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Expression unit, recombinant lentivirus expression vector, recombinant lentivirus and preparation method and application of recombinant lentivirus

A technology of recombinant lentivirus and expression vector, applied in the field of recombinant lentivirus and its preparation, can solve the problems of long course of disease, complex etiology, and easy emergence of drug resistance, etc.

Pending Publication Date: 2022-03-04
HENAN UNIVERSITY
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Patients with chronic pain have a long course of disease and complex etiology. During the use of traditional drugs, drug resistance is prone to occur and cause side effects on other organs. Obviously, this type of drug is not suitable for patients with chronic pain. Therefore, patients with chronic pain urgently need a long-term Effective drug analgesia to relieve psychological and physical unpleasant feelings

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  • Expression unit, recombinant lentivirus expression vector, recombinant lentivirus and preparation method and application of recombinant lentivirus
  • Expression unit, recombinant lentivirus expression vector, recombinant lentivirus and preparation method and application of recombinant lentivirus
  • Expression unit, recombinant lentivirus expression vector, recombinant lentivirus and preparation method and application of recombinant lentivirus

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preparation example Construction

[0042] The present invention also provides a method for preparing the above-mentioned recombinant lentivirus, which specifically includes the following steps:

[0043] It is obtained by co-transfecting the above-mentioned recombinant lentiviral expression vector with the lentiviral envelope vector and the packaging vector into a cell line.

[0044] In the present invention, the lentiviral envelope vector is preferably pMD2.G; the packaging vector is preferably psPAX2; and the cell line is preferably 293T cells. The present invention has no special limitation on the transfection method, and conventional transfection methods in the art can be used.

[0045] The present invention also provides the application of the above expression unit, recombinant lentiviral expression vector or recombinant lentivirus in the preparation of drugs for treating chronic pain.

Embodiment 1

[0050] 1. Construction of recombinant lentiviral expression vector 1

[0051] (1) Using the Ensembl tool to analyze the human PIRT gene, artificially synthesize the sequence SEQ ID NO.1 of the PIRT gene promoter, the 5' end restriction endonuclease Nsi I and the 3' end NheI;

[0052] (2) Digest the plasmid containing the artificially synthesized PIRT gene promoter element in step (1) with NsiI and NheI, and recover about 930 bp by gel electrophoresis to obtain the hPIRT promoter fragment.

[0053] (3) Ligate the hPIRT promoter fragment with T4 DNA ligase and the large fragment of the SYN-DsRed-SYN-GFP plasmid that was double-digested with Nsi I and Nhe I and recovered by gel electrophoresis, transform TOP 10 competent bacteria, and pick Bacterial culture was performed on a single colony and the plasmid was extracted for Nsi I and Nhe I digestion and sequencing identification to obtain the SYN-DsRed-hPIRT-GFP vector;

[0054] (4) In vitro synthesis sequence such as the botulin...

Embodiment 2

[0059] Example 2 Identification and Extraction of Recombinant Lentiviral Expression Vector

[0060] 1. Group double enzyme digestion to verify the correctness of the recombinant lentiviral expression vector construction

[0061] Take a 200 μL centrifuge tube, add each of the reagents in Table 1 to the centrifuge tube in turn, gently pipette to mix, and place it in a 37°C water bath for 3 hours. After 3 hours, add 10 μL 6×Loading Buffer to each centrifuge tube to stop the enzyme digestion and mix gently by pipetting. A 0.7% agarose nucleic acid gel was prepared and loaded for agarose gel electrophoresis. After electrophoresis, the nucleic acid gel was placed in G-BOX to take pictures, and the attached figure 2 .

[0062] Table 1 Add reagents

[0063] Reagent Volume (μL) Recombinant lentiviral expression vector DNA (1 μg / μL) 1 10×NEB Buffer 5 h 2 o

42 Enzyme 1 1 Enzyme 2 1 Total 50

[0064] Among them, different promo...

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Abstract

The invention relates to the technical field of biology, in particular to an expression unit, a recombinant lentivirus expression vector, a recombinant lentivirus and a preparation method and application of the recombinant lentivirus. According to the invention, a human PIRT gene promoter sequence and a rabbit RFT-I minimum promoter sequence are synthesized, and protease coding sequences for cutting SNARE are respectively inserted into the downstream of the two promoters to prepare two different recombinant lentiviral expression vectors and viruses. The lentivirus provided by the invention has the characteristics of specifically inhibiting peripheral pain generation and signal transduction, effectively cuts SNARE protein, inhibits secretion of pain neurotransmitters, and achieves the purpose of gene therapy of pain.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to an expression unit, a recombinant lentivirus expression vector, a recombinant lentivirus and a preparation method and application thereof. Background technique [0002] Pain is an important topic in the medical field, and many diseases and traumas are accompanied by pain. Pain will appear in one or more parts of the patient's body. After the onset, it will affect the patient's daily life and psychological state, thus bringing a series of unpleasant experiences to the patient. In 1979, the International Pain Society (IAPS) defined pain as: an unpleasant sensory and emotional experience accompanied by existing or potential tissue damage. According to the time and nature of pain, pain can be divided into acute pain and chronic pain. Among them, persistent or recurrent episodes of more than three months are called chronic pain. Chronic pain is very common, affecting approximately 20% ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/867C12N15/57A61K48/00A61K38/48A61P29/00
CPCC12N15/86C12N9/52A61K48/0008A61K48/005A61K38/4886A61K38/4893A61P29/00C12Y304/24069C12Y304/24068C12N2740/15043C12N2800/107
Inventor 王家富王婉至孟疆辉窦雨
Owner HENAN UNIVERSITY
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