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Construction and expression, separation and purification method of recombinant vector containing reindeer NADPH-cytochrome P450 reductase gene

A technology of cytochrome and recombinant vector, applied in the field of genetic engineering

Inactive Publication Date: 2018-12-11
嘉兴欣贝莱生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The NADPH-cytochrome P450 reductase of reindeer has a relatively high catalytic activity, but it has not been reported so far

Method used

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  • Construction and expression, separation and purification method of recombinant vector containing reindeer NADPH-cytochrome P450 reductase gene
  • Construction and expression, separation and purification method of recombinant vector containing reindeer NADPH-cytochrome P450 reductase gene
  • Construction and expression, separation and purification method of recombinant vector containing reindeer NADPH-cytochrome P450 reductase gene

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Embodiment 1 plasmid construction process:

[0020] According to the requirements of gibson technology, the primers needed in the present invention are designed (the 5F end of the primers has a homology arm of about 20-40bp), and the gene and the pET-28a carrier optimized according to the codon preference of Escherichia coli are linearized by PCR technology respectively. Then, add the linearized gene and vector into the gibson system, incubate, connect, transform E. coli DH5α, carry out plasmid amplification, pick a single clone, verify, and save the positive clone for future use.

Embodiment 2

[0021] Embodiment 2 expression vector transformation process:

[0022] Extract the plasmid from the above positive clone, take 200ng of the plasmid, mix it with 50μL of commercial Escherichia coli competent BL21(DE3), incubate on ice for 30min, then heat shock at 42°C for 45S, place it on ice for 2min, and then add 300μL of LB The culture medium was revived at 37°C for 1 hour, and spread on LB solid plate medium with kanamycin resistance (pET28a plasmid is kanamycin resistance). Cultivate overnight, pick a single clone for verification, and keep positive clones for future use.

Embodiment 3

[0023] Expression and purification of embodiment 3 protein:

[0024] 1) Seed liquid culture: Pick the above-mentioned BL21(DE3) strain containing the target plasmid, inoculate it into a small test tube containing 5 mL of LB liquid medium (Kan+, 100 μg / mL), and culture overnight at 37°C and 220 rpm as the seed liquid.

[0025] 2) Transplant: transfer the seed liquid into 800mL 2YT liquid medium (Kan+, 100μg / mL) at 1% inoculum amount, culture on a shaker at 37°C, 220rpm (about 4-6h) until the OD 600 is about 0.6- 0.8.

[0026] 3) Induction: lower the temperature of the shaker to 16°C, and after the temperature of the cultured bacterial solution is lowered, add isopropylthio-β-D-galactoside (IPTG) to a final concentration of 0.5mM to induce the expression of 14 -16h.

[0027] 4) Bacteria collection: After the expression is completed, collect the cultured bacteria solution into a bottle, pre-cool the centrifuge to 4° C., and centrifuge at 6000 rpm for 30 minutes.

[0028] 5) Cl...

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Abstract

The invention belongs to the technical field of genetic engineering, in particular to construction of a recombinant vector containing a reindeer NADPH-cytochrome P450 reductase gene and a method for expression, separation, purification and activity assay. The method comprises the following steps: 1) according to the NADPH-cytochrome P450 reductase gene annotated from the genome of a reindeer and E. coli codon preference, the optimized reindeer NADPH-cytochrome P450 reductase gene sequence is obtained, and the NADPH-cytochrome P450 reductase gene sequences of roe deer and sheep optimized according to the codon preference of E. coli are obtained to be as a control; and 2) the optimized reindeer, roe deer and sheep 25 hydroxyvitamin D-1 alpha hydroxylase gene are constructed into E. coli-expressed vector pET-28a by gibson connection method using the designed primers to obtain a recombinant vector. The catalytic activity of the strain containing the reindeer NADPH-cytochrome P450 reductasegene is 20 times that of the sheep and 5 times that of the roe deer.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a recombinant vector containing a reindeer NADPH-cytochrome P450 reductase gene and methods for expressing, separating and purifying the encoded protein and measuring its activity. Background technique [0002] NADPH-cytochrome P450 reductase (NADPH-cytochrome P450 reductase, CPR) is a cell microsomal complex flavoprotein with FAD and FMN as prosthetic groups, which can catalyze the generation of reduced cytochrome C from oxidized cytochrome C (see appendix figure 1 ), which is mainly distributed in the liver in the human body, with a molecular weight of about 78KD, and is an important component of the liver microsomal mixed function oxidase-cytochrome P450 enzyme system. Cytochrome P450 (cytochrome P450 or CYP450, referred to as CYP450) represents a large family of heme proteins that can be oxidized by itself, and belongs to the class of monooxygenases. I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N9/02
CPCC12N15/70C12N9/0042C12N2800/22C12Y106/02004
Inventor 陈贤情杨月林泽山王筱夏文豪蒿飞
Owner 嘉兴欣贝莱生物科技有限公司
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