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Silkworm nuclear polyhedron virus and its prepn process and application in gene expression

A technology of nuclear polyhedron and polyhedron gene, which is applied in the field of silkworm nuclear polyhedrosis virus and its preparation, can solve the problems of less viral DNA, difficult to distinguish blue spots, and atypical virus spots, etc., and achieve the effect of increasing the recombination rate

Inactive Publication Date: 2005-07-13
JIANGSU UNIV
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Problems solved by technology

The current common method is to infect BmN cells with silkworm bacilliform virus particles containing the lac Z gene, harvest the virus particles, and then purify them by ultracentrifugation. Requires large equipment such as an ultracentrifuge
Another shortcoming is that when doing plaque analysis, sometimes after adding X-gal, the blue spots that have not undergone recombination are also difficult to distinguish, and the added X-gal also affects the growth state of silkworm cells, causing virus plaques to be atypical, only Only experts who have been engaged in this field for a long time can distinguish, and the technical requirements are very high

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  • Silkworm nuclear polyhedron virus and its prepn process and application in gene expression
  • Silkworm nuclear polyhedron virus and its prepn process and application in gene expression
  • Silkworm nuclear polyhedron virus and its prepn process and application in gene expression

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Embodiment Construction

[0021] Such as figure 1 As shown in the figure, 1 indicates that there is an inserted characteristic sequence before the translation initiation codon of the polyhedron gene, including the restriction site of Bus 36 I; 2 indicates that there is an insertion after the translation termination codon of the polyhedron gene The characteristic sequence of , including the restriction site of Bus 36 I; 3 represents the reading frame of the polyhedron gene, from atg to the end of taa, the polyhedron gene fragment is amplified by PCR reaction with the above-mentioned pair of primers, and the reaction conditions For: 94°C, denaturation for 4 minutes; 94°C, 50 seconds, 50°C for 40 seconds, 72°C, 1 minute, a total of 3 cycles; 94°C, 50 seconds; 64°C for 30 seconds, 72°C, 1 minute, a total of 25 cycle, with a final extension at 72°C for 10 minutes.

[0022] Such as figure 2 As shown, the original Bombyx mori baculovirus does not contain a Bsu 36 I restriction site and cannot be digested b...

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Abstract

The present invention provides one modified silkworm baculovirus for expressing foreign gene, and has Bsu 36I restriction sites added to two sides of polyhedron gene. Thus modified silkworm baculovirus is fed to silkworm or injected to silkworm larva or pupa to obtain great amount of modified silkworm baculovirus polyhedron and to extract great amount of modified silkworm baculovirus DNA. Thus obtained DNA is Bsu 36I restricted for expressing foreign gene via homologous recombination. The recombinant silkworm baculovirus may be observed in microscope to select recombinant silkworm baculovirus spot without polyhedron. The present invention simplifies recombinant virus screening process.

Description

technical field [0001] The invention belongs to the production process in the field of virus bioreactors, in particular to the improvement of the silkworm baculovirus DNA used for gene expression, that is, the improved silkworm nuclear polyhedrosis virus with enzyme cleavage sites on both sides of the polyhedron gene and its preparation method and its application in gene expression. Background technique [0002] Baculovirus has been used as an expression vector for 20 years and has become one of the commonly used expression systems. There are two main forms of baculovirus in the developmental cycle of insects, one is the virus ion that shuttles infection between insect cells, also known as budding virus, and the other is the virus particle coated in a protein membrane polyhedron form. Polyhedrons can remain active in the environment and, when ingested by insects, the polyhedron membrane cleaves, releasing virions and causing infection. Since the polyhedron of the virus is...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N7/01C12N15/34C12N15/866
Inventor 王文兵王利群宋志秀于峰朱姗颖乌慧玲
Owner JIANGSU UNIV
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