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Cell line capable of realizing induced rescue of rhabdovirus as well construction method and application of cell line

A rhabdovirus and construction method technology, applied in the biological field, can solve the problems affecting the rescue efficiency of recombinant rhabdovirus particles, the rescue efficiency of the restricted four-plasmid system, and application limitations, so as to achieve improved rescue efficiency, high rescue efficiency, and operation Simple and fast effect

Inactive Publication Date: 2018-01-09
南京普菲科医药科技有限公司
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AI Technical Summary

Problems solved by technology

[0005] At present, the application of the traditional rhabdovirus rescue system in the world is relatively limited. Due to the problem of the rescue efficiency of the four-plasmid system in BHK cells (hamster kidney cells), it has not been widely used. The limitation lies in the difference between the plasmids. Competitive expression between rhabdoviruses and rhabdovirus RNP polymers need to be combined in a specific ratio in order to efficiently complete the operation of the virus reverse genetics system. On the contrary, the amount of transient transfection of the three helper virus genes determines the RNP complex. The stability of the compound, as well as the increased toxicity to the cell line used to package the rescue virus, seriously affects the rescue efficiency of the recombinant rhabdovirion

Method used

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  • Cell line capable of realizing induced rescue of rhabdovirus as well construction method and application of cell line
  • Cell line capable of realizing induced rescue of rhabdovirus as well construction method and application of cell line
  • Cell line capable of realizing induced rescue of rhabdovirus as well construction method and application of cell line

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Experimental program
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Effect test

Embodiment 1B

[0041] Example 1 BHK21 (IFNAR1KO) cells

[0042] Establish BHK21 cells with IFNAR1 gene deletion, referred to as BHK21 (IFNAR1KO) cells, and on the basis of this cell line, use lentivirus infection to stably express TetOn-3G protein.

[0043] Studies have shown that the production and expression of type I interferon at the background level of cells in vitro has a certain inhibitory effect on the integration efficiency of lentivirus. At the same time, the expression of interferon also has a certain impact on the rescue of rhabdovirus ( image 3 ), this discovery prompts us to establish a cell model with impaired type I interferon pathway, which is constructed as follows:

[0044] Step 1: Knock out mouse-derived IFNAR1 (mIFNAR1) by CRISPR-Cas9 method: According to CRISPR-Cas9 technology, first design a short-chain sgRNA through http: / / crispr.mit.edu website, and design the mIFNAR1 sgRNA sequence as shown in Table 1 Shown: 5'-GAGACGGGAACATGTGGGCAC-3'. The synthesized DNA duple...

Embodiment 2

[0052] On the basis of BHK21 (IFNAR1KO) cells, a BHK cell line stably expressing TetOn3G protein was constructed, and a new cell line was named BHK (TetOn).

[0053] The synthetic TetOn3G gene fragment (synthesized by Nanjing GenScript Genetics Co., Ltd., the sequence is SEQ ID NO: 13) was digested, connected to the lentiviral vector FG with T4DNA ligase (NEB Company), and transformed into Stable2-sensitive State cells (purchased from Invitrogen), and denoted as FG-TetOn-3G herein, this lentiviral vector system integrates the target gene into the host cell for stable and continuous expression. The lentivirus was rescued in 293FT cells through the third-generation lentivirus packaging system, namely pGag / Pol, pRSV-Rev and pMD2.G four-plasmid system, and the lentivirus FG-TetOn-3G was stabilized in BHK (IFNAR1KO) cells expression, the cells were named BHK (TetOn). The specific operation is divided into two steps: virus packaging and infection:

[0054] A) Lentiviral packaging:...

Embodiment 3

[0058] The BHK (IFNAR1KO) cell line that can induce the expression of pT7pol / pP / pN at the same time was constructed.

[0059] a. Construction methods of three different resistant lentivirus-inducible expression vectors:

[0060] Table 2 Primer sequence list

[0061]

[0062]

[0063] First, PCR amplifies the specific primers for the structures P, N, and L of the Rhabdovirus (take VSV-MuddSummer strain as an example). The entire gene sequence of VSV-MuddSummer was synthesized by Nanjing KingScript Biotechnology Co., Ltd. The specific sequence is shown in SEQ IDNO:14, the sequence of specific amplification P, N, L primer is SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO: 12. The conditions for amplification of the target gene in pT7pol, pP, and pN are pre-denaturation at 98°C for 2 minutes; and then 32 cycles. Extend at 72°C for 10 min.

[0064] Use NotI and AscI (purchased from NEB Company) to double-digest the PCR product and pEntry vecto...

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Abstract

The invention provides a cell line capable of realizing induced rescue of rhabdovirus as well a construction method and an application of the cell line. The cell line is a BHK(TetOn) cell line for performing lentivirus induced expression on T7pol / P / N. The rhabdovirus construction method comprises the following steps: transfecting dual plasmids, namely a virus polymerase gene plasmid pL and a rhabdovirus core framework plasmid pCore, into the BHK(T7pol / P / N-TetOn) cell line according to a specific ratio, thereby rapidly obtaining a recombinant rhabdovirus. A vector for performing induced expression on T7pol / P / N is integrated into a BHK(TetOn) cell genome through pGag / Pol, pRSV-Rev and pMD2.G lentivirus plasmid system. According to the cell line disclosed by the invention, the rhabdovirus assembly can be efficiently subjected to rapid rescue, and screening and identification can be completed. Compared with the traditional multi-plasmid rescue system, the plasmid system in the invention has the advantage that the rescue efficiency of the recombinant rhabdovirus is greatly improved.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a cell line capable of inducibly rescuing rhabdoviruses, a construction method and application thereof. Background technique [0002] Rhabdoviridae virions are rod-shaped or bullet-shaped. "Rhabdo" comes from the Greek word "Rhabdos", meaning rod-shaped. The size of the virus particle is (130~380)×70nm, the core is a helical symmetrical nucleocapsid, which contains a single-stranded negative-strand RNA. When the virus infects the host cell, it is transcribed into mRNA by the RNA polymerase in the virion. The outer layer of the core shell has a lipoprotein envelope, and there are glycoprotein protrusions on the membrane. Viruses proliferate in the cytoplasm and are released by budding. [0003] Some viruses only reproduce in mammals, fish, arthropods or other invertebrates, many viruses have both arthropods and vertebrates, and some infect plants and some herbivorous a...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/867C12N15/90C12N15/85C12N7/00C12R1/93
CPCC12N5/10C12N7/00C12N15/85C12N15/867C12N15/90
Inventor 王红伟楼建华徐章敏
Owner 南京普菲科医药科技有限公司
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