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Duck circovirus ORF3 nucleus location NLS sequence and application thereof

A nuclear localization signal and sequence technology, which is applied in the field of duck circovirus ORF3 protein nuclear localization NLS sequence, can solve problems such as homology differences of ORF3 nucleic acid sequences

Inactive Publication Date: 2017-06-27
LINYI UNIVERSITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ORF3 protein is a DuCV virus protein newly discovered by the applicant in 2012. Homology analysis of the ORF3 gene sequences of different DuCV representative strains belonging to two genotypes found that the ORF3 nucleic acid sequences of genotype I and II DuCV are identical There is a big difference in origin

Method used

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  • Duck circovirus ORF3 nucleus location NLS sequence and application thereof
  • Duck circovirus ORF3 nucleus location NLS sequence and application thereof
  • Duck circovirus ORF3 nucleus location NLS sequence and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 The C-terminal differential region of ORF3 was mutated to determine the precise NLS sequence: RRLRTCNCRACRTLKH.

[0030] 1 Materials and methods

[0031] 1.1 Plasmid

[0032] The pEGFP-N3, pcDNA-FJ-ORF3 and pcDNA-WF-ORF3 plasmids are commercially available or prepared by the laboratory. The plasmids are transformed into Escherichia coli strain DH5α, the plasmid DNA is extracted, and the DNA concentration is measured with a DNA / RNA quantifier, and placed in - Store at 20°C for later use.

[0033] 1.2 Main reagents

[0034] Pfu Turbo DNA Polymerase was purchased from Agilent; DpnI was purchased from NEB; Lipo2000 was purchased from Invitrogen.

[0035] 1.3 Location analysis of pEGFP-FJ-ORF3 and pEGFP-WF-ORF3

[0036] 1.3.1 ORF3 sequence analysis of genotype I and II DuCV

[0037] Compared with type I and type II DuCV ORF3 gene sequences, the 236th nucleotide of ORF3 of type I strains undergoes a T→A mutation, and the codon TTG (235-237) is converted into a ...

Embodiment 2

[0075] Example 2 Fusion expression of the NLS signal in the C-terminal differential region with GFP protein, and the ability of EGFP to obtain nuclear localization

[0076] 2.1 Construction of pNLS-GFP expression vector and its expression measurement

[0077] pNLS-GFP plasmid construction: synthesize the following two primers, the slash mark is the NLS sequence

[0078] Primer NLS-F1:

[0079] AATTCATGCGACGATTGCGAACTTGCAATTGTCGAGCGTGCAGAACTCTAAAACATG

[0080] Primer NLS-R1:

[0081] GATCCATGTTTTAGAGTTCTGCACGCTCGACAATTGCAAGTTCGCAATCGTCGCATG

[0082] Anneal the upstream and downstream, and clone the NLS into pEGFP-N3 using the EcoRI and BamHI restriction sites (plasmid map as shown in figure 1 Shown) in the carrier, the specific test steps are carried out according to routine tests.

[0083] 2.2 NLS-GFP localization analysis

[0084] 2.2.1 Fluorescence test

[0085] 1. Place the flyer in the culture plate

[0086] 2. DF-1 cells were subcultured in 6-well cell culture pla...

Embodiment 3

[0103] Example 3 Nuclear Localization Effects of Different Copy Number NLS Sequences

[0104] 3.1 Construction of p2*NLS-GFP and p3*NLS-GFP expression vectors

[0105] Synthesize the following two sets of primers, the slash mark is the NLS sequence

[0106] Primer NLS-F2:

[0107] AATTCATGCGACGATTGCGAACTTGCAATTGTCGAGCGTGCAGAACTCTAAAACATATGCGACGATTGCGAACTTGCAATTGTCGAGCGTGCAGAACTCTAAAACATG

[0108] Primer NLS-R2:

[0109] GATCCATGTTTTAGAGTTCTGCACGCTCGACAATTGCAAGTTCGCAATCGTCGCATATGTTTTGAGTTCTGCACGCTCGACAATTGCAAGTTCGCAATCGTCGCATG

[0110] Primer NLS-F3:

[0111] AATTCATGCGACGATTGCGAACTTGCAATTGTCGAGCGTGCAGAACTCTAAAACATATGCGACGATTGCGAACTTGCAATTGTCGAGCGTGCAGAACTCTAAAACATATGCGACGATTGCGAACTTGCAATTGTCGAGCGTGCAGAACTCTAAAACATG

[0112] Primer NLS-R3:

[0113] GATCCATGTTTTAGAGTTCTGCACGCTCGACAATTGCAAGTTCGCAATCGTCGCATATGTTTTAGAGTTCTGCACGCTCGACAATTGCAAGTTCGCAATCGTCGCATATGTTTTAGAGTTCTGCACGCTCGACAATTGCAAGTTCGCAATCGTCGCATG

[0114] Anneal the upstream and downstream, and clone the NLS int...

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Abstract

The invention discloses a nuclear localization signal polypeptide, the sequence of which is RRLRTCNCRACRTLKH, and the double repetition of the signal polypeptide sequence can increase the expression of the fusion protein in eukaryotic cells.

Description

technical field [0001] The invention relates to a duck circovirus ORF3 protein nuclear localization NLS sequence and the application of the NLS sequence in the field of expression vectors. Background technique [0002] The transgenic technology of artificially introducing exogenous genes into cells is an important technology not only because it is a basic technology for analyzing various biological phenomena, but also because of its application in aspects such as gene therapy and beneficial animal production. In general, there are two approaches to genetic modification. One is a biological method that uses viruses with foreign genes, and the other is a physical method that uses physical methods to introduce foreign genes into cells. [0003] Duck circovirus (DuCV) can cause immunosuppressive diseases in ducks. Infected ducks mainly manifest as disheveled feathers, growth retardation, weight loss, hemorrhage and swelling of thymus, bursa of Fabricius, spleen and liver in var...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/01C12N15/34C12N15/85
CPCC07K14/005C12N15/85C12N2750/10022C12N2800/106
Inventor 王鑫武专昌吴家强陈广艳孟凡生孙培明王娟井文倩王军于江陈智郭立辉任素芳张玉玉张萍
Owner LINYI UNIVERSITY
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