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Method for preparing lentiviral particle packaged EBOV RNA as positive reference substance for EBOV nucleic acid detection

A technology of lentiviral particles and reference materials, applied in the field of genetic engineering, can solve the problems of difficulty in preservation, insufficient simulation, inability to simulate extraction, etc., and achieve the effects of cost saving, high stability and high copy number.

Inactive Publication Date: 2020-01-10
WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] 1. If the inactivated virus needs to be prepared in a level 4 biosafety laboratory, and the safety of the inactivated virus cannot be guaranteed;
[0004] 2. The nature of the reverse-transcribed RNA is very unstable, easy to degrade, and difficult to store;
[0005] 3. Plasmid DNA, as a universal positive template, cannot simulate the whole process from RNA extraction to nucleic acid amplification detection, and the simulation is insufficient

Method used

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  • Method for preparing lentiviral particle packaged EBOV RNA as positive reference substance for EBOV nucleic acid detection
  • Method for preparing lentiviral particle packaged EBOV RNA as positive reference substance for EBOV nucleic acid detection
  • Method for preparing lentiviral particle packaged EBOV RNA as positive reference substance for EBOV nucleic acid detection

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preparation example Construction

[0057] Because the commonly used EBOV positive reference materials for nucleic acid detection (plasmid DNA, reverse transcription RNA, or inactivated virus) have shortcomings such as insufficient simulation, instability, or potential safety hazards. Therefore, the present invention provides a method for preparing a safe and stable EBOV nucleic acid reference material. The EBOV RNA gene is wrapped with lentiviral particles. The nucleic acid reference material can truly simulate the whole process from RNA extraction to detection, and has good stability and safety. High, it has a good application prospect in EBOV nucleic acid detection kits.

[0058] The EBOV nucleic acid reference material of the present invention is that the partial fragment RNA of NP, GP and L gene of EBOV is packaged in lentiviral particles, and the sequence of NP, GP and L gene of the EBOV tandem of this nucleic acid reference material is as SEQ ID No. 1.

[0059] Generally, the sequence of EBOV obtained by...

Embodiment 1

[0062] First, it is necessary to construct a lentiviral particle expression vector containing the EBOV gene. In this example, the gene expression sequence is prepared by the following operations:

[0063] Obtain the nucleotide sequence shown in SEQ ID NO: 1, clone the sequence, select the genes NP, GP and L genes commonly used in nucleic acid detection of EBOV, obtain the corresponding nucleic acid detection sequence through literature search, and analyze the sequence of these three genes Conserved sequences, selected NP (560-1839 in the KJ660346.2 Zaire ebolavirus genome), GP (6053-7100 in the KJ660346.2 Zaire ebolavirus genome), L gene (10348 in the KJ660346.2 Zaire ebolavirus genome Position-13589 and 13900-14471) a total of 3453bp nucleotide sequence, entrusted to a gene synthesis company to synthesize, with restriction endonuclease sites of XbaI and BamHI added at both ends.

[0064] The lentiviral packaging vector of four plasmids (including lentiviral expression vector:...

Embodiment 2

[0078] For the expression of pseudoviral particles packaged with EBOV gene sequences, the lentiviral packaging system used in the present invention is the third-generation four-plasmid packaging system, which has an expression vector and three packaging plasmids: the packaging system puts the Rev gene in a single plasmid In order to prevent homologous recombination to produce a virus with self-replication ability; the second plasmid expresses Gag and Pol structural proteins; the third plasmid uses the herpetic stomatitis virus glycoprotein VSV-G to replace its own envelope protein Env, corresponding to Compared with the first-generation two-plasmid packaging system and the second-generation three-plasmid packaging system, it has higher security. The construction process of the pseudovirion particles is as follows: the exogenous gene is constructed into the lentiviral expression vector, together with the three packaging plasmids VSV-G, PLP1, and PLP2, the cells are transfected a...

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Abstract

The invention discloses a method for preparing lentiviral particle packaged EBOV RNA as a positive reference substance for EBOV nucleic acid detection. The preparation method comprises the following steps: connecting part of segments of three genes NP, GP and L of EBOV in series and then integrating the segments into an expression vector of lentivirus, separately amplifying the constructed lentiviral expression plasmid and packaging plasmid in an engineering escherichia coli strain to obtain a large number of plasmids, then jointly transfecting the constructed lentiviral expression plasmid andpackaging plasmid into HEK293T cells, and carrying out cell expression to obtain a large number of lentiviral particles packaged with RNA genes of EBOV. The invention relates to the technical field of gene engineering. According to the method for preparing lentiviral particle packaged EBOV RNA as a positive reference substance for EBOV nucleic acid detection. The obtained particles are used as apositive reference substance for EBOV nucleic acid detection and have the advantage of well simulating the whole process from virus nucleic acid extraction to nucleic acid detection in an actual sample, and freeze-dried powder obtained by freeze-drying pseudovirus particles of EBOV through a freeze-drying protective agent is very stable.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to a lentiviral particle packaging RNA of partial fragments of three genes NP, GP and L of Ebola virus (EBOV), as a preparation method for a positive reference product for EBOV nucleic acid detection, and Methods are involved to enable stable storage of the positive reference. Background technique [0002] Ebola virus (EBOV) has a very high lethality and infectivity, and is a potent pathogen with high global concern. Its commonly used detection method is mainly RT-PCR, and the corresponding kit must match the positive reference material. The positive reference substances of EBOV nucleic acid detection kits are generally plasmid DNA, reverse transcription RNA, or inactivated viruses. These positive reference substances currently have the following different defects: [0003] 1. If the inactivated virus needs to be prepared in a level 4 biosafety laboratory, and the safet...

Claims

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Application Information

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IPC IPC(8): C12N15/867C12N15/40C07K14/155C12Q1/70C12Q1/6851
CPCC07K14/005C12N15/86C12N2740/15023C12N2740/15043C12N2760/14122C12Q1/6851C12Q1/701C12Q2531/113C12Q2545/113
Inventor 危宏平余军平熊进
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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