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Lentiviral packaging vector system, lentivirus and its construction method, kit

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

Active Publication Date: 2022-04-12
YUNZHOU BIOSCIENCES (GUANGZHOU) INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 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

Method used

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  • Lentiviral packaging vector system, lentivirus and its construction method, kit
  • Lentiviral packaging vector system, lentivirus and its construction method, kit
  • Lentiviral packaging vector system, lentivirus and its construction method, kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Preparation of cell lines expressing ACE2 receptors

[0043] (1) pLV[Exp]-Bsd-CMV>hACE2 (from Yunzhou Biology) was packaged into lentivirus with VSV-G;

[0044] (2) The lentivirus in step (1) was transduced into HKE293T cells and screened with blasticidin Bsd (Blasticidin) at an appropriate concentration until the blank cells died to obtain a stable transfected strain expressing the ACE2 receptor, denoted as HEK293T- ACE2 stable transfected strain. The image of HEK293T-ACE2 stable transfected strain under 200× microscope is as follows figure 1 shown. Depend on figure 1 It can be seen that the HEK293T-ACE2 stably transformed strain grows well.

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

[0046] The results of qRT-PCR showed that com...

Embodiment 2

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

[0051] (1) Transfect HEK293T cells with the same amount of 10 kinds of S protein expression vectors using Lipofectamine 2000 as transfection reagent, respectively, 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, encoding the wild-type S protein; the nucleotide sequence of S_c is shown in SEQ ID No: 9, encoding the D614G mutant S Protein; the nucleotide sequence of S_b is shown in SEQ ID No: 10, which is the nucleic acid fragment corresponding to the purification tag 3×Flag after optimization on the basis of S_a (that is, the nucleotide sequence is shown in SEQ ID No: 3). The nucleic acid fragment of S_d+3×Flag correspon...

Embodiment 3

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

[0059] Part of the S protein expression vector of Example 2 was selected for lentiviral packaging test, and the materials used in the test included 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): containing lentivirus gag gene and pol gene; rev expression plasmid ( hereinafter referred to as PLV5): encoding lentivirus Rev regulator; RIPA lysate (strong), Biyuntian P0013B; Sars SpikeProtein Antibody (antibody against SARS-S protein), (novusbio NB100-56578SS); horseradish peroxidase labeled goa...

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Abstract

The invention relates to a lentivirus packaging vector system, a lentivirus, a construction method thereof, and a kit. The lentiviral packaging vector system consists of two or more plasmids, which can produce lentiviruses with only one infection ability but no replication ability. One of the plasmids contains sequentially linked promoters, enhancers, and encoding envelope proteins A nucleic acid fragment, the nucleic acid fragment encoding the envelope protein comprises a nucleic acid fragment for expressing the S protein of SARS-CoV-2 or a nucleic acid fragment for expressing a mutant of the S protein of SARS-CoV-2, for expressing SARS-CoV-2 The mutant of the S protein of CoV-2 is that the 614th amino acid at the C-terminus of the S1 subunit of the S protein of SARS-CoV-2 is mutated from aspartic acid to glycine. The lentivirus prepared by the above-mentioned lentiviral packaging vector system can be used to screen drugs against SARS-CoV-2 or its mutant strains, and has high safety.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a lentivirus packaging vector system, a lentivirus and a construction method and kit thereof. Background technique [0002] Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel betacoronavirus with high pathogenicity to humans discovered after SARS-CoV and MERS-CoV. Segmented single-stranded positive-stranded RNA enveloped virus. [0003] However, since SARS-CoV-2 is a biosafety level 3 (BSL-3) pathogen of a severe infectious disease, the isolation and culture of SARS-CoV-2 must be carried out in the presence of BSL-3, which limits the Efficiency of drug development against SARS-CoV-2. SUMMARY 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 SARS-CoV-2, and the lentivirus is used to develop and treat ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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