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Method for targeted knockout of non-essential genes for Bombyx mori nuclear polyhedrosis virus replication

A karyotype polyhedron and gene technology, applied in the field of DNA recombination technology and gene targeting, can solve problems such as missing target genes, troublesome virus screening, and impact on virus functions.

Active Publication Date: 2014-02-19
TIANJIN YAOYU BIOLOGICAL TECH
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  • Abstract
  • Description
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Problems solved by technology

However, the problem of the marker gene left on the virus genome has not been fully improved, which has brought a huge impact on the function of the virus itself and brought a lot of trouble to the subsequent virus screening, so we used two simultaneous The method of source recombination not only deletes the target gene but also solves the problem of the marker gene on the viral genome, which is of great significance for future scientific experiments

Method used

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  • Method for targeted knockout of non-essential genes for Bombyx mori nuclear polyhedrosis virus replication
  • Method for targeted knockout of non-essential genes for Bombyx mori nuclear polyhedrosis virus replication
  • Method for targeted knockout of non-essential genes for Bombyx mori nuclear polyhedrosis virus replication

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of transfer vector pUC19-IE1-EGFP-SV40polyA:

[0037] 1. The recombinant plasmid PSK-IE1-EGFP constructed by Gao Zhen et al. (Gao Zhen, Hong Yeting, Zhou Fang et al. Cloning, expression analysis and isolation and identification of the BmAGO1 gene of the silkworm [OL]. [2012-01 -09]. China Science and Technology Papers Online, http: / / www.paper.edu.cn / index.php / default / releasepaper / content / 201201-252) are designed as templates:

[0038] (IE-1)F:5-TTT GTC GAC AGTCGTTTGGTTGTTCACGATCG-3 (The bold italic part is Sal restriction site for I)

[0039] (IE-1)R: 5-CTCGCCCTTGCTCACCATGATTTGCAGTTCGGGACATAAAT-3

[0040] (EGFP)F: 5-TTATGTCCCGAACTGCAAATCATGGTGAGCAAGGGCGAG-3

[0041] (EGFP)R: 5-AATGTGGTATGGCTGATTATGATCTTACTTGTACAGCTCGTC-3

[0042] (SV40polyA) F: 5- CATGGACGAGCTGTACAAGTAAGATCATAATCAGCCATACC-3

[0043] (SV40polyA)R: 5-CGG GGTACC ATCCAGACATGATAAGATACATTG-3 (The bold italic part is KpnI enzyme cutting sites) (for the PCR results of the...

Embodiment 2

[0069] Example 2: Construction of recombinant transfer vector pUC19-lef7-IE1-EGFP-SV40polyA-gp64

[0070] Using the BmNPV viral genome (wherein, the viral DNA extraction kit was purchased from Sangon Bioengineering Co., Ltd.) as a template, the homologous sequences lef7 and gp64 were amplified, and the genes lef7 and gp64 were cloned into From the transfer vector pUC19-IE1-EGFP-SV40polyA, the recombinant transfer vector pUC19-lef7-IE1-EGFP-SV40polyA-gp64 was obtained.

[0071] Then, the recombinant transfer vector pUC19-lef7-IE1-EGFP-SV40polyA-gp64 was identified by PCR and double enzyme digestion, and the electrophoresis results are shown in Figure 5 , Image 6 , Figure 5 The identification results showed that the homologous sequence at one end of the target gene was cloned into the transfer vector pUC19-IE1-EGFP-SV40polyA, Image 6 The identification results showed that the homologous sequences at both ends of the target gene had been successfully cloned into the trans...

Embodiment 3

[0072] Embodiment 3: Extraction of BmNPV virus genome:

[0073] 1) Collect about 100ml of the cell fluid infected by the silkworm nuclear polyhedrosis baculovirus, freeze and thaw three times at -80°C and 37°C respectively, centrifuge at 12000rpm for 1h at 4°C, and take the supernatant;

[0074] 2) Centrifuge at 30,000 rpm for 1 hour at 4°C, discard the supernatant, collect virus particles, dissolve in 1 x TE, add 20 μL, 20 mg / ml proteinase K (purchased from Invitrogen), 20 μL, 10% SDS, 50°C Water bath overnight (at least 12h);

[0075] 3) Add 10 μL of 10mg / ml RNaseA (purchased from Beijing Dingguo Biotechnology Company), and bathe in water at 37°C for 1 hour;

[0076] 4) with phenol, chloroform extraction virus genome:

[0077] a. Gently mix with (equal volume) Tris-balanced phenol for 5 minutes, and centrifuge at 12000rpm for 10 minutes;

[0078] b. Extract again with phenol, chloroform and isoamyl alcohol with a volume ratio of 25:24:1 (the sum of the three volumes is th...

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Abstract

The present invention relates to a method for targeted knockout of non-essential genes for replication of the Bombyx mori nuclear polyhedrosis virus BmNPV. The method is as below: using BmNPV as a material to amplify a homologous sequence at both ends of a non-essential region for replication by PCR and cloning it into a vector pUC19; splicing successively an IE1 early promoter, a marker gene (EGFP) and a termination sequence SV40polyA of BmNPV by an overlapping PCR method and cloning them to the abovementioned vector pUC19 to obtain a recombinant transfer vector pUC19-lef7-IE1-EGFP-SV40polyA-gp64; co-transfecting BmN cells using the vector and the genome of wild BmNPV and carrying out homologous recombination to obtain a recombinant virus RBmNPV-EGFP with a fluorescent marker; and co-transfecting BmN cells using genomic DNA thereof and a transfer vector pUC19-lef7-gp64 without marker gene and carrying out homologous recombination to obtain a recombinant virus RBmNPV without fluorescent marker gene. The present invention solves the problem of the presence of marker genes in recombinant virus genomes and improves the screening efficiency of positive recombinant viruses, and thus the marker genes can be reused.

Description

technical field [0001] The invention relates to the fields of DNA recombination technology and gene targeting technology, in particular to a method for targeting and knocking out non-essential genes for Bombyx mori nuclear polyhedrosis virus replication. Background technique [0002] Gene knockout was developed in the second half of the 1980s by applying the principle of DNA homologous recombination. In the early 1980s, the success of the isolation and in vitro culture of embryonic stem cells (ES cells) laid the technical foundation for gene knockout. In 1985, the first demonstration of the existence of homologous recombination in mammalian cells laid the theoretical foundation for gene knockout. By 1987, Thompsson established for the first time a complete ES cell gene knockout mouse model. Until now, gene knockout by gene homologous recombination is still the most commonly used method in the construction of gene knockout animal models. It is also an important means of st...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/65C12N15/66C12N15/63C12N15/09
CPCC12N2710/14143C12N15/09C12N15/86C12N15/62C12N15/63
Inventor 田凤鸣陈剑清舒特俊张耀洲
Owner TIANJIN YAOYU BIOLOGICAL TECH
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