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Phi C31 site specific recombinase mutant, preparation method thereof and application

A mutant and recombinase technology, applied in the field of genetic engineering, can solve the problem of low efficiency of mediating site-specific integration, and achieve the effect of improving recombination activity and specificity

Inactive Publication Date: 2009-06-17
FUDAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, the ΦC31 integrase system still has basic problems to be solved, such as the efficiency of mediating site-specific integration in mammalian cells is much lower than that in prokaryotic cells, and it can recognize multiple pseudo-sites on chromosomes of mammalian cells

Method used

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  • Phi C31 site specific recombinase mutant, preparation method thereof and application
  • Phi C31 site specific recombinase mutant, preparation method thereof and application

Examples

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Effect test

Embodiment 1

[0018] Example 1 Verification of the activity of ΦC31 site-specific recombinase mutants on prokaryotic cells

[0019] After loading the ΦC31 site-specific recombinase mutant gene sequence into the pINT-T expression vector, transform the E.coli cells carrying the pRES-MpsL1 vector, and spread it on the X-gal plate. After induction at different temperatures (30°C to 37°C first), observe the appearance time (3-12h), color depth and quantity of blue clones.

[0020] The result is as figure 1 with figure 2 As shown, 4 hours after temperature induction, almost all clones on the plate expressing the ΦC31 site-specific recombinase mutant were blue, and the blue color was darker, while the plate with the ΦC31 site-specific recombinase as the control On , there are fewer blue clones and they are lighter in color. This shows that the ΦC31 site-specific recombinase mutant has higher activity.

Embodiment 2

[0021] Example 2 Verification of the activity of ΦC31 site-specific recombinase mutants on eukaryotic cells

[0022] Construct eukaryotic expression vectors of ΦC31 site-specific recombinase mutants and eukaryotic expression vectors carrying attB sites, transfect mouse 3T3 cells, and apply 200 μg / ml hygromycin B to 30% of transfected cells 48 hours later. After 14 days of drug screening, clones were counted; single clones were amplified, and DNA was extracted by DNEasy; the recombination results were detected by PCR. The sequencing results of the PCR product showed that the first 10 bp of the obtained sequence was the attB site sequence, and the exchange center TTG (nt.11-13) was followed by the mouse genome mpsL1 site sequence. This shows that the ΦC31 site-specific recombinase mutant of the present invention can specifically mediate gene recombination at the mpsL1 site.

Embodiment 3

[0023] Example 3 Verification of integration efficiency of ΦC31 site-specific recombinase mutants on eukaryotic cells

[0024] 50ng of plasmid pHZ-attB and 5μg of expression mutant plasmids were co-transfected into mouse 3T3 cells, and the plasmid pCmv-Int of φC31 integrase was used as a control, and the Lipofectamine method was used to complete. 48 hours after transfection, 70% of the transfected cells were subjected to tissue DNA extraction. Genomic DNA was extracted with the DNEasy tissue kit. These DNAs were used for quantitative PCR detection to find the frequency of recombination at the mpsL1 locus. Design PCR forward primers and reverse primers based on the attB sequence and mpsL1 site sequence, and design a probe attB sequence integrated in the mpsL1 site binding region, which is mainly used to detect site-specific reactions at the mpsL1 site. Quantitative detection of PCR was accomplished by SYBR Green kit (Finzymes, F-430S), and the amplification reaction was carri...

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Abstract

The invention belongs to the genetic engineering field, especially to a site-specific recombinase, a preparation method thereof and applications. A phi C31 site-specific recombinase is an effective tool for mammalian cells genetic operation, but its mediated integration site specificity and integration efficiency on mammalian cells await further improvement. Thus the invention provides a phi C13 site-specific recombinase mutant capable of identifying a genome MpsL1 site with specificity and having higher activity. Thereby the site-specific recombinase of the invention can be an effective tool for the genetic engineering operation, especially the gene fixed-point insertion and recombination, and open up a novel path for researching the gene function analysis and the gene therapy.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and specifically relates to a site-specific recombinase, its preparation method and application. Background technique [0002] ΦC31 integrase belongs to the serine recombinase family, the genome size is about 2.1Kb, the open reading frame encodes 613 amino acids, and the molecular weight is about 67Kda. ΦC31 integrase can recognize the specific attachment point (attB) consisting of at least 34bp on the bacterial genome and the specific attachment point (attP) consisting of at least 39bp on the phage genome, and can mediate attB and attP without cofactors DNA recombination between. Studies have found that in mouse and human cell lines modified by attP sites, the site-specific integration efficiency mediated by ΦC31 integrase is 10-20 times higher than the efficiency of random integration spontaneous background; The gene targeting efficiency is 2-3 orders of magnitude higher; 10-100 times highe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/00C12N15/09C12N5/10C12Q1/68
Inventor 朱焕章辛清婷马金芳刘韶辉唐丽莎彭斯曼余龙
Owner FUDAN UNIV
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