Construction method of HIV-1 integrase mutant library

A HIV-1, 1.HIV-1 technology, applied in the field of development and design of AIDS treatment drugs, can solve the problems of good specificity, low efficiency and coverage, uneven distribution, etc., and achieve the effect of high resolution

Inactive Publication Date: 2015-10-14
ZHEJIANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The technical problem to be solved in the present invention is to provide a method of using TDEM (transposon-directed base substitution mutation method) to develop a saturated mutation library with high resolution, so as to solve the problem of low efficiency and coverage of conventional mutation methods or specific mutations. Good sex but uneven distribution Insufficient

Method used

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  • Construction method of HIV-1 integrase mutant library
  • Construction method of HIV-1 integrase mutant library
  • Construction method of HIV-1 integrase mutant library

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Experimental program
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Embodiment Construction

[0068] Table 1. Primer sequences and templates used for amplifying fragments when constructing plasmids

[0069]

[0070]

[0071] 1. Plasmid construction

[0072] 1.1 pCompact-Kana-IN

[0073] 1.1.1 pCompact-Kana

[0074] Plasmid origin of replication (Origin) and Kanamycin resistance gene (Kanamycin R) are represented by Table 1 and figure 1 The indicated primers and templates were amplified by polymerase chain reaction (PCR), and the obtained PCR product was a blunt-ended fragment, and the two fragments were ligated by T4 DNA ligase to obtain the plasmid pCompact-Kana.

[0075] (1) Amplified Fragment Origin

[0076] PCR reaction system: template pUC19 (Takara, 2ng / μl), primer Ori-up / Ori-lo (0.5μM), 1*Pfu DNA polymerase Buffer, dNTP (0.2mM each), Pfu DNA polymerase (Promega, 0.05U / μl), add double distilled water to a total volume of 50μl.

[0077] (2) Amplified fragment Kanamycin R:

[0078] PCR reaction system: template pCR Blunt II-TOPO (Invitrogen, 2ng / μl), pr...

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Abstract

The present invention discloses a construction method of an HIV-1 integrase mutant library. The method comprises the following elements: (1) manufacturing gaps on random sites of a target gene by using transposition of transposon and restriction enzyme on both ends of the transposon, inserting mutation insertion boxes, using the restriction enzyme sites on the box to achieve replacement of three bases on the gene through digestion self-connection, and translating the target gene into protein to cause mutation o one or two consecutive amino acids on the target protein; and (2), using site-directed mutagenesis PCR to realize mutation of three bases, for the replacement of the original target gene and on the inserted mutation insertion boxes, from codons encoding cysteine into codons encoding other 19 amino acids, thereby increasing the diversity of the final mutant library.

Description

technical field [0001] The invention belongs to the field of development and design of AIDS treatment drugs, and the constructed integrase mutant library can be used to screen the drugs with inhibitory effect on HIV-1 integrase. Background technique [0002] Highly Active Antiretroviral Therapy (HAART) is currently the standard program for the treatment of AIDS approved by the FDA, but so far there has been no case of cure. At the same time, most of the vaccines may only prevent but not treat; even if the vaccine is used, most of today's HIV-1 infected people will eventually develop into AIDS patients. Therefore, it is of great significance to develop new chemical drugs to prevent the development of the disease. [0003] Integrase (IN) is one of the three basic enzymes necessary for HIV-1 replication and plays an important role in its life cycle. Currently, raltegravir and eltgravir are the only two FDA-approved drugs for IN. Other existing more than 30 approved HIV inhib...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C40B50/06C12N15/66C12N15/63
Inventor 周亚夫杨立莉沈学彬叶剑
Owner ZHEJIANG UNIV
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