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cDNA sequence coding fibrinolysin, amino acid sequence and applications of sequences

A fibrinolytic and amino acid technology, applied in the fields of biotechnology and biomedicine, can solve the problems of not disclosing the molecular weight, amino acid sequence and related DNA sequence of thrombolytic enzymes

Inactive Publication Date: 2011-09-28
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In this invention patent, the inventor did not disclose the molecular weight, amino acid sequence and related DNA sequence of the thrombolytic enzyme

Method used

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  • cDNA sequence coding fibrinolysin, amino acid sequence and applications of sequences
  • cDNA sequence coding fibrinolysin, amino acid sequence and applications of sequences

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1: Extraction of total RNA from Echinochloa japonicus

[0031] Collect about 1Kg of fresh single-ringed spiny moth from the beach of Qingdao seaside intertidal zone (collected by: Bi Qingqing; Tel: 13698687750), temporarily raise it with fresh sea water, and take appropriate amount of single-ringed spiny moth body wall and intestinal tissue with isothiocyanate The guanidine method is used to extract total RNA from worms.

[0032] (1) Prepare solution D: Weigh 48g of guanidine isothiocyanate, 0.5g of sodium laurate creatinine, put it in a 250ml beaker, add 3.33ml of 0.75M sodium citrate solution, add a small amount of DEPC water 20ml, stir to dissolve, and transfer all to In a 100ml volumetric flask, add DEPC water to a constant volume to obtain solution D.

[0033] [(2) Extraction of total RNA: add 1ml solution D, 225ul chloroform, 5ul β-mercaptoethanol to a 5ml centrifuge tube, pre-cool on ice for 5 minutes, add 100mg of the body wall and intestinal tissue of the mon...

Embodiment 2

[0034] Example 2: Synthesis of the first strand of cDNA

[0035] Design and synthesize 3'RACE Adaptor: 5'-TACCGTCGTTCCACTAGTGATTTCACTATAGGTTTTTTTTTTTTTTTT-3', and use a synthetic 3'RACE Adaptor to replace Oligo dT Primer in PrimeScript lst Strand cDNA Synthesis Kit (TaKaRa) as reverse transcription primers, and extract the total RNA as For the template, perform RT-PCR operation according to the kit instructions to obtain the first strand of cDNA, and store it at -80°C for later use.

Embodiment 3

[0036] Example 3: Cloning of cDNA of Fibrinolytic Enzyme Gene of Echinochloa striata

[0037] (1) Primer design and synthesis: According to the determined protein N-terminal sequence ICGGSPADIT of fibrinolytic enzyme, design and synthesize forward degenerate primers: 103,5'-TCNCCNGCNGAYATHAC-3'; 104,5'-AGYCCNGCNGAYATHAC-3' . Design and synthesize reverse primer according to 3’RACEAdaptor: OP, 5’-TACCGTCGTTCCACTAGTGATTT-3’

[0038] (2) Degenerate primer PCR: Using the first strand of cDNA in Example 2 as a template, perform degenerate PCR with the designed and synthesized primer combination. Add sequentially to 0.2ml PCR tube: cDNA template, 0.5ul; 10×buffer, 2.5ul; 25mM MgCl 2 , 3ul; 2.5mM dNTP, 2ul; 10μM forward primer 103 / 104, 2.5ul; 10μM reverse primer OP, 1ul; ddH 2 O, 13.3ul; rTaq DNA polymerase, 0.2ul, constitute a 25ul reaction system. The above reaction system was subjected to PCR reaction on a PCR machine, and the reaction conditions were: 94°C pre-denaturation for 2 min...

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Abstract

The total RNA of Urechis unicinctus, which is used as template, is cloned to obtain the full-length cDNA sequence of the Urechis unicinctus fibrinolysin gene, wherein the open reading frame (ORF) of the sequence has 792 basic bases and is from the initiation codon ATG to the stop codon TAA. The cDNA original coding sequence of the fibrinolysin gene is translated to obtain the amino acid sequence of the Urechis unicinctus fibrinolysin, wherein the amino acid sequence has 263 amino acids in all. Different recombinant plasmid vectors can be constructed to construct fibrinolysin gene engineering bacteria for different receptors such as Escherichia coli and Saccharomyces and ensure that the cDNA sequence of the Urechis unicinctus fibrinolysin gene is expressed in the gene engineering bacteria and prepare fibrinolysin. The prepared fibrinolysin can be utilized to prepare the anticoagulant medicines or antithrombotic medicines for curing cardiovascular diseases and have very good research and development prospects.

Description

Technical field [0001] The present invention relates to a cDNA sequence and amino acid sequence encoding fibrinolytic enzyme, in particular to a cDNA sequence and amino acid sequence encoding fibrinolytic enzyme of monocyclic spine moth, and its application, belonging to the fields of biotechnology and biomedicine. Background technique [0002] Urechis unicinctuss is a protozoan eutropha, distributed in Russia, Japan, Korea, and my country’s Yellow and Bohai Sea coasts. It is a benthic animal living in coastal sediments. It is classified as Echiurioidea (Echiurioidea). Class (Echiurida), Xenopneusta (Xenopneusta), Urchidae (Urchidae). At present, the research on the single ring thorn moth is still in the basic biological research stage, such as morphology, embryo development, life history, artificial seedling technology and nutrient composition analysis, etc. Because it can tolerate high concentrations of sulfide, its metabolism research is also academic An aspect of world resear...

Claims

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Application Information

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IPC IPC(8): C12N15/57C12N9/68C12N15/81A61K38/48A61P7/02C12R1/84
CPCY02A50/30
Inventor 韩宝芹刘万顺冯伊琳杨艳董文
Owner OCEAN UNIV OF CHINA
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