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Cholesterol esterase and its nucleotide sequence, recombinant vector, recombinant host cell, preparation method and kit

A technology of recombining host cells and cholesterol esterase, applied in the direction of recombinant DNA technology, using vectors to introduce foreign genetic material, bacteria, etc., can solve problems such as poor heat resistance, and achieve good heat resistance.

Active Publication Date: 2012-01-18
BEIJING LEADMAN BIOCHEM
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] An object of the present invention is to overcome the shortcoming of poor heat resistance of existing cholesterol esterases, and provide a cholesterol esterase with good heat resistance and high stability and activity in a wide temperature range

Method used

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  • Cholesterol esterase and its nucleotide sequence, recombinant vector, recombinant host cell, preparation method and kit
  • Cholesterol esterase and its nucleotide sequence, recombinant vector, recombinant host cell, preparation method and kit
  • Cholesterol esterase and its nucleotide sequence, recombinant vector, recombinant host cell, preparation method and kit

Examples

Experimental program
Comparison scheme
Effect test

preparation Embodiment 1

[0051] A) Obtaining the existing cholesterol esterase COE gene (that is, obtaining the nucleotide sequence shown in sequence 3)

[0052] A-1) Culture of Streptomyces venezuelae

[0053] Prepare LB medium:

[0054] Peptone: 10g / L

[0055] Yeast powder: 5g / L

[0056] NaCl: 10g / L

[0057] Sterilize at 121°C for 20 minutes.

[0058] Inoculate the Streptomyces venezuelae strain in the frozen tube into LB medium with an inoculation needle, and culture overnight at 26°C.

[0059] A-2) Extraction of Genomic DNA

[0060] Take the overnight culture obtained in step 1), centrifuge at 8000 rpm for 5 min at room temperature, discard the supernatant, and resuspend the pellet in 1 ml TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Add 6 μl of 50 mg / ml lysozyme, act at 37°C for 2 hours, then add 50 μl of 2mol / L NaCl, 110 μl of 10% SDS, 3 μl of 20 mg / ml proteinase K, and act at 50°C for 15 minutes. Afterwards, the obtained bacterial solution was divided into 10ml centrifuge tubes, and an equa...

preparation Embodiment 2-5

[0132] Prepare reagents according to the formula in Table 2:

[0133] Table 2

[0134]

[0135] The reagents of Preparation Examples 5 and 6 can be prepared in liquid form respectively. The reagents of Preparation Examples 7 and 8 were respectively freeze-dried under the following conditions: frozen at -10°C for 2 hours in a common refrigerator, then pre-frozen in a deep-freezing refrigerator at -40°C for 8 hours, at CHRIST, Germany In the ALPHA 1-4LSC type freeze dryer, freeze-dry for 10 hours under the conditions of vacuum degree 0.04mbar, safety pressure 0.100mbar and cold trap temperature -60°C. Then wait until the temperature difference between the material temperature and the freeze-dryer partition is zero, and observe that the pressure indication remains unchanged within 15 seconds, then end the freeze-drying. The dry powder of the reagent obtained by lyophilization can be reconstituted with distilled water to the volume of the original liquid form before use. The...

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Abstract

The invention discloses a cholesterol esterase. An amino acid sequence of the cholesterol esterase is an amino acid sequence shown in the formula of SEQ ID NO: 1, or is an amino acid sequence which is obtained by processes of deletion, addition and / or replacement of one or more amino acids on the amino acid sequence shown in the formula of SEQ ID NO: 1 and maintains original cholesterol esterase functions. The invention also discloses a nucleotide sequence for coding the cholesterol esterase, a recombinant vector of the nucleotide sequence, a recombinant host cell of the recombinant vector, amethod for purifying the cholesterol esterase from the recombinant host cell, and a kit containing at least one of the cholesterol esterase, the nucleotide sequence, the recombinant vector and the recombinant host cell. The cholesterol esterase has good heat resistance and high stability. The kit can carry out rapid, effective and accurate detection on genes to guarantee prompt case diagnosis andtreatment, accurate aetiology investigation, and establishment of scientific prevention and control policies.

Description

technical field [0001] The present invention relates to an enzyme, a nucleotide sequence encoding the enzyme, a recombinant vector comprising the nucleotide sequence, a recombinant host cell comprising the recombinant vector, a method for purifying the enzyme from the aforementioned recombinant host cell, and A kit comprising at least one of the above enzymes, nucleotide sequences, recombinant vectors and recombinant host cells. More specifically, the present invention relates to a cholesterol esterase, a nucleotide sequence encoding the cholesterol esterase, a recombinant vector comprising the nucleotide sequence, a recombinant host cell comprising the recombinant vector, and the aforementioned recombinant host cell A method for purifying the enzyme, and a kit including at least one of the above-mentioned cholesterol esterase, nucleotide sequence, recombinant vector and recombinant host cell. Background technique [0002] Cholesterol esterase is an enzyme that catalyzes th...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/18C12N15/55C12N15/63C12N1/21C12Q1/68C12Q1/60C12Q1/44
Inventor 不公告发明人
Owner BEIJING LEADMAN BIOCHEM
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