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Method for obtaining human copper and zinc superoxide dismutase genes from human body

A technology of superoxide and human tissue, applied in the biological field, can solve the problems of difficulty in obtaining, high cost, and difficulty, and achieve the effect of clear amplification bands and low cost

Inactive Publication Date: 2004-04-21
JIANNAN RESISTANCE GENE XIAMEN CITY
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  • Abstract
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  • Claims
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Problems solved by technology

This method is difficult and expensive
The other is to extract total RNA from healthy human liver tissue with guanidine isothiocyanate / phenol / chloroform, and the cDNA obtained by reverse transcription of RNA by AMV reverse transcriptase is used as a template for PCR amplification. After 30 PCR thermal cycles, Amplified hCu, Zn-SOD cDNA (Xiang Hua, Wei Wenzhong, Tan Huarong et al. Cloning of human copper zinc superoxide dismutase and expression in Lactococcus lactis. Acta Biological Engineering, 2000, 16(1): 6~9), but healthy human liver tissue is difficult to obtain

Method used

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  • Method for obtaining human copper and zinc superoxide dismutase genes from human body
  • Method for obtaining human copper and zinc superoxide dismutase genes from human body
  • Method for obtaining human copper and zinc superoxide dismutase genes from human body

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preparation example Construction

[0027] 2) Preparation of gel:

[0028] Weigh 0.187g of agar powder into a treated alkane, add 12.5ml of DEPC-treated water, dissolve, cool to 60°C, add 3.6ml of 37% formaldehyde and 4.0ml of 5-fold formaldehyde gel electrophoresis buffer, mix ,Glue.

[0029] 3) Processing of samples:

[0030] Mix the reagents in the following proportions:

[0031] RNA 4.5μl

[0032] 5x formaldehyde gel electrophoresis buffer 2.0μl

[0033] Formaldehyde 3.5μl

[0034] Formamide 10.0μl

[0035] Total 20.0μl

[0036] Collect by centrifugation, take a water bath at 65°C for 15 minutes, cool in an ice bath, and collect by centrifugation.

[0037] 4) Electrophoresis:

[0038] 50V pre-electrophoresis for 5min, take 10μl of the treated sample and 1μl of loading buffer, dip the tip of the sampling pipette with a little ethidium bromide, apply the sample, and electrophoresis at 40V for 2 to 3 hours.

[0039] 5) Gel imaging analysis system scanning analysis:

[0040] fi...

Embodiment 2

[0143] Its method step is similar to embodiment 1. In the extraction of total RNA, the human embryo tissue just produced was quickly put into a liquid nitrogen container, and 100 mg of fresh human embryo tissue was taken for the water phase, and quickly put into a glass homogenizer. Move the tissue homogenate to a centrifuge tube and place it at room temperature for 5 minutes; add 0.2ml chloroform to the tissue homogenate, shake vigorously for 15 seconds, and place it at room temperature for 3 minutes; centrifuge at 15,000g for 20 minutes at 4°C, then transfer to another centrifuge tube, and Add 0.5ml of isopropanol, let stand at room temperature for 10min; centrifuge at 12000g for 15min at 4°C. Discard the supernatant, add 1ml 75% ethanol, shake and wash; centrifuge at 10000g at 4°C for 8min. After adding DEPC-treated water and pumping, place in a water bath at 55°C for 12 minutes.

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Abstract

A process for extracting the human Cu, Zn superoxide dismutase (hCu, Zn-SOD) gene from human tissue includes such steps as extracting the common RNA from human embryo tissue, and reverse transcription-PCR for amplifying cDNA of hCu, Zn-SOD. Its advantages are simple process, low cost, and high sequence homology up to 99.8%.

Description

(1) Technical field [0001] The invention relates to a biotechnology, in particular to a method for extracting human copper-zinc superoxide dismutase gene from human tissue. (2) Background technology [0002] Superoxide dismutase (Superoxide dismutase, SOD) is a specific scavenger for superoxide anion free radicals, and plays an important role in maintaining the dynamic balance of free radicals in organisms (Kushleika J.Checkoway H.Woods J.S.et al. Ann Neurol, 1996, 39(3): 378-381; Noda J. Otagiri M. Akaike T et al. J Pharmacol Exp Ther, 1996, 279(1): 162-171). SOD has broad application prospects in medical treatment, health care, food and other aspects. [0003] The research on recombinant human SOD can solve the problem of the source of human SOD on the one hand, and on the other hand, can avoid the safety problems that may be caused by the application of non-human SOD. At present, there are mainly two methods for obtaining hCu, Zn-SOD genes: one is artificial synthesis (...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/34
Inventor 刘仁海章军周克夫徐虹楼士林
Owner JIANNAN RESISTANCE GENE XIAMEN CITY
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