Virus expression veclor of tomato flower-leaf virus low virulent strain K

A technology of tomato mosaic virus and expression vector, which is applied in the field of virus expression vector and its construction of tomato mosaic virus attenuated strain K, which can solve the problems of unusable agricultural crop traits, improved nutrition and medicinal value, and decreased yield of host plants, etc. problems, to achieve the effect of broad application prospects

Inactive Publication Date: 2003-04-23
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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Problems solved by technology

So far, plant virus vectors are all constructed with strong viruses, so they cannot be used to improve the traits of agricultural crops or increase...

Method used

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  • Virus expression veclor of tomato flower-leaf virus low virulent strain K
  • Virus expression veclor of tomato flower-leaf virus low virulent strain K
  • Virus expression veclor of tomato flower-leaf virus low virulent strain K

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Embodiment

[0017] Example: 1. Construction of the intermediate cloning vector pTOMV-KC:

[0018] 1.1 Use BamH I to cut pBluescript plasmid with single restriction enzyme, fill in DNA polymerase I Klenow fragment, ligate with T4DNA ligase, transform E. coli DH5a, extract the plasmid, confirm that the BamH I site disappears, and make it pBluescriptΔBamH I . Then pBluescriptΔBamH I was digested with Spe I, after filling in with DNA polymerase I Klenow fragment, ligated with T4DNA ligase, transformed into E. coli DH5a, the plasmid was extracted, and the digestion was performed to confirm that the Spe I site disappeared , Making it pBluescriptΔBamH I-Spe I.

[0019] 1.2 Use the plasmid pNBH (5.7kb) with the cDNA clone of the attenuated vaccine ToWK as a template for PCR.

[0020] 1.2.1 PCR amplification of ToMV-K gene (5461-5711) sequence fragment:

[0021]Design primers according to the published complete sequence of ToMV-K genome, and design a Kpn I restriction site at the 5'end of the upstream...

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Abstract

The present invention relates to the field of plant virus gene engineering technology. It utilizes gene insertion recombination mode to make the cDNA genome of low virulent strain K of tomato mosaic virus with low virulent protecting function implement recombination to construct recombinant virus vector capable of high-effectively and stably expressing exogenous gene, after it is transcripted into RNA, it can directly infect plant or use transformed agrobacterium to infect plant to obtain recombinant virus. Said recombinant virus is separated and purified, then can be inoculated into host plant again, and can continuously and high-effectively express exogenous gene in plant body for several generations. The target protein expressed by natural form can be made into oral preparation.

Description

Technical field [0001] The invention relates to the field of plant virus genetic engineering, in particular to a virus expression vector of the tomato mosaic virus attenuated strain K of the tobacco mosaic virus genus and a construction method thereof. Background technique [0002] The construction of expression vectors using plant viruses began in the 1980s. In 1980, Shepherd et al. discovered that the DNA clone of cauliflower mosaic virus (CaMV) can infect plants by mechanical inoculation to have the characteristics of an expression vector; after obtaining the cDNA clone of plant RNA virus infectivity, it was also used for development Expression vector (Ahlguist et al., 1983; Kumagai et al., 2000). In the past ten years, the use of plant viruses as vectors to express foreign proteins has received considerable attention, and some RNA virus vectors have made considerable progress. (Hendy et al., 1999; McCormick et al., 1999; Gopinath et al., 2000; Zhang et al., 2000). [0003] Pl...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/34C12N15/83
Inventor 邱并生李勇刘广超
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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