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An essential element for nucleocapsid assembly and its application

A nucleocapsid and element technology, applied in the field of virus nucleocapsid assembly, can solve the problem that positioning features cannot be well explained, etc.

Active Publication Date: 2020-06-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the positioning characteristics of Ac83 cannot well explain its function of participating in viral genome assembly

Method used

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  • An essential element for nucleocapsid assembly and its application
  • An essential element for nucleocapsid assembly and its application
  • An essential element for nucleocapsid assembly and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1Ac83

[0056] Example 1Ac83 protein is not necessary for baculovirus nucleocapsid assembly

[0057] 1.1.1 Blocking the expression of Ac83 by inserting a large fragment does not affect the normal proliferation of the virus in insect cells

[0058] 1.1.1.1 Preparation of linear fragment Ac83US2-CmR-Ac83DS2 for homologous recombination

[0059] The first step is to construct a linear fragment for Red homologous recombination. Using bMON14272 as a template, primer pairs Ac83KO2-US-U / Ac83KO2-US-D and Ac83KO2-DS-U / Ac83KO2-DS-D were used to PCR amplify the upstream and downstream sequences of the region to be deleted, respectively, and named as Ac83US2 and Ac83DS2. pUC18-38KUS-CmR-38KDS is a recombinant plasmid constructed by predecessors, and its chloramphenicol resistance (chloramphenicol resistance, CmR) gene upstream and downstream contains the upstream and downstream sequences of the 38K gene. The Ac83DS2 fragment was digested with restriction endonuclease PstI / HindIII and cloned int...

Embodiment 2

[0081] Example 2 Identification of the chemical nature of ac83 by detecting progeny virus DNA

[0082] To test the chemical nature of ac83, experiments were designed to test whether ac83 has the characteristics of a cis-acting element. If ac83 participates in viral nucleocapsid assembly through its cis-acting element, theoretically only when ac83 exists on the viral genome, the genome can be assembled into the progeny virus.

[0083] In this experiment, the 38K deletion virus v38KKO was used as a positive control. 38K is a structural protein on the nucleocapsid of baculovirus. During the process of baculovirus infection of cells, 38K is involved in the assembly of the viral nucleocapsid, and the deletion of 38K does not affect the replication of the viral genome, but it will cause the viral genome to be unable to be compressed and packaged into the capsid precursor. Therefore, the 38K protein could rescue the nucleocapsid assembly ability of v38KKO by acting in trans. In ad...

Embodiment 3

[0093] Example 3 Identification of specific regions of NAE

[0094] 3.1.1ac83 truncated virus construction

[0095] The 1351-2011nt of ac83 may contain the complete sequence of NAE, and its 1651-1800nt may be the core region of NAE. Therefore, the present invention designed a series of ac83 truncated viruses, and tried to determine the specific region of NAE by comparing their ability to produce progeny BV.

[0096] First, use primer pairs ac831351-U / ac832011-D, ac831451-U / ac832011-D, ac831551-U / ac832011-D, ac831651-U / ac832011-D, ac831351-U / ac831850-D, ac831351-U / ac831900: FLAG-D and ac831351-U / ac831950-D, using bMON14272 as a template for PCR amplification, obtained ac83 truncated fragments ac83(1351-2011), ac83(1451-2011), ac83(1551-2011), ac83(1651-2011), ac83(1351-1850), ac83(1351-1900) and ac83(1351-1950). Then, the ac83 (1351-1900) fragment was digested with restriction endonuclease XbaI / PstI, and connected to the corresponding site of pUC8-SV40, and the positive clo...

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Abstract

The invention relates to the assembling field of virus nucleocapsid, particularly to a baculovirus nucleocapsid assembling necessary element and an application thereof in nucleocapsid assembling, establishment of a carrier comprising a target nucleotide sequence, and the like. The nucleocapsid assembling necessary element disclosed by the invention comprises a 45-65 map unit positioned in a viral genome of a hepatitis A baculovirus, preferably, a DNA sequence in the position of a 47-61 map unit. An NAE sequence plays an essential role in the nucleocapsid assembling process. The baculovirus has wide application prospect in the gene transduction field, so that the NAE sequence found in the invention can improve a baculovirus gene-transduced carrier, so that the establishment process is simpler and more convenient, the target DNA is higher in capacity, and higher biosecurity is achieved; and therefore, the nucleocapsid assembling necessary element is applicable to a required gene transduction operation, and requirements of clinical application, such as gene therapy and the like, can be better satisfied.

Description

technical field [0001] The invention relates to the field of virus nucleocapsid assembly, in particular to a baculovirus nucleocapsid assembly essential element and its application in nucleocapsid assembly and construction of a vector containing a target nucleotide sequence. Background technique [0002] Baculovirus (baculovirus) is a kind of dsDNA virus that specifically infects insects. The genome of baculovirus is a covalently closed circular dsDNA molecule with a size of 80-180kb, encoding 89-183 genes, packaged in a rod-shaped and enveloped protein capsid. Among them, Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is a representative species of baculovirus. [0003] Baculoviruses can produce two types of virions, occlusion-derived virion (ODV) and budded virion (BV). Among them, there is an efficient membrane fusion protein GP64 on the envelope of BV, which can enable BV to efficiently enter different types of cells, so baculovirus can be used as a gene...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/113C12N15/866C12Q1/6883
CPCC12N15/113C12N15/86C12N2310/10C12N2710/14043C12Q1/6883C12Q2600/136
Inventor 黄智宏潘梦佳吴文碧袁美妗杨凯
Owner SUN YAT SEN UNIV
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