue specificity. Cystatin C can be freely filtered from the glomerulus. After being reabsorbed in the proximal convoluted tubule, it is completely metabolized and decomposed by the epithelial cells and does not return to the blood. Therefore, the kidney is the only organ that clears CysC in the circulation. The glomerulus Filtration rate (GFR) determines the concentration of CysC in serum. The generation of CysC is not affected by age, gender, body weight, inflammation and other factors, and it is an ideal endogenous marker to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/68G01N33/96G01N33/543
CPCG01N33/6893G01N33/96G01N33/54313G01N2333/8139G01N2800/347
Inventor 马永杨芸赵利利王安良
Owner ZONHON BIOPHARMA INST
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