Rana chensinensis functional gene Rd-RNase3 sequence, construction method and amino acid sequence and application thereof

A technology of functional genes and construction methods, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc.

Inactive Publication Date: 2010-07-14
BIOCHEM ENG COLLEGE OF BEIJING UNION UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Anti-tumor major issues: immunogenicity, selectivity of cytotoxicity, stability

Method used

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  • Rana chensinensis functional gene Rd-RNase3 sequence, construction method and amino acid sequence and application thereof
  • Rana chensinensis functional gene Rd-RNase3 sequence, construction method and amino acid sequence and application thereof
  • Rana chensinensis functional gene Rd-RNase3 sequence, construction method and amino acid sequence and application thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0104] Embodiment 1: The specific construction method of the Chinese Rana functional gene Rd-RNase3 sequence of the present invention:

[0105] 1. Primer design

[0106] Gene-specific primers:

[0107] Forward primer N275: TAT TTG CAG TTG TTT TGA GTC TCA C

[0108] Reverse primer C715: CTG CTT ATC ACA TCC CTG TTG TC

[0109] 2. PCR amplification of the target gene

[0110] (1) Genomic DNA was extracted from the fresh blood of Rana using a blood genome extraction kit, and the molecular weight and concentration were detected by 1% agarose electrophoresis.

[0111] (2) Add 100ng of genomic template, 1μL of dNTPs (10mM), 5μl of 10×Taq Buffer, 3U of Taq DNA Polymerase and 30pmol of primers (N275, C715) into a sterile PCR tube, and add sterile ultrapure water to a total volume of 50μl ,well mixed;

[0112] (3) Put the PCR tube containing the sample into the preheated PCR instrument for PCR reaction. PCR amplification conditions: 94°C for 30s, 48°C for 30s, 72°C for 50s, 35 cyc...

Embodiment 2

[0162] Embodiment 2: Biological experiment of the Chinese forest functional gene Rd-RNase3 sequence and amino acid sequence constructed by the present invention

[0163] 1. Materials and methods

[0164] 1. Materials

[0165] (1) Strain and carrier

[0166] Escherichia coli BL21 (DE3) competent cells were purchased from TIANGEN Biotechnology Co., Ltd., and pFLAG-CTS vector was purchased from sigma-aldrich.

[0167] (2) Tool enzymes and biochemical reagents:

[0168] Tryptone, Yeast Extract, Agarose, ampicillin, X-Gal, IPTG, etc. (all purchased from TIANGEN Biotechnology Co., Ltd.), and other reagents were commercially available analytical grade.

[0169] (3) Equipment and equipment

[0170] equipment

[0171] THZ-D desktop constant temperature oscillator, Taicang Experimental Equipment Factory;

[0172] SPN202F electronic balance, Mettler-Toledo Weighing Equipment Systems Co., Ltd.;

[0173] LRH-250 biochemical incubator, Shanghai Yiheng Technology Co., Ltd.;

[0174] ...

Embodiment 3

[0185] Embodiment 3: Preparation of the recombinant fusion protein of the Chinese Rana functional gene Rd-RNase3 sequence of the present invention

[0186] 1. Materials and equipment

[0187] 1. Strain and carrier

[0188] Escherichia coli BL21 (DE3) competent cells were purchased from TIANGEN Biotechnology Co., Ltd., and pFLAG-CTS vector was purchased from sigma-aldrich.

[0189] 2. Tool enzymes and biochemical reagents

[0190] Tryptone, Yeast Extract, Agarose, ampicillin, X-Gal, IPTG, etc. (all purchased from TIANGEN Biotechnology Co., Ltd.), mouse anti-FLAG monoclonal antibody (purchased from Sigma Company), goat antibody linked to alkaline phosphatase Mouse secondary antibody, BCIP / NBT chromogenic kit (purchased from Beijing Zhongshan Jinqiao Biological Co., Ltd.), and other reagents were commercially available analytical grade.

[0191] 3. Equipment

[0192] THZ-D desktop constant temperature oscillator, Taicang Experimental Equipment Factory;

[0193] LRH-250 bioch...

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Abstract

The invention discloses a rana chensinensis functional gene Rd-RNase3 sequence which is characterized by being a nucleotide sequence shown in a sequence table SEQ ID No.1. In the invention, a tumor cell agglutination mediated by the rana chensinensis ribonuclease is not relevant to the selective cytotoxicity of tumor cells and the existence of the wild-type p53 tumor restraint substance, cannot produce gene mutation, and has better stability compared with the prior art. Long-term tumor restraint experiment proves that the Rd-RNase3 gene recombination fusion protein of the invention has strong cytotoxicity on the tumor cells under extremely low concentration, but has no cytotoxicity on normal cells, and thereby having the great advantage on preparing antitumor drugs compared with the existing gene drugs.

Description

technical field [0001] The invention relates to a functional sequence of Rana chinensis and its coded protein sequence, in particular to a functional gene Rd-RNase3 sequence of Rana chinensis and its coded amino acid sequence and its application. Background technique [0002] There are 100 million newly diagnosed cancer cases in the world every year, and 250 people die of cancer in every 100,000 people. Therefore, defeating cancer is one of the most important challenges of modern medicine. Although surgery can cure resectable cancers, unresectable cancers can only be treated with chemotherapy or radiation. The main problems of conventional drugs for treatment are lack of selectivity, strong toxicity and immunogenicity of foreign proteins, which often cause side effects, such as temporary diarrhea, nausea, hair loss, and susceptibility to infection. Sometimes long-term effects on the heart, lungs, kidneys, and bone marrow. [0003] Apoptosis caused by most DNA-degrading dru...

Claims

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Application Information

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IPC IPC(8): C12N15/55C12N9/22C12N15/10C12N15/11C12N15/63A61K38/46A61P35/00
Inventor 陶凤云赵伟
Owner BIOCHEM ENG COLLEGE OF BEIJING UNION UNIV
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