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Method for determining baculovirus titer

A baculovirus and titer technology, applied in the direction of measuring devices, instruments, scientific instruments, etc., can solve the problems of insignificant cell lesions, subjective factors, and inability to distinguish between dead and alive viruses, and achieve good repeatability and accurate detection results Effect

Inactive Publication Date: 2013-10-23
WUHAN CHOPPER BIOLOGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] 4) Fluorescent quantitative PCR method quantifies baculovirus by quantifying viral nucleic acid, and cannot distinguish between dead and alive viruses
The so-called deficiency mainly refers to the fact that the cytopathy caused by the low concentration of baculovirus-infected cells is not obvious, and the result judgment is easily affected by the subjective factors of the operator.

Method used

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  • Method for determining baculovirus titer
  • Method for determining baculovirus titer

Examples

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Effect test

Embodiment 1

[0031] Example 1 Determination of Bac-HANA strain recombinant baculovirus titer

[0032] (1) Material

[0033] Sf9 cell is a commonly used insect cell, purchased from Invitrogen Company; AcMNPV-based recombinant baculovirus Bac-HANA (a recombinant baculovirus expressing avian influenza HA and NA genes, the construction method is shown in the application number CN200910104637.4 patent); gp64 monoclonal antibody is the product of ebioscience; goat anti-mouse fluorescent antibody is the product of Thermo; SF900Ⅱ serum-free medium is the product of Gibco; PBS (0.01M, pH7.2).

[0034] (2) Operation method

[0035] 1) Preparation of Sf9 cell plate: Sf9 cells were cultured in suspension in shake flasks with SF900II serum-free medium, the rotation speed of the shaker was 110 rpm, and the culture temperature was 27°C. Sf9 cells in the logarithmic growth phase and in suspension culture were diluted with SF900Ⅱ serum-free medium, and the diluted cell solution was diluted with 3×10 4 S...

Embodiment 2

[0044] Example 2 Determination of Bac-2ORF2 strain recombinant baculovirus titer

[0045] (1) Material

[0046] Sf9 cell is a commonly used insect cell, purchased from Invitrogen Company; AcMNPV-based recombinant baculovirus rBac-2ORF2 (a recombinant baculovirus expressing circovirus Cap protein, the construction method sees the application number CN201210270504.6 patent); gp64 monoclonal antibody is a product of ebioscience; cap protein monoclonal antibody is a product of American RTI; goat anti-mouse fluorescent antibody is a product of Thermo; SF900Ⅱ serum-free medium is a product of Gibco; PBS (0.01M, pH7.2) .

[0047] (2) Operation method

[0048] 1) Preparation of Sf9 cell plate: Sf9 cells were cultured in suspension in shake flasks with SF900II serum-free medium, the rotation speed of the shaker was 110 rpm, and the culture temperature was 27°C. Sf9 cells in the logarithmic growth phase and in suspension culture were diluted with SF900Ⅱ serum-free medium, and the dil...

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Abstract

The invention discloses a method for determining baculovirus titer. The method comprises the following steps: paving a cell culture plate with insect cells in the logarithmic phase at the cell density of 2*104-4*104 / 0.1mL / hole, placing the cell culture plate into an incubator for culture and sticking the insect cells to hole bottom; inoculating baculovirus to be detected into the insect cell plate and culturing in the incubator; removing a medium in the cell plate, fixing, washing the cell plate, adding gp64 McAb for incubation, washing the cell plate, adding goat-anti-mouse fluorescent antibody for incubation, washing the cell plate, and observing under a fluorescence microscope; and calculating TCID50 of the virus. The method for determining baculovirus titer is combined with indirect immunofluorescence, is a TCID50 determination method for directly judging whether each hole cell is infected and has good repeatability. Obtained detection results are more accurate. It is not necessary to combine cytopathic effect for determination, and there is no influence from aspects of subjective and experience factors.

Description

[0001] technical field [0002] The invention relates to virus titer (content) measurement, in particular to a method for measuring baculovirus titer. Background technique [0003] Insect baculovirus expression vector system (BEVS) is a eukaryotic expression vector system that can efficiently express foreign genes. BEVS, based on Autographa californica nuclear polyhedrosis virus (AcMNPV) as the basic research model, is one of the most versatile and widely used eukaryotic cell expression vector systems. BEVS with AcMNPV as the basic research model has been fully commercialized, including a variety of commercial vectors, efficient transfection reagents, culture media, and mature construction methods and successful cases. Optimizing the optimal virus infection (MOI) is an important means to increase the expression of BEVS exogenous genes, so it is necessary to accurately quantify the baculovirus titer. Commonly used baculovirus titer determination methods include plaque metho...

Claims

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Application Information

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IPC IPC(8): G01N33/577G01N33/569
Inventor 李晶梅朱薇温文生漆世华谢红玲靖志强李建秦红刚廖园园
Owner WUHAN CHOPPER BIOLOGY
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