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Expression separation and purification method for Chinese Phytolacca acinosa cDNA mutant and TAT gene recombinant in prokaryon and application therefor

A recombinant and mutant technology, applied in DNA/RNA fragments, recombinant DNA technology, chemical instruments and methods, etc., can solve the problems of easy generation of drug resistance, large toxic and side effects, and achieve the effect of good biological activity

Inactive Publication Date: 2007-04-25
THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the anti-AIDS drugs in clinical use are chemical compounds, such as 3' azidodeoxy-thumidine AZT (azidodeoxy-thumidine), which is an inhibitor of reverse transcriptase, and it is the first drug approved by the US FDA. Antiviral drugs used for AIDS treatment have relatively high toxicity and side effects, and are prone to drug resistance

Method used

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  • Expression separation and purification method for Chinese Phytolacca acinosa cDNA mutant and TAT gene recombinant in prokaryon and application therefor
  • Expression separation and purification method for Chinese Phytolacca acinosa cDNA mutant and TAT gene recombinant in prokaryon and application therefor
  • Expression separation and purification method for Chinese Phytolacca acinosa cDNA mutant and TAT gene recombinant in prokaryon and application therefor

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Tat obtained:

[0060] Using PCV1 as a template, using tat outer primers PA (5'GGGGTACCTCGAGAAATGGAGCCAG 3') and PB (5'GAAGATCTGGATCCGTTCACTA 3'), the normal tat cDNA gene fragment was obtained by PCR amplification;

Embodiment 2

[0062] Cloning of Phytophthora chinensis Antiviral Protein Gene

[0063] Take pokeweed leaves, add a small amount of liquid nitrogen, grind and break the wall. Add TRIZOL reagent, and extract total RNA according to conventional methods. Reverse transcription was carried out using a kit from PROMEGA, and the method is shown in the kit instructions. 5ug pokeweed leaf total RNA was added to 2ug random hexamer primers, M-MLV reverse transcriptase, Rnasin and dNTP, and reacted at 42°C for 1 hour to synthesize cDNA. Using this cDNA as a template, the CAP gene was amplified. The primers used were

[0064] RT-PCR: 5' Primer: ATGAAGTCGATGCTTGTGGTG

[0065] 3' Primer: TCAGAATCCTTCAAATAGATC

[0066] The conditions of the PCR reaction were: 94 degrees for 5 minutes, 94 degrees for 30 seconds, 56 degrees for 30 seconds, 72 degrees for 2 minutes, 30 cycles, using Pfu enzyme.

[0067] The PCR product was tailed with Taq enzyme at 72°C for 30 minutes. The PCR product was identi...

Embodiment 3

[0069] Construction of Tat-CAP (C-terminal deletion of 22 amino acids and N-terminal deletion of 29 amino acids) fusion gene expression vector

[0070] The full-length fragment was amplified by PCR from the cDNA template of TAT, and the upstream and downstream primers were respectively

[0071] TAT 1#(Nde I) 5'GGA ATT CCA TAT GGA GCC AGT AGA TCC TAG ACT AG 3',

[0072] TAT 2# Linker

[0073] 5' GCC CAC CCC GGG CAC TTC CTT CGG GCC TGT CGG 3'.

[0074] CAP was amplified from the cDNA of CAP (deleted 22 amino acids at the C-terminal and 29 amino acids at the N-terminal), and the upstream and downstream primers were

[0075] PAP 3# Linker

[0076] 5’GTG CCC GGG GTG GGC GTG AAT ACA ATC ATC TAC AAT G 3’

[0077] PAP 4# Sal I

[0078] 5’ACGCGTCGAC TCA AGT TGT TTG GCA GCT CCC ACC AAC GTAGTT TAA GAG3’

[0079] Bridge PCR: Recover the first round of PCR reaction products to obtain two PCR products with overlapping regions, mix them in equal amounts, and use them as templates for t...

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Abstract

The invention concerns a Ribosome inactibating protein (RIPS) Chinese mainland protein separated from plants which plays a role in anti-AIDS through catalyzing rRNA depurination of prokaryotic and eukaryotic ribosome with highly conserved stem-loop structure at the surface. The invention involves a Tat protein, the basic amino acid enrichment domain of which is protein transduction domain (PTD) and able to carry many exogenous protein directly into cells as protein orientation of cytokines or toxin. The invention involves a fusion protein of Tat and Chinese antiviral protein mutant. Performing deletion mutation to CAP using Quick-mutagenesis site-directed mutagenesis technology: 22 amino acid deletion in N-terminal, 29 amino acid deletion in C-terminal and site-specific mutagenesis: 151KI152 / 151AA152, 191FN192 / 191AA192. The recombinant protein of the invention has biological activity of anti-HIV-1 in vitro. It is found that CAP deletion and site-specific mutagenesis in fusion gene can significantly reduce its ability to combine with rRNA (cytotoxicity), however, without influence to its ability of combination of HIV RNA (anti-HIV activity), has more clinical application.

Description

Technical field: [0001] The present invention relates to two genes / proteins related to AIDS virus, the gene of Pokeweed antiviral protein and its encoded protein (CAP) mutant and a trans-acting activator (TAT) encoded by human immunodeficiency virus HIV-1 gene ) gene and its coded protein relate to the field of development and application of new gene and new protein in biomedical high technology. Background technique: [0002] Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus (HIV). Twenty years since the virus / disease was discovered, it has spread rapidly throughout the century, seriously threatening human health and social development. Since the first HIV patient was found in Peking Union Medical College Hospital on June 23, 1985, the prevalence of AIDS in my country has shown an accelerated upward trend in recent years. It is estimated that the actual number of HIV-infected people in my country has exceeded 600,000. [0003] As one...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C07K19/00C12N15/63C12N15/62C12N1/21A61K38/16A61K48/00A61P31/18
Inventor 温见燕阴彬王先平文中伟樊峥彭小忠袁建刚强伯勤
Owner THE INST OF BASIC MEDICAL SCI OF CHINESE ACAD OF MEDICAL SCI
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