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Small-ring type DNA (deoxyribonucleic acid) recombinant vector for blocking HIV (human immunodeficiency virus)-1 membrane fusion and application thereof

A DNA recombination, HIV-1 technology, applied in the field of virology, can solve difficult and other problems

Inactive Publication Date: 2013-09-11
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, using known technology, it is possible to clone the gene capable of expressing HR212 into a small circle vector and express HR212 protein, but it is quite difficult to secrete HR212 protein outside the cell
So far, no one has been able to achieve the expression of HR212 protein secreted outside eukaryotic cells

Method used

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  • Small-ring type DNA (deoxyribonucleic acid) recombinant vector for blocking HIV (human immunodeficiency virus)-1 membrane fusion and application thereof
  • Small-ring type DNA (deoxyribonucleic acid) recombinant vector for blocking HIV (human immunodeficiency virus)-1 membrane fusion and application thereof
  • Small-ring type DNA (deoxyribonucleic acid) recombinant vector for blocking HIV (human immunodeficiency virus)-1 membrane fusion and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1: Effects of adding different labels on HR212 to the effect of virus suppression

[0060] In order to facilitate detection, purification and analysis, tags are often added to the N-terminus and C-terminus of a protein. However, the addition of tags will sometimes affect the functional effect of the target protein. The purpose of this example is to analyze whether the function of the tagged HR212 protein is affected.

[0061] Experimental design: Add the Flag tag and the Strep II tag to both sides of the HR212 protein (the amino acid sequence is SEQ ID No.1; the codon-optimized nucleotide sequence encoding HR212 in the present invention is SEQ ID No.2), After the protein is purified and digested in the form of GST fusion protein, the membrane inhibitory activity of HIV-I virus with recombinant HR212 proteins with different tags is detected by HIV pseudovirus inhibition. The following four types of HR212 were designed in the experiment: HR212-Flag (that is, the ...

Embodiment 2

[0134] Example 2: Pseudovirus effect when HR212 uses linkers of different lengths

[0135] Experimental design: The length of the joint between HR2 (SEQ ID No.34) and HR1 (SEQ ID No.33) may affect the membrane translocation ability of proteins expressed in eukaryotic cells. Different The eukaryotic expression vector was constructed in the form of a joint combination of length, and after the plasmid was extracted and purified, it was transfected into Hela cells, and the supernatant was collected for pseudovirus inhibition experiments to compare the effects of different joints on the function of HR212. The combination pattern of the experiment is as follows:

[0136] Clone 05: HR212-5-5: HR2-link05a-HR1-link05b-HR2-Flag

[0137] Clone 06: HR212-15-5: HR2-link15a-HR1-link05b-HR2-Flag

[0138] Clone 07: HR212-5-15: HR2-link05a-HR1-link15b-HR2-Flag

[0139] Clone 08: HR212-15-15: HR2-link15a-HR1-link15b-HR2-Flag

[0140] Where link represents a linker, and its amino acid sequen...

Embodiment 3

[0201] Example 3: Effects of using GPI-anchored and non-anchored expression

[0202] Experimental design: A GPI anchor sequence was added behind the HR2 gene of clone 06, and the ability of its exocrine protein and membrane protein to inhibit HIV virus invasion was compared when the anchor sequence was used and without the anchor sequence.

[0203] Clone 09: HR212-GPI: HR2-link05-HR1-link05-HR2-Flag

[0204] Experiment content:

[0205] 1: Preparation of clone 09:

[0206] Synthesize NewHR2 segment-Flag-GPI as follows

[0207] Cgtacgggaggttcaggtggatcaagcggaggttggatggagtgggacagagagattaataactacaccagcctgattcactcactcatcgaagagtcccagaaccaacaggagaagaatgagcaggaattgctcggtagcgctggaagcgattacaaggatgacgacgataagggaggttctccccccgccggcaccaccgacgccgcgcatccggggcggtccgtggtccccgcgttgctgcctctgctggccgggaccctgctgttgctcgagacggccactgctcccggaggttctgctccagctaagc

[0208] The restriction site is BsiWI-BlpI, which was ligated to clone 05 digested with the same enzyme to form a new clone. The new clone ...

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Abstract

The invention provides a small-ring type DNA (deoxyribonucleic acid) recombinant vector capable of realizing eukaryotic excrine expression of HIV (human immunodeficiency virus)-1 membrane fusion inhibition protein HR212, which comprises a nucleotide sequence coding HR212 and a small-ring DNA vector. The invention also provides a small-ring type DNA gene therapeutic agent for blocking an HIV-1 membrane fusion process; the small-ring type DNA gene therapeutic agent comprises a small-ring type DNA recombinant vector capable of realizing eukaryotic excrine expression of HIV-1 membrane fusion inhibition protein HR212; the active ingredient is nucleotide coding heptad repeat triple-helix protein HR212; and after transfecting eukaryotic cells through a small-ring type DNA vector mode, the nucleotide is expressed in the cells and is released outside the cells through an excrine or cell outer membrane expression mode, the process of virus entering a host cell is blocked, and then an effect on suppressing HIV-1 virus infection is realized. The invention also provides an application of the small-ring type DNA recombinant vector coding heptad repeat triple-helix protein HR212 in preparing a gene therapeutic agent for resisting HIV-1 infection.

Description

technical field [0001] The invention belongs to the field of virology, and specifically relates to a small circular DNA recombination vector capable of eukaryotic exocrine expression of HIV-1 membrane fusion inhibitor protein HR212, which comprises a nucleotide sequence encoding HR212 and a small circular DNA vector. The present invention also relates to a group of small circular DNA gene therapy agents for blocking the process of HIV-1 membrane fusion, which comprises a small circular DNA recombinant vector capable of eukaryotic exocrine expression of HIV-1 membrane fusion inhibitor protein HR212, and its active ingredient is Nucleotides encoding the heptad repeat triple helix protein HR212. The invention also relates to the application of the small circular DNA recombination vector encoding the heptapeptide repeating triple helix protein HR212 in the preparation of the gene therapy agent for anti-HIV-1 infection. Background technique [0002] HIV infection is already a gl...

Claims

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Application Information

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IPC IPC(8): C12N15/79C12N15/85A61K48/00A61P31/18
Inventor 田波潘煦文孟颂东
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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