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A kind of prbe-hcr-haat-hfixml plasmid and its construction and application

A prbe-hcr-haat-hfixml, pt-hcr-haat technology, applied in the direction of recombinant DNA technology, introduction of foreign genetic material using a vector, etc., can solve the problem that the amount of gene expression product needs to be further improved, and the efficiency of in vivo mediated integration is not high. problem, to achieve high expression, increase integration efficiency, and improve the effect of expression

Inactive Publication Date: 2016-01-20
YANGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the simple site-directed integration transgenic system containing this 16bpRBE still has the disadvantages that the efficiency of mediating integration in vitro is acceptable, the efficiency of mediating integration in vivo is not high, and the amount of gene expression products needs to be further improved (GeneTher.2009; 16( 5):589-95.)

Method used

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  • A kind of prbe-hcr-haat-hfixml plasmid and its construction and application
  • A kind of prbe-hcr-haat-hfixml plasmid and its construction and application
  • A kind of prbe-hcr-haat-hfixml plasmid and its construction and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1: The acquisition and purification of pRBE-CMV-FIXml without the CMV promoter vector

[0026] According to pRBE-CMV-FIX sequence (GeneTher.2009;16(5):589-95.JMolBiol.2006;358(1):38-45.), design specific primers (see Table 1-primers), meanwhile, in Restriction sites BglII and NheI were added to the 5' and 3' ends, respectively. Using pRBE-CMV-FIX as a template, the target band was amplified.

[0027] Table 1 - Primers

[0028]

[0029]

[0030] Prepare the PCR reaction system as shown in the table below for cloning pRBE-CMV-FIXml without the CMV promoter vector sequence:

[0031] Element

volume

wxya 2 o

72μl

PCR Buffer

10μl

dNTP

2μl

Mg 2+

8μl

Forward Primer (TEST-Bgl II)

2μl

Reverse Primer (TEST-Nhe I)

2μl

template

3μl (~100ng)

KOD plus neo

1μl

total

100μl

[0032] Carry out the PCR reaction according to the program shown in the ta...

Embodiment 2

[0036] Embodiment 2: "NheI-hAAT-BglII" fragment amplification

[0037] Using human genomic DNA (AmpFlSTR standard DNA, Applied Biosystems, USA) as a template, the NheI-hAAT-BglII DNA fragment was amplified with an upstream primer containing NheI and a downstream primer containing BglII. Prepare the PCR reaction system as shown in the table below for the amplification of the -NheI-hAAT-BglII-fragment:

[0038] Element

volume

wxya 2 o

2μl

Buffer

20μl

[0039] dNTP

2.5μl

Forward Primer (hAAT-F)

1μl

Reverse Primer (hAAT-R)

1μl

template

1μl

HS Taq

25μl

total

50μl

[0040] Carry out the PCR reaction according to the program shown in the table below:

[0041]

[0042] Prepare agarose gel with a concentration of 2%, and conduct constant voltage electrophoresis with a voltage of 120V. Use the AxyPrepDNA Gel Extraction Kit to recover the target DNA fragment.

[...

Embodiment 3

[0044] Embodiment 3: "SpeI-HCR-SpeI" fragment amplification

[0045] Using plasmid human genomic DNA (AmpFlSTR standard DNA, Applied Biosystems, USA) as a template, the SpeI-HCR-SpeI DNA fragment was amplified with SpeI-containing upstream primers and SpeI-containing downstream primers. Prepare the PCR reaction system as shown in the table below for the amplification of the -SpeI-HCR-SpeI-fragment:

[0046] Element

volume

wxya 2 o

14.3μl

Buffer

2μl

dNTP

0.4μl

Forward Primer (F-Spe I)

0.4μl

Reverse Primer (R-Spe I)

0.4μl

Mg +

1.6μl

template

0.5μl

Taq

0.4μl

total

20μl

[0047] Carry out the PCR reaction according to the program shown in the table below:

[0048]

[0049]Prepare agarose gel with a concentration of 2%, and conduct constant voltage electrophoresis with a voltage of 120V. Use the AxyPrepDNA Gel Extraction Kit to recover the target DNA frag...

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Abstract

The invention relates to pRBE-HCR-hAAT-hFIXml plasmid as well as construction and application thereof. The sequence of the plasmid is represented as SEQ ID NO.17, and the plasmid contains an hAAT promoter, an RBE (rep binding element) and an HCR (hepatic locus control region) control element. The plasmid and rep expression plasmid are injected into a mammal body at high pressure and can be used for targeted integration of an exogenous gene onto a specific site of AAVS1 (adeno-associated virus integration site1) on the No.19 chromosome of a human so as to express a coagulation factor IX of the human. With application of the expression plasmid, the coagulation factor IX of the human can be expressed stably, and the integration efficiency of an integration system in a liver is improved, so that higher expression quantity and longer expression time are achieved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a pRBE-HCR-hAAT-hFIXml plasmid and its construction and application. Background technique [0002] Among the many natural genetically modified events in nature, there are some events that have been shown to be safe during long-term evolution. Among them, the most meaningful human genome is an integration site called AAVS1 (adeno-associated virus integration site 1) on chromosome 19. Under natural circumstances, AAV is only integrated in AAVS1; under in vitro experimental conditions, more than 75% of the additional genes of AAVITR (inverted terminal repeat) can be integrated in AAVS1 under the guidance of rep protein. [0003] So far, a series of research results at home and abroad have not found any known human diseases related to AAV infection. The importance of this conclusion is that this conclusion is based on the premise that the AAV infection rate of adults is grea...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/85C12N15/66
Inventor 许正新陈金中叶娟张阿敏谢林俊
Owner YANGZHOU UNIV
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