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Construction method and application of antheraea pernyi multinucleocapsid nucleopolyhedrovirus shuttle vector

A kind of nuclear polyhedrosis, construction method technology, applied in the construction field of tussah nuclear polyhedrosis virus shuttle vector

Active Publication Date: 2019-12-20
LIAONING OCEAN & FISHERIES SCI RES INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no technical method for constructing AnpeNPV shuttle vector

Method used

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  • Construction method and application of antheraea pernyi multinucleocapsid nucleopolyhedrovirus shuttle vector
  • Construction method and application of antheraea pernyi multinucleocapsid nucleopolyhedrovirus shuttle vector
  • Construction method and application of antheraea pernyi multinucleocapsid nucleopolyhedrovirus shuttle vector

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0076] Example 1 Construction of bacterial artificial chromosome transfer vector pSMART BAC-N1 / lacZα:mini-attTn7:lacZα / phΔN

[0077] (1) Modification of bacterial artificial chromosome cloning vector pSMART BAC

[0078] The commercially available bacterial artificial chromosome cloning vector pSMART BAC (42030-1) has restriction sites such as BamH I, Hind III, EcoR I, and Not I on both sides of the gene cloning site, and at the same time, 3 sites of the chloramphenicol resistance gene There is also an Avr II (Bln I) restriction site sequence (CCTAGG) at the ' end ( figure 1 -a), not suitable for direct use in the construction of transfer vectors. Two pairs of primers were designed according to the pSMART BAC vector sequence (GenBank number: EU101022.1): SEQ ID NO:1 / SEQ ID NO:2 and SEQ ID NO:3 / SEQ ID NO:4. With pSMART BAC vector plasmid DNA as template, carry out PCR amplification respectively with primer SEQ ID NO:1 and SEQ ID NO:2, SEQ ID NO:3 and SEQ ID NO:4, wherein: SEQ...

Embodiment 2

[0084] Embodiment 2 Obtaining of AnpeNPV shuttle vector

[0085] (1) Acquisition of recombinant AnpeNPV with bacterial artificial chromosome vector pSMART BAC

[0086] 1) Preparation of linearized transfer vector pSMART BAC-N1 / lacZα:mini-attTn7:lacZα / phΔN plasmid DNA: use Avr II (Bln I) to digest transfer vector pSMART BAC-N1 / lacZα:mini-attTn7:lacZα / phΔN plasmid DNA was recovered by agarose gel electrophoresis to prepare a certain amount of linearized transfer vector plasmid DNA ( Figure 4 -a, b), after aseptic treatment, use ultraviolet spectrophotometer to quantify, and set aside.

[0087] 2) AnpeNPV genomic DNA preparation for transfection: use a linearizable AnpeNPV mutant (ApNPV-Δph / Avr II + / egfp + )[Patent application number: 201910517434.1.] Genomic DNA( Figure 4 -a). AnpeNPV mutant (ApNPV-Δph / Avr II) was digested with Avr II (Bln I) + / egfp + ) genomic DNA, agarose gel electrophoresis recovery to prepare a certain amount of linearized AnpeNPV genomic DNA ( ...

Embodiment 3

[0099] Example 3 AnpeNPV-bacmid is used for the knockout of a certain gene in the AnpeNPV genome

[0100] (1) Construction of targeting gene cloning vector pUC57 / FRT-Zeo-FRT

[0101] In this example, the bleomycin (Zeocin, Zeo) resistance gene was selected as the selectable marker gene to construct a gene targeting vector. At the same time, in order to facilitate the insertion of the expression gene and further remove the selection marker gene after gene knockout or insertion, a flippase binding site (flipase recognition target, FRT) sequence and multiple Cloning site sequence. The DNA sequence is shown in SEQ ID NO: 11. The DNA fragment was synthesized by Beijing Saibaisheng Gene Technology Co., Ltd. and cloned into the vector pUC57 to construct a general targeting gene cloning vector pUC57 / FRT-Zeo-FRT( Figure 8 -a).

[0102] (2) Construction of cathepsin and chitinase gene targeting vectors in AnpeNPV-bacmid genome

[0103] In this example, partial DNA fragments of cath...

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Abstract

The invention discloses a construction method and application of an antheraea pernyi multinucleocapsid nucleopolyhedrovirus shuttle vector, and belongs to the field of a biological technology. A bacterium artificial chromosome cloning vector is used for constructing a transfer vector comprising a DNA fragment containing a lacZ alpha report gene and a bacterial transposon Tn7 transposition target point sequence, a DNA fragment containing an AnpeNPV polyhedrin gene promoter sequence and an upstream gene partial sequence, and a DNA fragment containing a polyhedrin gene 3' terminal partial codingregion sequence and a downstream gene partial sequence, and linearized transfer vector plasmid DNA and linearized AnpeNPV genomic DNA are subjected to cotransfection with Tn-High Five cells, so that recombinant AnpeNPV is obtained. The recombinat AnpeNPV genomic DNA is transformed to escherichia coli, so that an AnpeNPV shuttle vector AnpeNPV-bacmid is obtained. The shuttle vector can be reproduced in the manner of plasmid DNA in the escherichia coli, can also be reproduced in the virus DNA manner in Tn-High Five insect cells and antheraea pernyi, and has infectivity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a construction method and application of a tussah nuclear polyhedrosis virus shuttle vector. Background technique [0002] Baculoviruses, also known as Nucleopolyhedroviruses (NPVs), are insect viruses that can form inclusion bodies in the nucleus of insect cells. More than 1,000 species of baculoviruses have been reported to infect various insects, most of which infect Lepidoptera. Baculovirus has become an important research tool in pest control, establishment and application of the Baculovirus Expression Vector System (BEVS), viral gene function analysis, virus-insect host interaction, etc. [0003] Autographa californica nucleopolyhedrovirus (AcNPV) was first used in 1983 to establish a baculovirus expression vector system. In 1993, Luckow et al [Luckow et al. Journal of Virology, 1993, 67:4566-4579] integrated the bacterial F plasmid DNA replicon (mini-F replica) i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866C12N15/65C12N15/66
CPCC12N15/65C12N15/66C12N15/86C12N2710/14143
Inventor 范琦叶博赵振军李佩佩岳冬梅王林美张波
Owner LIAONING OCEAN & FISHERIES SCI RES INST
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