ck-mb fusion protein and its preparation method and detection kit

A fusion protein, CK-M technology, applied in biochemical equipment and methods, chemical equipment and methods, hybrid peptides, etc., can solve the problems of multiple inclusion bodies and poor activity, and achieve high sensitivity, stable labeled products, reliable results

Active Publication Date: 2021-03-26
郑忠亮
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0016] Another object of the present invention is to provide a method for obtaining highly active CK-MB recombinant protein for the existing genetic engineering method to prepare CK-MB recombinant protein with many inclusion bodies and poor activity

Method used

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  • ck-mb fusion protein and its preparation method and detection kit
  • ck-mb fusion protein and its preparation method and detection kit
  • ck-mb fusion protein and its preparation method and detection kit

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Embodiment 1: Cloning, expression and purification of CK-MB fusion protein

[0051] ① Synthesize the whole gene sequence of "CKM-Connection Loop-CKB" according to the direction from the N-terminal to the C-terminal of the amino acid sequence. Among them, the gene sequence and protein sequence of CK-M, connecting Loop and CK-B are as follows:

[0052] CK-M amino acid sequence: SEQ ID NO.3;

[0053] CK-M coding gene sequence: SEQ ID NO.4;

[0054] Loop amino acid sequence: SEQ ID NO.1;

[0055] Loop coding gene sequence: SEQ ID NO.5;

[0056] CK-B amino acid sequence: SEQ ID NO.6;

[0057] CK-B coding gene sequence: SEQ ID NO.7.

[0058] ②Clone the "CKM-Joint Loop-CKB" gene sequence into the multiple cloning insertion sites of BamH I and XholI of the pET28a plasmid expression vector.

[0059] ③ The cloned expression plasmid pET28a-CKMB was transformed into Escherichia coli BL21(DE3), plated and cultured overnight.

[0060] ④ The next morning, pick a single colony a...

Embodiment 2

[0063] Embodiment 2: ELISA quantitative detection of CK-MB fusion protein

[0064] ①The total protein content of the purified CK-MB fusion protein was measured using the BCA protein content assay kit produced by Thermoelectric Corporation, and the three batches of purified proteins were detected and error analysis was performed. The results are shown in Table 1.

[0065] ②Use the CK-MB ELISA kit produced by CloneTech to detect the active protein content of CK-MB fusion protein. The above three batches of purified proteins were tested and analyzed for errors. The results are shown in Table 1.

[0066] Table 1. Determination of total protein content by BCA method and determination of CK-MB fusion protein by ELISA method

[0067] white content

[0068]

Embodiment 3

[0069] Embodiment 3: the preparation of immunochromatography kit

[0070] (1) Fluorescent microsphere-labeled antibody:

[0071] ① Take 0.5mL rare earth fluorescent microspheres, centrifuge to get the precipitate, add pH 6.0 MES buffer to wash twice.

[0072] ②Activation: 114 μL of 50 mM carbodiimide (EDAC) and 114 μL of 50 mM hydroxysulfosuccinimide (Sulfo-NHS) were added to the washed microspheres respectively, and shaken at room temperature for 1 h.

[0073] ③ Centrifuge the microspheres and wash twice with PB buffer.

[0074] ④ Add an appropriate amount of mouse-derived CK-MB monoclonal antibody and shake at room temperature for 2 hours.

[0075] ⑤ Centrifuge the microspheres, add 50mM hydroxylamine buffer to quench the reaction, shake at room temperature for 5min; then centrifuge the microspheres, add 50mM hydroxylamine buffer to completely quench the reaction, shake at room temperature for 30min

[0076] ⑥ Centrifuge the microspheres, add blocking solution (0.5% casei...

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Abstract

The invention discloses a CK-MB fusion protein, a preparation method thereof, and a detection kit. The fusion protein is formed by connecting a CK-M protein and a CK-B protein through Loop. The functions of the fusion protein are the same as those of natural CK-MB protein. The preparation method comprises the following steps: coupling the coding gene sequences of human CK-M and CK-B by nucleotidebases of Loop, cloning the coupled coding gene sequences to an expression vector to obtain expression plasmids, transferring the expression plasmids to escherichia coli BL21(DE3), carrying out IPTG inducible expression, subjecting the cultured product to ultrasonic cell wall breaking, carrying out centrifugation, collecting the supernate, and finally making the supernate go through a nickel column, a strong anion exchange column Q column, and a Superdex G200 molecular sieve chromatographic column to obtain high purity protein having an active protein content of 85%. Rare earth fluorescence microspheres are taken as the labeling carrier to prepare a kit for detecting CK-MB, and the detection kit has the characteristics of stability, sensitiveness, simpleness, quantitative detection, rapidness, and reliable results.

Description

technical field [0001] The invention relates to CK-MB fusion protein, its preparation method and detection kit. Specifically, it relates to the genetic engineering expression of a new creatine kinase MB type (CK-MB) standard product and its time-resolved fluorescence quantitative detection kit. Background technique [0002] Creatine Kinase (Creatine Kinase, CK) usually exists in the cytoplasm and mitochondria of animal heart, muscle, and brain tissues. It is an important kinase that is directly related to intracellular energy operation, muscle contraction, and ATP regeneration. It is reversible It catalyzes the transphosphoryl reaction between creatine and ATP. Creatine kinase has four isozyme forms: muscle (MM), brain (BB), hybrid (MB) and mitochondrial (MiMi). The MM type mainly exists in various muscle cells, the BB type mainly exists in brain cells, the MB type mainly exists in cardiomyocytes, and the MiMi type mainly exists in myocardial and skeletal muscle mitochondr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K19/00C12N9/12C12N15/62C12N15/70G01N33/543G01N33/573
CPCC12N9/1223C12N15/70C12Y207/03002G01N33/54313G01N33/573
Inventor 郑忠亮
Owner 郑忠亮
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