Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method

A technology of shuttle expression vector and karyotype polyhedron, applied in the fields of molecular biology and bioengineering, can solve the problems of low yield of recombinant virus, time-consuming, complicated operation of plaque purification method, etc.

Inactive Publication Date: 2003-07-16
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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Problems solved by technology

Due to the low production rate of recombinant virus and the complex and time-consuming operation of plaque purification, it often takes several months or even longer to construct a complete recombinant virus

Method used

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  • Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method
  • Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method
  • Cotton bollworm single particle embedded nuclear polyhedrosis virus shuttle expression carrier and preparation method

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Experimental program
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Embodiment Construction

[0015] The steps are as follows:

[0016] 1. Activation of DH10B recipient strain 1 containing HZ8 (HaBacmid) and helper plasmid. Inoculate DH10B recipient bacteria stored at -80°C on LB solid medium containing kanamycin (50 μg / ml) and tetracycline (50 μg / ml), culture at 37°C for 12-16 hours, pick a single colony and inoculate into On 3 mL of LB liquid medium containing kanamycin (50 μg / ml) and tetracycline (50 μg / ml), culture at 37° C. for 24 hours, and prepare competent recipient bacteria according to conventional methods.

[0017] 2. Activation of DH5α bacteria containing HapFastPhP10 plasmid and extraction of HapFastPhP10 plasmid 2. Inoculate DH5α recipient bacteria stored at -80°C on LB solid medium containing gentamicin (50 μg / ml), culture at 37°C for 12-16 hours, pick a single colony and inoculate into 3 mL containing gentamicin ( 50 μg / ml) LB liquid medium, cultured at 37° C. for 16 hours, and extracted the HapFastPhP10 plasmid according to the conventional method fo...

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Abstract

A single embedded polyhedronvirus shuttle expression carrier of bollworm is configured through inserting the low-copy bacterium F replicon, kanamycine resistant gene and beta galactosidase gene to the polyhedrom gene site of the virus genom. The doner plasmid is used to transfer the exogenous gene to the insetion site of transposon in the said shuttle expression carrier. After bollworm cell is transfected by said DNA, the infections recombinant virus particles are formed to express exogenous gene under control of polyhedron promoter and P10 promoter.

Description

technical field [0001] The invention belongs to the technical fields of molecular biology and bioengineering, and in particular relates to a nuclear polyhedrosis virus shuttle expression vector embedded in single grain of cotton bollworm, and also relates to a preparation method of the shuttle expression vector. Background technique [0002] Baculoviruses are widely used in biological control of agricultural and forestry pests. At the same time, the baculovirus-insect (cell) system is also a very good eukaryotic expression system, which has been used in the research and production of foreign genes, diagnostic reagents and vaccines. Like other baculoviruses, the Chinese cotton bollworm single nucleocapsid nucleopolyhedrovirus (Helicoverpaarmigera single nucleocapsid nucleopolyhedrovirus, hereinafter referred to as wild-type virus or HaSNPV) as an insecticide has the disadvantage of slow insecticidal speed, causing its large area The role of control...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N63/00C12N7/01C12N15/33C12N15/79
Inventor 王汉中陈新文胡志红
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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