Gene for coding catalase as well as preparation method and application thereof

A catalase and gene technology, applied in the field of gene encoding catalase and its preparation, can solve the problems of difficult to penetrate cell membrane, difficult application, large molecular weight of natural enzyme, etc., and achieves the improvement of catalase activity. Effect

Inactive Publication Date: 2012-07-11
FUZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006]CAT has now become a medicinal enzyme with a wide range of curative effects, but there are still some defects, such as the natural enzyme has a large molecular weight and is difficult to penetrate the cell membrane, poor stability, The short half-life in vivo, immunogenicity, and relatively single function make it difficult to apply clinically, thus limiting its application

Method used

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  • Gene for coding catalase as well as preparation method and application thereof
  • Gene for coding catalase as well as preparation method and application thereof
  • Gene for coding catalase as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Cloning of CAT and CAT-TAT genes

[0041] (1) Isolation and extraction of total RNA:

[0042] (2) Synthesis of the first strand of cDNA:

[0043] Reverse transcription into the first-strand cDNA: refer to the product description of (First-straind cDNA Synthesis KIT), in a 20ul system, add 1ug total RNA, 1ul Oligo dT18 (500ug / ml) and Rnase-free water, at 70℃ water bath temperature After incubating for 10 minutes, immediately place it in an ice bath to cool; continue to add 4ul 5×Buffer, 2ul 0.1M DTT and 1ul 10mM dNTPs to the cooled tube, mix well and keep in a 42℃ water bath for 2min; add 1ul (200U) Keep the reverse transcriptase at 42°C for 50 minutes; finally, place it in a water bath at 70°C for 15 minutes to inactivate the reverse transcriptase to obtain the first strand cDNA. Set up a negative control without RNA during the experiment.

[0044] (3) Get the target gene by PCR

[0045] Design primers based on the human CAT gene sequence published by NCBI: P1 (SEQ ...

Embodiment 2

[0054] Example 2 Expression of CAT-TAT encoding gene in E. coli

[0055] The plasmid pMD-18T-CAT-TAT1 obtained in Example 1-(4) was passed through Nde I and Hind Ⅲ Double enzyme digestion to obtain CAT-TAT fragment, and after Nde I and Hind Ⅲ Connect the double digested pET21a(+) plasmid, transfer the linker into the prepared competent cell DH5ɑ, screen for blue and white spots, and extract the plasmid for identification (the method is the same as in Example 1), to obtain the recombinant plasmid pET21a(+)- CAT-TAT (such as figure 1 Shown in A).

[0056] Take 1ul of the constructed recombinant plasmid pET21a(+)-CAT-TAT, add it to 100ul of prepared competent cells (E. coli Rosetta2(DE3)), spread on LB plate (containing 100ug / ml ampicillin), culture at 37℃ Incubate overnight, pick the positive clones that grow on the resistant plate and insert them into 2ml LB medium (containing 100ug / ml ampicillin), cultivate overnight at 37℃ and 250rpm with shaking, and transfer to 50ml LB cultu...

Embodiment 3

[0057] Example 3 Construction of yeast recombinant expression vector of CAT-TAT encoding gene

[0058] The plasmid pMD-18T-CAT-TAT2 obtained in Example 1-(4) was used SnaB I and Not I Carry out restriction digestion. The digested sample is tested by nucleic acid electrophoresis and the target gene is recovered with a gel recovery kit (Shanghai Shenggong). And Jing SnaB I and Not I double digested pPIC9k plasmid was ligated with T4 ligase in a 16°C water bath for 3 hours, and the ligation was transferred to DH5α (the method is the same as in Example 1). Pick the resistant strains and cultivate, and extract the plasmids. SnaB I and Not I Carry out restriction digestion and nucleic acid electrophoresis detection to obtain the recombinant plasmid pPIC9k-CAT-TAT (such as figure 1 Shown in B).

[0059] Using the obtained pPIC9k-CAT-TAT recombinant plasmid as a template, PCR amplification was performed with CAT-TAT specific primers P3 and P4, and the amplification procedure was...

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Abstract

The invention belongs to the biotechnical field and particularly relates to a gene for coding catalase (CAT) as well as a preparation method and an application of the gene. The gene for coding catalase has a nucleotide sequence as shown in SEQ NO.1. The invention relates to a coding sequence of CAT-TAT (catalase-tetanus antitoxin) cloned from human liver cells. The codling sequence is obtained by the steps of firstly extracting total RNA (ribonucleic acid) of human liver cells L-02, then cloning to a full-length sequence and a CAT-TAT fusion gene for coding CAT by RT-PCR (reverse transcription-polymerase chain reaction) and PCR methods. A CAT-TAT protein is obtained by using the gene expression, the production industrialization of CAT-TAT is realized, CAT-TAT products with good stability are achieved, and the activity of catalase in cells is improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and more specifically relates to a gene encoding catalase and its preparation method and application. Background technique [0002] Catalase (CAT) is a protein scavenger, and it is a conjugated enzyme with iron porphyrin as a prosthetic group. It can promote H 2 o 2 Decomposes into molecular oxygen and water to remove hydrogen peroxide in the body, thus protecting cells from H 2 o 2 It is one of the key enzymes in the biological defense system. The scavenging of oxygen free radicals can be divided into two steps: the first step is that the superoxide anion, which is a harmful substance, reacts with hydrogen ions under the action of SOD to generate H 2 o 2 ; The second step is H 2 o 2 Under the action of catalase, it reacts with hydrogen ions, and finally converts H 2 o 2 degraded into H 2 O and O 2 . In fact, SOD, GSH, vitamin E, CAT, etc. are a mutual protection defense group. When the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N9/08C12N15/63C12N1/21C12N1/19C12N15/10A61K38/44A61K47/48C12R1/19C12R1/84
Inventor 饶平凡李仁宽刘树滔
Owner FUZHOU UNIV
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