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Recombination bacillary viral vector skeleton plasmid and application thereof

A technology for recombining baculovirus and backbone plasmid, applied in the field of animal genetic engineering

Inactive Publication Date: 2010-12-22
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] For a long time, due to its host specificity, this highly expressed vector was only used to mediate the expression of foreign genes in its host insect cells

Method used

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  • Recombination bacillary viral vector skeleton plasmid and application thereof
  • Recombination bacillary viral vector skeleton plasmid and application thereof
  • Recombination bacillary viral vector skeleton plasmid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] by figure 1 The shown pFastBac-Dual (purchased from Invitrogen Company) and pSFV1 (purchased from Invitrogen Company) are materials, according to figure 1 Shown flow process, construction said plasmid of the present invention, concrete steps are as follows:

[0025] 1. The pFastBac-Dual and pSFV1 plasmids were digested with SphI and SpeI respectively, and the large fragment containing the backbone of the baculovirus transfer vector and the nonstructural protein nsPs1-4 of SFV and the linearized pFastBac-Dual were respectively recovered. The two fragments were ligated to obtain the intermediate plasmid I, namely pFB-G-SFV1;

[0026] 2. Use pCI-neo as a template to amplify the HCMV IE Enhancer / Promoter element with SphI restriction sites at both ends, such as figure 1 Middle Fragment I shown;

[0027] 3. Insert the HCMV IE Enhancer / Promoter element obtained in step 2 into the SphI site of the intermediate plasmid I to obtain the intermediate plasmid II, namely pFB-G...

Embodiment 2

[0031] 1. Construction of pFB-G-SFV-EGFP recombinant plasmid

[0032] In this example, the fluorescent reporter gene EGFP (Genebank serial number: 1377915) will be used to directly and objectively evaluate whether the modified vector obtained in the present invention can correctly express foreign genes, and compare the modified plasmid with the currently commonly used baculovirus backbone Plasmids vary in transfection efficiency and expression levels. Using plasmid pEGFP-C1 (purchased from ClonTech Company) as a template to amplify the EGFP sequence (Genebank sequence number: 1377915) with BamHI and SmalI restriction sites at both ends, insert the plasmid pFB-G- obtained in Example 1 The BamHI and SmalI restriction sites of SFV were cut to obtain the recombinant plasmid pFB-G-SFV-EGFP. The results are attached image 3 As shown in A.

[0033] 2. Transposition of recombinant plasmid containing pFB-G-SFV-EGFP

[0034] 2.1 Take out DH10Bac TM Competent cells (purchased from ...

Embodiment 3( application Embodiment 1

[0061]1. In a six-well plate with 9×10 5 The density inoculation number of each cell / 2ml / hole is GDC010 Syrian hamster kidney cell line BHK-21 (purchased from the commercial cell material of the Chinese Type Culture Collection Center in Wuhan University, China, see http: / / www.bio-equip.com / showlequip.asp? equipid=14757&division=2604 ) cells, cultured at 37°C for 1 h to allow the cells to adhere to the wall.

[0062] 2, with Ca 2+ , Mg 2+ Phosphate buffered saline solution (PBS, purchased from Sigma Company) was used to wash the cells three times, and the baculovirus Bac-G-SFV-EGFP was inserted into the six-well plate cells at a dose of 50 multiplicity of infection (MOI=50). After infection for 4 hours at °C, the supernatant was discarded, and fresh DMEM containing 10% newborn bovine serum (purchased from Gibico) was added.

[0063] 3. Place the six-well plate in CO at 37°C 2 In the incubator, cultivate for 48 hours, and detect the expression of exogenous gene EGFP by flu...

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Abstract

The invention belongs to the animal genetic engineering technical field, in particular relating to a construction for a framework plasmid of a recombinant baculovirus vector and application thereof. The invention acquires a framework plasmid pFB-G-SFV of a recombinant baculovirus transfer vector by cloning, and the plasmid is collected in China Center for Type Culture Collection (CCTCC) with the accession number of CCTCC NO: M207070. The invention also discloses the application of the plasmid pFB-G-SFV on the package of the recombinant baculovirus.

Description

technical field [0001] The invention belongs to the technical field of animal genetic engineering, and in particular relates to a recombinant baculovirus vector backbone plasmid and an application thereof. Background technique [0002] Baculovirus vectors are one of the most widely used viral vectors, and are widely used in gene therapy, gene function research, vaccine development and other fields. Baculovirus is the largest family of insect viruses, and its host range is mainly concentrated in the insect class Lepidoptera. At present, the insect baculoviruses used for exogenous gene expression are limited to the nuclear polyhedrosis virus genus, among which Autographa californica multiple nucleopolyhedrosis virus (AcMNPV) and Spodoptera frugiperda cells The system is a research model of baculovirus expression vector system commonly used in the world. In 1983, Smith et al. first reported the successful expression of human β-2 interferon in Spodoptera frugiperda cells by us...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/866C12N7/01
Inventor 肖少波方六荣赵骞江云波潘永飞
Owner HUAZHONG AGRI UNIV
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