In-fusion cloning method for recombinant baculovirus

A recombinant baculovirus and seamless cloning technology, applied in the biological field, can solve the problem of no new breakthrough in the baculovirus recombination method, and achieve the effect of simple operation, simple operation and high efficiency

Inactive Publication Date: 2016-02-03
杭州洪晟生物技术股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But in general, these methods either still require plaque screening, or require specific strain recombinant cloning, or rely on special recombinases and marker genes, and there is no new breakthrough in the baculovirus recombination method

Method used

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  • In-fusion cloning method for recombinant baculovirus
  • In-fusion cloning method for recombinant baculovirus
  • In-fusion cloning method for recombinant baculovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1. Construction of replication-deficient baculovirus vector BmBacmid-CAT-KO1629:

[0063] According to the tetragonal polyhedron BmNPV virus genome (accession number in GenBank database: JQ991009.1; or refer to http: / / www.paper.edu.cn / releasepaper / content / 201202-788) orf1629 gene, BAC fragment sequence and pKD3 plasmid Primer KO-1629F (5′- atgggaattagccatggtcc-3′) and KO-1629R (5′- AAAGACGCTGAAAATCATTTGATTTTCGCTCTAACATACCAC tgtaggctggagctgcttc-3'), wherein the sequence in the box is the Bsu36I site, the part marked by the wavy line is the terminal sequence of the BAC fragment, and the underlined part is the 3' terminal sequence of the orf1629 gene, using the pKD3 plasmid as a template, using KOD-FX DNA polymerase Amplify the CAT expression cassette and synthesize the targeting fragment CAT-KO1629.

[0064] The PCR reaction system is:

[0065]

[0066] The PCR reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 98°C for ...

Embodiment 2

[0069] Example 2, Construction of linearizable replication-deficient baculovirus vector BmBacmid-KO1629

[0070] BmBacmid-CAT-KO1629DNA was extracted according to the BacmidDNA extraction method of Invitrogen Company. BmBacmid-CAT-KO1629DNA was digested with Bsu36I, and the digested product was extracted with phenol / chloroform, precipitated with ethanol and washed, then dissolved in water, added an appropriate amount of T4 DNA ligase and ligation buffer, and ligated overnight at 12°C. About 100ng of the ligation product was taken to electrotransform DH10B cells, and then recombinant clones were screened by Kan resistance and Cm sensitivity. The recombinant BmBacmid DNA was extracted by GenopurePlasmidMidiKit of Roche Company, and the recombinant BmBacmid was identified by Bsu36I digestion. Such as figure 1 , Bsu36I digestion of recombinant BmBacmidDNA produced only a 23kb fragment, indicating that CAT was successfully excised. The obtained recombinant BmBacmid is the linear...

Embodiment 3

[0072] Example 3, Synthesis of Recombined Rescue DNA Fragments

[0073] The recombinant rescue DNA fragment includes a promoter, a target gene and an essential gene rescue DNA sequence. This example uses the green fluorescent protein (AcGFP) gene as the target gene as an example to illustrate the synthesis of the recombinant rescue DNA fragment.

[0074] First, according to the nuclear polyhedrosis virus genome sequence, chemically synthesized the polyhedron gene promoter fragment (Pph): 5'-TGGAAATAATAACCATCTCGTAAATAAATAAGTATTTTACTGTTTTCGTAACGGTTTTGTAATAAAAAAACCCTATAAATATTCCGAATTATTCATACACCCCCCACCATC-3' and the essential gene rescue DNA fragment (orf16293'-HA50) containing a 50bp homology arm: 5 '-AACACTATACATTGTTATTAGTACATTTATTAAGCGTTAGATTCTGTGCGTTGTTGATTTACAGACAATTGTTGTACGTATTTTAATAACTCATTAAATTTATAATCTTTAGGGTGGTATGTTAGAGCGAAAATCAAATGATTTTCAGCGTCTTTGTAT-3'. Primers GFP-F (5'-GAATTATTCATACACCCCACCATCATGTCGTACTACCATCACCATC-3') and GFP-R (5'-TGTACTAATAACAATGTATAGTGTTGAATTCTCACTT...

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Abstract

The invention discloses an in-fusion cloning method for recombinant baculovirus. The method employs two main constitution members: a linearizable replication-deficient baculovirus vector and a rescued recombination DNA fragment, the above two members are directly subjected to recombination in an insect cell for generating recombinant baculovirus. DNA recombination of the linearizable replication-deficient baculovirus vector and the rescued recombinant DNA fragment in the insect cell relates to homologous recombination and / or non-homologous end joining of 10-50 bp homologous arms. The method is applicable to high-flux construction of recombinant virus and one-step cloning of multiple genes for multiple expression.

Description

technical field [0001] The invention belongs to genetic engineering and protein engineering in the field of biotechnology, and specifically relates to a method for recombination and construction of baculovirus, which can be used for expressing recombinant proteins, constructing gene therapy vectors, preparing pseudovirus-like particle vaccines, and the like. Background technique [0002] Baculovirus Vector Expression System (BEVS) is a eukaryotic expression system that uses insect baculovirus as a vector to express foreign proteins in insects or host insect cells. Because the promoters of very late genes (ph and p10) can strongly drive the expression of foreign genes, they are often used to construct recombinant viruses and efficiently express target proteins. Since Smith et al. used AcMNPV as an expression vector to express human β-interferon at a high level in 1983, BEVS has expressed tens of thousands of foreign proteins in the past three decades, and thus has become the ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/866
Inventor 陈健钱月忠费伟强郝学敏陈琴
Owner 杭州洪晟生物技术股份有限公司
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