Recombination cell line and method for determining titer of baculovirus

A technology of recombinant cell line and baculovirus, applied in the biological field, can solve the problems of difficult to judge the nucleus, inaccurate results of virus titer determination, and enlargement of the nucleus.

Active Publication Date: 2014-02-12
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

[0004] However, since Sf9 insect cells are round cells, when they are infected by baculovirus, the lesion is mainly manifested as nuclear enlargement; and Sf9 insect cells are also prone to nuclear enlargement

Method used

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  • Recombination cell line and method for determining titer of baculovirus
  • Recombination cell line and method for determining titer of baculovirus
  • Recombination cell line and method for determining titer of baculovirus

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Embodiment 1

[0021] In an embodiment of the present invention, a method for constructing a recombinant cell line for baculovirus titer determination is provided, comprising the following steps:

[0022] Construction of reporter plasmids;

[0023] Wherein, the reporter plasmid includes a reporter gene encoding green fluorescent protein inserted therein, a promoter P gene of the late gene of the baculovirus that induces the expression of the reporter gene;

[0024] Using the reporter plasmid to transfect Sf9 insect cells;

[0025] Selecting the transfected Sf9 insect cells with a medium containing bleomycin and fetal bovine serum to obtain single cell clones;

[0026] The single cell clone is further screened with a medium containing bleomycin to obtain a recombinant cell line.

[0027] The recombinant cell line used for the determination of baculovirus titer provided by the embodiment of the present invention, which integrates the reporter gene capable of encoding green fluorescent protei...

Embodiment 2

[0032] Please refer to figure 1 , the construction method of this embodiment includes the following steps:

[0033] Step 101: constructing a reporter plasmid;

[0034] Since baculovirus gene expression is a sequential process, baculovirus genes can be divided into early genes and late genes according to the sequence of expression time. Considering the requirement of the promoter on the timing and expression quantity of the reporter gene expression, it is preferable to use the baculovirus very late gene containing the polyhedrin gene promoter P gene to regulate the expression of the reporter gene in the process of designing the reporter plasmid.

[0035] Specifically, the construction of the reporter plasmid can be obtained by the following methods:

[0036] 1. Starting from the transient expression vector plasmid pIZ / V5-His (Invitrogen, Life Science), use restriction endonucleases BsmI and SpeI to digest and recover a 2248bp band to obtain the vector framework pIZ / V5.

[00...

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Abstract

The invention relates to the biotechnical field, and in particular relates to a recombination cell line and a method for determining the titer of baculovirus. A construction method of the recombination cell line for determining the titer of the baculovirus comprises the following steps: constructing reporter plasmids; transfecting Sf9 insect cells through the reporter plasmids; screening the transfected Sf9 insect cells through a culture medium containing bleomycin and fetal bovine serum to obtain single cell clones; continuously screening the single cell clones through the culture medium containing the bleomycin to obtain the recombination cell line. According to the recombination cell line and the method for determining the titer of the baculovirus, the titer of the baculovirus can be quickly determined, and a determination result is accurate.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant cell line for measuring the titer of baculovirus and a measuring method thereof. Background technique [0002] Baculoviruses are a class of DNA viruses that widely exist in nature and mainly infect insects of Lepidoptera, Diptera and Hymenoptera, and their infection can lead to liquefaction death of insects. With the deepening of research, baculovirus has been more and more widely used in biological insecticide, eukaryotic expression, surface display and gene therapy. In order to make the baculovirus exert its maximum efficacy in the application, it is necessary to quantify the baculovirus, that is, to measure the virus titer. [0003] Virus titer is the virulence or price of the virus. In related technologies, the titer of baculovirus is generally measured by the endpoint dilution method (endpoint dilution), which refers to the virus dilution that can make half of th...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/85C12Q1/70C12Q1/02C12R1/93
Inventor 邓菲邓马平张艳芳王华林胡志红
Owner WUHAN INST OF VIROLOGY CHINESE ACADEMY OF SCI
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