Nucleocapsid assembling necessary element and application thereof

A nucleotide and nucleotide sequence technology, applied in the field of virus nucleocapsid assembly, can solve the problem that the positioning characteristics cannot be well explained

Active Publication Date: 2017-04-19
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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  • Nucleocapsid assembling necessary element and application thereof
  • Nucleocapsid assembling necessary element and application thereof
  • Nucleocapsid assembling necessary element and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1Ac83

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

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

[0058] 1.1.1.1 Preparation of the 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, and named Ac83US2 and Ac83DS2. pUC18-38KUS-CmR-38KDS is a recombinant plasmid constructed by predecessors, and its chloramphenicol resistance (CmR) gene contains upstream and downstream sequences of 38K gene. The Ac83DS2 fragment was digested with restriction enzymes PstI / HindIII, and cloned into the corresponding position in pUC18-38KUS-CmR-38KDS, replacing the 38KDS fragment. T...

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, an experiment was designed to detect whether ac83 has the characteristics of a cis-acting element. If ac83 participates in viral nucleocapsid assembly through its cis-acting elements, theoretically, only when ac83 exists on the viral genome, the genome can be assembled into progeny viruses.

[0083] In this experiment, the 38K deletion virus v38KKO was used as a positive control. 38K is a structural protein on the baculovirus nucleocapsid. In the process of baculovirus infection of cells, 38K participates in the assembly of the viral nucleocapsid. The deletion of 38K does not affect the replication of the viral genome, but will cause the viral genome to be compressed and packaged into the capsid precursor. Therefore, the 38K protein can rescue the nucleocapsid assembly ability of v38KKO through trans-action. In addition, vCNEKO containing th...

Embodiment 3

[0093] Example 3 Identification of specific regions of NAE

[0094] 3.1.1 Construction of truncated ac83 virus

[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 truncated ac83 viruses and tried to determine the specific area of ​​NAE by comparing their ability to produce progeny BV.

[0096] First, use the 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 template for PCR amplification to obtain 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 enzymes XbaI / PstI and ligated into the corresponding site of pUC8-SV40. The positive clone was n...

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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 present invention relates to the field of viral 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 is a type of dsDNA virus that specifically infects insects. The genome of a baculovirus is a covalently closed circular dsDNA molecule with a size of 80-180kb, encoding 89-183 genes, packaged in a rod-shaped and envelope-coated protein capsid. Among them, Autographacalifornica multiple nucleopolyhedrovirus (AcMNPV) is a representative species of baculovirus. [0003] Baculovirus can produce two types of virions, namely 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 enter different types of cells efficiently, so baculovirus can be used as a gene ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/113C12N15/866C12Q1/68
CPCC12N15/113C12N15/86C12Q1/6883C12N2310/10C12N2710/14043C12Q2600/136
Inventor 黄智宏潘梦佳吴文碧袁美妗杨凯
Owner SUN YAT SEN UNIV
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