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Method for easily and efficiently constructing recombinant baculovirus

A technology of recombinant baculovirus and construction method, which is applied in the field of simple and efficient construction of recombinant baculovirus, can solve the problems of complicated time-consuming, low efficiency, and low transposition efficiency, and achieves the effects of convenient storage and convenient construction.

Inactive Publication Date: 2009-02-25
NANYANG NORMAL UNIV
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Problems solved by technology

[0003] The most widely used at present is the Bac-to-Bac system. Although this system does not require homologous recombination in insect cells, the transposition efficiency in bacteria is not very high, usually only about 10% of the white spot, and the white spot colonies Further streak culture and PCR verification are required to remove false positives, which is time-consuming and complicated
Existing methods can basically meet the requirements of constructing a single or a small number of recombinant viruses, but once it is necessary to construct dozens or even more recombinant viruses to express foreign genes in large quantities, the efficiency is too low
And because the efficiency of obtaining recombinant virus is less than 90%, it is not suitable to construct a baculovirus cDNA library with high quality and large library capacity

Method used

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  • Method for easily and efficiently constructing recombinant baculovirus
  • Method for easily and efficiently constructing recombinant baculovirus
  • Method for easily and efficiently constructing recombinant baculovirus

Examples

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Embodiment 1

[0017] Embodiment 1: the method for constructing recombinant AcBacmid

[0018] (1) Construction of donor plasmid

[0019] Such as figure 1 As shown, the plasmid contains two strong baculovirus promoters, Pp10 and Ppolh, which can simultaneously drive the expression of two target genes. The CpoI / NotI site in MCS1 and the two different Bsu36I sites in MCS2 are suitable for directional cloning of PCR products. R6kγori is a conditional replicon, which can only replicate and reproduce in the strain expressing the pir gene (the product is π protein). The cis-activating element oriT originates from the F factor and is responsible for the transfer of the donor plasmid. The specific construction process is as follows: Zeocin resistance gene (Em7-ZeoR) was amplified by PCR, and two homology arm sequences (HL and HR), two I-SceI sites, and BstBI were added to its two segments respectively , SacI, pmeI, AvrII and other enzyme cutting sites. The upstream and downstream primers are: ZeoR-...

Embodiment 2

[0031]Embodiment 2: Construction method carrying reporter gene AcBacmid

[0032] (1) Construction of donor plasmids carrying reporter genes

[0033] The donor vector pHTdual was obtained according to the method of step (1) in Example 1, which contains dual promoters (p10 and Ppolh), so it is very suitable for cloning a reporter gene downstream of the P10 promoter, and cloning the other target gene into the polyhedron Downstream of the promoter (Ppolh), the reporter gene can trace the production of recombinant virus, the process of infecting cells and the expression of foreign proteins to facilitate the determination of specific cell collection time. Using pDsRed2-1 (Clontech) as a template, the DsRed gene was amplified by PCR, and the primers were Red-F (5'-TTACCATGGCCTCCTCCGA-3', Red-R (5'-TGACTACAGGAAC AGGTGGT-3'). The PCR product was purified by T4DNA After polymerase and dGTP treatment, it was cloned into the Bsu36I site of the donor vector pHTdual to generate the recombi...

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Abstract

The invention discloses a method for conveniently and efficiently constructing a recombinant baculovirus. The method mainly comprises the following steps: a donor plasmid which contains dual-promoter and can directionally and rapidly clone PCR product is constructed; (2) an I-SceI cassette is introduced into the genome of E.coliDH10B, simultaneously two I-Scel sites and a resistance gene are introduced into the baculovirus Bacmid, and the plasmid which can express Red-Gam recombinase is transformed into E.coliDH10B to construct a new receptor strain CTRDH10B / Bacmid / PML104; (3) the recombined donor plasmid is introduced into the CTRDH10B / Bacmid / pML104 to induce the expression of I-SceI endonuclease and the Red-Gam recombinase, and the recombinant Bacmid is obtained by homologous recombination in the bacterium; (4) the recombinant Bacmid DNA transfection insect cells are prepared by the conventional method and analyzed. The method needs no complex PCR verification and fussy plaque purification, and is really efficient, rapid and convenient. The method is especially suitable for constructing high-quality baculovirus cDNA library with large storage capacity.

Description

Technical field [0001] The invention belongs to the fields of genetic engineering and protein engineering, and is specifically a simple and efficient method for constructing recombinant baculovirus. Background technique [0002] Smith et al. (1983) used Autographa californica multiple nucleocapsid nucleopolyhedrovirus (AcMNPV, Autographa californica multiple nucleocapsid nucleopolyhedrovirus) as a vector to efficiently express human β-interferon in Spodoptera frugiperda cells Sf-21. The research results have opened up a new field of baculovirus as a vector for expressing foreign genes. With the advent of the post-genomic era, the field of rapid and high-throughput expression of various eukaryotic cDNA and functional proteins is full of challenges and opportunities. Because it has a super strong polyhedron promoter (Ppolh) that is safe for humans and mammals to drive the efficient expression of foreign genes in insect cells, convenient insect cell culture, simple virus opera...

Claims

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Application Information

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IPC IPC(8): C12N7/01C12N15/866
Inventor 姚伦广刘宗才阚云超张祥满刘飞张二辉
Owner NANYANG NORMAL UNIV
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