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PAlb-uPA slow virus vector and preparation method and application thereof

A lentiviral vector and vector technology, applied in the field of genetic engineering, can solve the problems that hinder the construction, popularization and use of human chimeric liver mouse models, the difficulty of screening, and the high mortality rate of offspring newborn mice, so as to achieve convenient regulation, The effect of simple screening and simple preparation method

Inactive Publication Date: 2012-08-08
THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the urokinase-type plasminase transgenic mice obtained by transgenic methods have high mortality rate and difficult screening of offspring, which greatly hinders the construction, promotion and use of human chimeric liver mouse models

Method used

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  • PAlb-uPA slow virus vector and preparation method and application thereof
  • PAlb-uPA slow virus vector and preparation method and application thereof
  • PAlb-uPA slow virus vector and preparation method and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0033] Embodiment 1, PAlb-uPA lentiviral vector construction

[0034] Lenti-X used in the present invention TMTet-on Advanced Inducible Expression System Kit was purchased from Clontech Company (containing pLVX Tight Puro vector and pLVX-Tet-On vector); pMD19-T Simple vocter vector, PCR reaction reagent, reverse transcription kit, in-Fusion Dry-Down PCR cloning kit and DNA polymerase primeSTAR HS were purchased from TaKaRa Company; RNA extraction kit RNAprep pure Tissue Kit and gel recovery kit were purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd.; plasmid extraction kit was purchased from Qiagen Company; Trizol extract, RNase H and FV membrane were purchased from Invitrogen; pLKO.1 cloning vector, polybrene Ploybrene and trypsin were purchased from Sigma; DMEM medium, ethylenediaminetetraacetic acid (EDTA) and Opti-MEM medium Purchased from GIBCO; 293T cells, P-eGFP plasmid and H2.35 mouse hepatocytes (ATCC) were preserved by our Institute.

[0035] 1. JG...

Embodiment 2

[0041] Example 2, PAlb-uPA lentiviral vector functional verification

[0042] 1. PAlb-uPA lentiviral vector virus packaging

[0043] Take well-grown 293T cells, digest and wash them, and use Opti-MEM selection medium to make cell suspension, then transfer them to different culture dishes for further culture, and set them aside; then take two 50mL sterile centrifuge tubes, of which Add 0.5 mL of the PAlb-uPA lentiviral vector JGY-PLV at a concentration of 0.5 mg / mL, 1 mL of the viral envelope protein expression plasmid pMD2.G at a concentration of 0.2 mg / mL and 0.5 mL of the Viral structural protein expression plasmid psPAX2, then add Opti-MEM medium to make up to 18mL, and set aside; add 0.5 mL Fran-EZ solution and 17.5 mL Opti-MEM medium to the other tube, mix well, then add to the first test tube, Shake while adding until it is completely added to obtain plasmid transfection solution. Then, the obtained plasmid transfection solution was added dropwise to the cell culture...

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Abstract

The invention discloses a PAlb-uPA slow virus vector, which contains an albumin promoter PAlb, a urokinase plasminogen activator gene uPA, a tetracycline trans-acting factor rtTA / rtTA2S-M2 of the albumin promoter PAlb and a PTight promoter for regulating and controlling the urokinase plasminogen activator gene uPA. The invention further discloses a preparation method and an application of the PAlb-uPA slow virus vector. The preparation method is simple; the obtained slow virus vector can be used for preparing a urokinase fibrinolysis protease mouse liver injury model; and an effective research platform is provided for the clinical research of a human hepatitis virus infection mechanism and an anti-virus medicament.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to a PAlb-uPA lentiviral vector, and also to a preparation method and application of the PAlb-uPA lentiviral vector. Background technique [0002] In addition to humans and primates, human hepatitis virus does not have a natural small animal infection host, which seriously hinders the research on the mechanism of human hepatitis virus infection and antiviral drugs. At present, only chimpanzees are the best in vivo experimental model for studying human hepatitis virus infection, but the use of protected rare primates is subject to many restrictions such as ethics and funding. Therefore, it is an urgent problem to establish cheap and easy-to-obtain small animal models. The mouse animal model developed in recent years that can carry human liver tissue provides a good platform for the research on the mechanism of hepatitis virus infection and antiviral drugs. The study found that th...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/66A01K67/027
Inventor 毛青白家驷李俊刚
Owner THE FIRST AFFILIATED HOSPITAL OF THIRD MILITARY MEDICAL UNIVERSITY OF PLA
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