Lentivirus packaging vector system, lentivirus, construction method of lentivirus and kit

A technology of lentiviral packaging and vector system, applied in the field of lentiviral packaging vector system, can solve the problems of limiting the efficiency of SARS-CoV-2 drug development and other issues

Active Publication Date: 2021-03-05
YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] However, since SARS-CoV-2 is a pathogen of severe infectious diseases of Biosafety Level 3 (BSL-3), the isolation and cultiva...

Method used

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  • Lentivirus packaging vector system, lentivirus, construction method of lentivirus and kit
  • Lentivirus packaging vector system, lentivirus, construction method of lentivirus and kit
  • Lentivirus packaging vector system, lentivirus, construction method of lentivirus and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of cell lines expressing ACE2 receptor

[0043] (1) Pack pLV[Exp]-Bsd-CMV>hACE2 (from Yunzhou Bio) into a lentivirus with VSV-G;

[0044] (2) Transduce the HKE293T cells with the lentivirus in step (1), screen with a suitable concentration of blasticidin Bsd (Blasticidin) until the blank cells die, and obtain a stable transfection strain expressing the ACE2 receptor, which is denoted as HEK293T- ACE2 stable transfection strain. The image of HEK293T-ACE2 stable transfection strain under 200× microscope is as follows: figure 1 shown. Depend on figure 1 It can be seen that HEK293T-ACE2 stable transfection plants are in good growth state.

[0045] (3) Extract the genomic DNA of the HEK293T-ACE2 stable transfection strain and perform qRT-PCR detection, wherein the qPCR primers are as follows: ACE2-F: 5'-GCAGCCCACCTAAGCATT-3' (SEQ ID No: 6); ACE2-R: 5' - CCATCCACCTCCACTTCTCT-3' (SEQ ID No: 7); HKE293T was used as blank control.

[0046] The qRT-PCR results s...

Embodiment 2

[0050] Detect the expression level of S protein after transiently transfecting HEK293T cells with different S protein expression vectors (the vector is used as an envelope protein expression plasmid during the virus packaging process)

[0051] (1) Transient HEK293T cells with the same amount of 10 kinds of S protein expression vectors, each using Lipofectamine 2000 as a transfection reagent, to obtain HEK293T cells corresponding to each S protein expression vector. Among the 10 S protein expression vectors, the nucleotide sequence of S_a is shown in SEQ ID No: 8, which encodes the wild-type S protein; the nucleotide sequence of S_c is shown in SEQ ID No: 9, which encodes the D614G mutant S Protein; the nucleotide sequence of S_b is as shown in SEQ ID No: 10, is optimized on the basis of S_a and adds the nucleic acid fragment corresponding to the purification tag 3×Flag (that is, the nucleotide sequence is as shown in SEQ ID No: 3 The nucleic acid fragment corresponding to the ...

Embodiment 3

[0058] Comparison of lentiviral packaging effects of different S protein expression vectors

[0059] The partial S protein expression vector of embodiment 2 is selected to carry out the lentiviral packaging test, and the materials used in the test include the following:

[0060] Vector 1: pRP[Exp]-CMV>S_a; Vector 2: pRP[Exp]-CMV>S_b; Vector 4: pRP[Exp]-CMV-human beta globin intron>S_b; Vector 6: pRP[Exp]-CAG >S_b; Vector 7: pRP[Exp]-CMV-humanbeta globin intron>S_c; Vector 8: pRP[Exp]-CMV-human beta globin intron>S_d; Vector 9: pRP[Exp]-CAG>S_c; Vector 10 : pRP[Exp]-CAG>S_d; lentiviral packaging expression plasmid: pLV[Exp]-CMV>EGFP; gag-pol expression plasmid (hereinafter referred to as PLV4): gag gene and pol gene containing lentivirus; rev expression plasmid ( Hereinafter referred to as PLV5): encoding lentiviral Rev regulatory factor; RIPA lysate (strong), Biyuntian P0013B; Sars SpikeProtein Antibody (SARS-S protein antibody), (novusbio NB100-56578SS); horseradish peroxida...

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Abstract

The invention relates to a lentivirus packaging vector system, a lentivirus, a construction method of the lentivirus and a kit. The lentivirus packaging vector system comprises two or more plasmids; the plasmids can generate the lentivirus with only one-time infection capability and without replication capability, and each plasmid includes a promoter, an enhancer and a nucleic acid fragment used for encoding envelope protein, wherein the promoter, the enhancer and the nucleic acid fragment are connected in sequence; the nucleic acid fragment used for encoding the envelope protein includes a nucleic acid fragment used for expressing S protein of the SARS-CoV-2 or a nucleic acid fragment used for expressing a mutant of the S protein of the SARS-CoV-2; and the mutant of the S protein of the SARS-CoV-2 is a mutant obtained after the 614 amino acid at the C end of an S1 subunit of the S protein of the SARS-CoV-2 is mutated into glycine from aspartate. The lentivirus prepared by utilizing the lentivirus packaging vector system can be used for screening drugs aiming at SARS-CoV-2 or mutant strains thereof, and is high in safety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentivirus packaging vector system, lentivirus and its construction method, and kit. Background technique [0002] Severe acute respiratory syndrome coronavirus 2 (Severe acute respiratory syndrome coronavirus 2, SARS-CoV-2) is a new type of betacoronavirus with high pathogenicity to humans discovered after SARS-CoV and MERS-CoV. Segmented positive-sense single-stranded RNA enveloped virus. [0003] However, since SARS-CoV-2 is a pathogen of severe infectious diseases of Biosafety Level 3 (BSL-3), the isolation and cultivation of SARS-CoV-2 must be carried out under conditions with BSL-3, which limits Efficiency in the development of drugs against SARS-CoV-2. Contents of the invention [0004] Based on this, it is necessary to provide a lentiviral packaging vector system. The lentiviral envelope protein constructed by the lentiviral packaging vector system is the S protein of S...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/50
CPCC12N15/86C07K14/005C12N2740/15043C12N2740/15052C12N2800/107C12N2770/20022
Inventor 施金秀罗燕兰胜叶知晟
Owner YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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