Construction and function test of double-long-hairpin RNA (Ribonucleic Acid) expression element for inhibiting HIV-1 (Human Immunodeficiency Virus-1)

A technology of HIV-1 and hairpin structure, applied in the field of genetic engineering, can solve problems such as rapid replication, high mutation rate, and no inhibitory effect of RNAi

Inactive Publication Date: 2012-02-15
NANKAI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

But at the same time, due to the rapid replication and high mut...

Method used

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  • Construction and function test of double-long-hairpin RNA (Ribonucleic Acid) expression element for inhibiting HIV-1 (Human Immunodeficiency Virus-1)
  • Construction and function test of double-long-hairpin RNA (Ribonucleic Acid) expression element for inhibiting HIV-1 (Human Immunodeficiency Virus-1)
  • Construction and function test of double-long-hairpin RNA (Ribonucleic Acid) expression element for inhibiting HIV-1 (Human Immunodeficiency Virus-1)

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Experimental program
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Effect test

Embodiment 1

[0076] Construction and functional detection of the expression element sequence of double long hairpin structure RNA inhibiting HIV-1

[0077] 1. Construction of expression plasmid pGMT-dlhRNA-VGTE containing double long hairpin structure RNA expression element (dlhRNA-VGTE) sequence

[0078] The method is characterized in that the (long hairpin RNA, lhRNA) long hairpin RNA expression original is obtained by the Two-step PCR method, and then the (double long hairpin RNA, dlhRNA) double long hairpin RNA expression original is obtained by the Two-step PCR method using lhRNA as a template ; The specific method is as follows:

[0079] 1) Construction of expression plasmid pGMT-lhRNA-VG containing lhRNA expression elements:

[0080] First round of PCR

[0081] Upstream sequence: 5'-AAGATCTGGGCAGGAAGAGGG-3'

[0082] Downstream sequence: 5'-CTGGGTCAGGGACATAAATCTTGTGGGGGTGGCTCCCTATTGCCACTGTCTTCTGCTCCGACTCTCGTCCTTTTCCACAAG-3'

[0083] PCR reaction system and conditions:

[0084] ...

Embodiment 2

[0134] Construction of FG12-dlhRNA-VGTE lentivirus to inhibit HIV-1 (NL4.3 standard strain) replication

[0135] 1. Construction of lentiviral expression vector pFG12-dlhRNA-VGTE

[0136] Upstream sequence: 5'-GCTCTAGAGCAAGATCTGGGCAGGAAGAGGG-3'

[0137] Downstream sequence: 5'-CCGCTCGAGCGGAAAAAAGGCATTCCTCTATGGCAG

[0138] GAAGGTTGGAGAAGTGAATTATATATGG-3'

[0139] All the above sequences are synthesized by chemical synthesis method to synthesize single-stranded oligonucleotides.

[0140] dlhRNA fragment obtained by PCR method

[0141]

[0142] The PCR product was recovered and purified into 40 μl of PCR product recovery kit by BIOMIGA Company (DC3511 / DC3514-01). The purified DNA fragment and the plasmid vector FG12 were double-digested with XbaI (NEB, R0145) and XhoI (NEB, R0146), respectively. The double digestion product was recovered by BIOMIGA company (DC3511 / DC3514-01) product recovery kit. Takara company (D2011A) T4DNALigase ligase was used for ligation o...

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Abstract

The invention relates to construction and function test of a double-long-hairpin RNA (Ribonucleic Acid) expression element for inhibiting HIV-1 (Human Immunodeficiency Virus-1), belonging to the technical field of gene engineering. In accordance with the design principle of siRNA and the conservative analysis of the HIV-1 sequence, an HIV-1 capsid protein gag gene (532-552), an envelope protein env gene (1428-1448), a non-structural protein tat gene (131-151) and a vpu gene (143-161) are selected and designed into a corresponding siRNA sequence; through a two-step PCR (Polymerase Chain Reaction) method, a double-long-hairpin RNA expression element (the sequence list is shown as SEQ ID No.1) is finally constructed; and through microscopic fluorescent observation and flow cell analysis quantitative test, the inhibition effect of the double-long-hairpin RNA expression element on the eukaryotic expression of an HIV gene EGFP (Enhanced Green Fluorescent Protein) fusion protein is tested. Experiments prove that the dlh RNA expression element can effectively inhibit the expression of multiple HIV genes.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering. Background technique [0002] AIDS, Acquired Immunodeficiency Syndrome (AIDS), caused by Human Immunodeficiency Virus (HIV) infection, is one of the most serious epidemics. Inhibiting the replication of HIV-1 can effectively improve the life conditions of AIDS patients, and it is also a necessary experimental method for HIV-1 related research. Currently, methods for inhibiting HIV-1 include: reverse transcriptase inhibitors, protease inhibitors, receptor analogs, and RNA interference. [0003] RNA interference (RNA interference, RNAi) is a method of inhibiting the gene expression of HIV-1 at the gene transcription level. Methods for realizing RNAi include: chemically synthesizing small siRNA fragments to directly perform RNAi, or expressing artificial RNAi-inducing precursors through gene vectors to perform RNAi. Chemically synthesized siRNA is difficult to be widely used due to it...

Claims

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Application Information

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IPC IPC(8): C12N15/113C12N15/10C12N15/85C12Q1/68
Inventor 刘畅孔晓红梁之聘袁青刁丹红
Owner NANKAI UNIV
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