Building method of silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system

The technology of bac-to-bac and acmnpvbac-to-bac is applied in the field of baculovirus gene expression system, which can solve problems such as affecting the expression efficiency of silkworm baculovirus and inability of oral infection of recombinant virus.

Inactive Publication Date: 2010-06-09
ZHEJIANG UNIV
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Problems solved by technology

However, the recombinant viruses constructed by the above-mentioned system are all recombinant baculoviruses constructed by using the strong promoter of baculovirus-polyhedron promoter, all of which are generally polyhedron-deleted (polh - ) virus, this type of rec...

Method used

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  • Building method of silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system
  • Building method of silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system
  • Building method of silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system

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Embodiment Construction

[0023] 1) Construction of recombinant transfer vector:

[0024] Using the silkworm wild-type BmNPV genomic DNA as a template, P10-upF / P10-upB and P10-downF / P10-downB were respectively used as primers (Table 1) to obtain flanking sequences p10-up (including p10 promoter) and p10-down. The cleavage sites designed by the primers are underlined in the table. PCR amplification conditions were: denaturation at 94°C for 3 min; 30 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 2 min, and finally extension at 72°C for 7 min. After ethanol precipitation and purification, the amplified p10-up and p10-down fragments were digested with HindIII / PstI and PstI / BamHI respectively and inserted into the BamHI-PstI-HindIII site of pUC19 to obtain p10 gene-containing Recombinant plasmid pUC19-p10-up-down with flanking sequences head-to-tail.

[0025] Polyhedron gene fragments were obtained by PCR using wild-type BmNPV genomic DNA of silkworm a...

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Abstract

The invention discloses a building method of a silkworm BmNPV Polh+Bac-to-Bac rhabdovirus expression system, comprising: taking silkworm wild type BmNPV genome DNA as the template; respectively taking P10-upF/P10-upB and P10-downF/P10-down B as a primer PCR for amplification to obtain p10-up and p10-down; after processed, inserting into BamHI-PstI-HindIII locus in pUC 19 to obtain recombinant plasmid pUC19-p10-up-down; using pUC-19-p10-up-polh-down, liposome lipofectin and MilliQ H2O to prepare DNA-Lipofectin mixed liquor; adding the mixed liquor into BmN cells for cultivating; collecting transfection cell supernatant; inoculating BmN cells, and recovering a polyhedral body; extracting virus DNA from the polyhedral body and electrically converting DH10 beta competent cells; screening locus ceruleus and cultivating; after cultivating PCR positive bacterial plaque, extracting macro-molecular DNA to transfect to the BmN cells; separating to obtain helper plasmids from DH10Bac culture bacteria of AcMNPV Bac-to-Bac; converting the helper plasmids into E.coli DH10 beta containg Ploh+BmBacmid; and screening the DH10 beta bacterial strain of the helper plasmid containg Ploh+BmBacmid. The invention can produce recombinant virus capable of infecting by eating with mouth, and recombinant virus does not need to infect by intracutaneous inoculation, thus improving the production efficiency of the silkworm rhabdovirus expression system.

Description

technical field [0001] The invention relates to a baculovirus gene expression system in biotechnology, in particular to a silkworm BmNPV Polh + A method for constructing a Bac-to-Bac baculovirus expression system. Background technique [0002] The silkworm is the largest economic resource insect in my country. With the development of modern biotechnology, the development of silkworm as a new type of bioreactor has a good market development prospect. [0003] The silkworm baculovirus gene expression system is one of the most efficient eukaryotic expression systems. We used the principle of bacterial transposon to construct a Bac-to-Bac baculovirus expression system specially for silkworm (see our previous authorized patent ZL200310108781.8). The advantage of this system is that the recombinant baculovirus can achieve gene transfer and recombination in Escherichia coli through the gene positioning and transfer of bacterial transposons, quickly obtain recombinant viruses, an...

Claims

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Application Information

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IPC IPC(8): C12N15/866
Inventor 相兴伟吴小锋于少芳杨锐
Owner ZHEJIANG UNIV
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