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DNA sequence for expressing 3 alpha-hydroxysteroid dehydrogenase in Pseudomonas aeruginosa

A technology of hydroxysteroid and DNA sequence, applied in the field of DNA sequence of polypeptide chain and its complementary chain sequence, can solve problems such as high cost, and achieve the effect of high activity and cost reduction

Inactive Publication Date: 2015-03-04
SICHUAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these assays require expensive 3α-hydroxysteriod dehydrogenase (3α-HSD)
Expensive enzyme reagents are the main factor affecting the popularization and application of these SBAs detection techniques

Method used

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  • DNA sequence for expressing 3 alpha-hydroxysteroid dehydrogenase in Pseudomonas aeruginosa
  • DNA sequence for expressing 3 alpha-hydroxysteroid dehydrogenase in Pseudomonas aeruginosa
  • DNA sequence for expressing 3 alpha-hydroxysteroid dehydrogenase in Pseudomonas aeruginosa

Examples

Experimental program
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Effect test

Embodiment 1

[0053] This embodiment is to clone Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) 3α-Hydroxysteroid dehydrogenase 3-HSDA gene fragment, cloned from Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) The 3α-hydroxysteroid dehydrogenase 3-HSDA gene fragment was sequenced and identified to be 1134 bp in length.

Embodiment 2

[0055] The present embodiment is Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) The 3α-hydroxysteroid dehydrogenase 3-HSDA gene fragment is recombined with the pET30b(+) expression vector to form the pET30b(+)-hsdA recombinant vector. The steps are as follows:

[0056] A. Pseudomonas aeruginosa ( Pseudomonas aeruginosa ), the size of the original clone of its 3α-hydroxysteroid dehydrogenase gene 3-hsdA is 1134bp, and its polymerase chain reaction (polymerase chain reaction, PCR) system is as follows:

[0057]

[0058] After mixing, carry out PCR reaction according to the following reaction conditions.

[0059]

[0060] Detected by 1% agarose gel electrophoresis, and cut the gel to recover the target fragment.

[0061]

[0062] B. Single digestion of cloning vector pUC19

[0063] The size of the cloning vector pUC19 used for the 3α-hydroxysteroid dehydrogenase gene 3-hsdA is 2686 bp, pUC19 was digested with SmaⅠ, and the enzyme digestion reaction system was as foll...

Embodiment 3

[0081] In this embodiment, after pET30b(+)-hsdA recombinant is transformed into Escherichia coli BL21(DE3), the experimental steps for obtaining BL21(DE3) engineering bacteria of 3α-hydroxysteroid dehydrogenase through screening with kanamycin are as follows:

[0082] Get through kanamycin selection, pET30b (+)-hsdA recombinant vector transforms Escherichia coli BL21 (DE3) the single bacterium colony that forms in the culture medium, inoculate in the 5mlLB culture fluid that contains 50μg / ml kanamycin, 37 Shake culture at ℃ overnight; take 1ml of the culture solution, and extract the plasmid according to the "Plasmid Small Extraction Kit Instructions" of Tiangen Biochemical Technology (Beijing) Co., Ltd.; take 10μl of plasmid DNA, add 4μl of distilled water, 2μl of buffer, 2μl 0.1%BSA and 1μl NcoⅠ, 1μl Eco RⅠ After the enzyme was mixed, it was placed in a water bath at 37°C and incubated for 5 hours; the recombinant DNA and the digested plasmid DNA were electrophoresed at th...

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Abstract

The invention discloses a DNA sequence of a polypeptide chain with 3 alpha-HSD (3 alpha-hydroxysteroid dehydrogenase) activity. The DNA sequence comprises a complementary chain sequence. The DNA sequence is characterized in that an expression product of the DNA sequence has 3 alpha-hydroxysteroid dehydrogenase activity and is a novel Pseudomonas aeruginosa 3 alpha-hydroxysteroid dehydrogenase. The DNA sequence comprises the following contents: a pseudomonas aeruginosa 3 alpha-hydroxysteriod dehydrogenase gene sequence obtained by utilizing bioinformatics analysis, a recombinant expression vector which contains the pseudomonas aeruginosa 3 alpha-hydroxysteroid dehydrogenase gene sequence, a genetically engineered bacterium which expresses the 3 alpha-hydroxysteroid dehydrogenase 3-hsdA and an enzyme kinetics parameter of the 3 alpha-hydroxysteroid dehydrogenase.

Description

technical field [0001] The present invention relates to a DNA sequence of a polypeptide chain with 3α-hydroxysteroid dehydrogenase (3α-hydroxysteriod dehydrogenase, 3α-HSD) activity, including its complementary chain sequence. It is characterized in that the expression product of the DNA sequence has 3α-hydroxysteroid dehydrogenase activity, and is a new type of Pseudomonas aeruginosa ( Pseudomonas aeruginosa ) 3α-hydroxysteroid dehydrogenase. The enzymatic property of the protein is identified for the first time, has the practicality of being applied to the clinical detection of sulfonated bile acids, and belongs to the field of bioengineering. Background technique [0002] 3α-hydroxysteroid dehydrogenase (3α-hydroxysteriod dehydrogenase, 3α-HSD) is a class of oxidoreductase widely present in mammalian cells and prokaryotic cells. Bacterial 3α-HSD is an indispensable enzyme reagent in the quantitative analysis of bile acids (BAs), sulfated bile acids (SBAs) and androstero...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/04C12N15/63C12Q1/32
Inventor 高秀峰陈建敏李永生
Owner SICHUAN UNIV
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