Quantitative determination method for titer of recombinant insect baculovirus

A technology for quantitative determination of baculovirus, applied in measuring devices, color/spectral characteristic measurement, instruments, etc., can solve the problems of no quantitative determination of recombinant insect virus titers, etc., and achieve easy-to-master technology, short time-consuming, and simple operation Effect

Active Publication Date: 2014-01-22
WATERSTONE PHARMA WUHAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

There is currently no simple and rapid method for quantitative determination of recombinant insect virus titers

Method used

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  • Quantitative determination method for titer of recombinant insect baculovirus
  • Quantitative determination method for titer of recombinant insect baculovirus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: Cell Seeding

[0028] (1) Materials Sf9 cells were purchased from China Center for Type Culture Collection; TC-100 medium and fetal bovine serum were purchased from GIBCO; 96-well plates were purchased from NUNC.

[0029] (2) Operation method

[0030] Take the Sf9 adherent growth cells in the logarithmic growth phase, and gently pipette to resuspend the cells. Centrifuge at 1,000g (horizontal rotor) for 8min. Discard the supernatant, and resuspend the cells in TC-100 medium containing 10% (V / V) fetal bovine serum to make a cell suspension. Samples were stained with trypan blue and counted.

[0031]Dilute the cells to 3 × 10 with TC-100 medium containing 10% (V / V) fetal bovine serum 4 cells / mL, added to a 96-well cell culture plate, 100 microliters per well. Stand at 27°C for 2h.

Embodiment 2

[0032] Example 2: Dilution of recombinant baculovirus titer standard and sample to be tested

[0033] (1) Materials TC-100 medium and fetal bovine serum were purchased from GIBCO; micropipettes were purchased from EPPENDORF; EP tubes and tips were purchased from AXYGEN.

[0034] (2) Operation method

[0035] Dilute the recombinant baculovirus titer standard with TC-100 medium, the highest concentration is 320-fold dilution, and then use 2-fold gradient dilutions such as 640-fold dilution and 1280-fold dilution, a total of 8 dilutions; use TC-100 medium Dilute the sample to be tested, the highest concentration is 20-fold dilution, the following 4-fold dilutions such as 80-fold dilution, 320-fold dilution, etc., are used for a total of 5 dilutions.

Embodiment 3

[0036] Example 3: Quantitative determination of recombinant insect baculovirus titer

[0037] (1) Materials Fetal bovine serum was purchased from GIBCO; pNPP was purchased from Amersco; Triton X-100, sodium acetate, and NaOH were purchased from Sinopharm; EDTA was purchased from Shanghai Test.

[0038] (2) Operation method

[0039] Aspirate the culture medium in the 96-well cell culture plate, add 50 μL of serially diluted virus solution to each well, and incubate at room temperature for 2 hours to allow the virus to adsorb. Add 50 μL of TC-100 medium containing 20% ​​(V / V) fetal bovine serum to each well, and culture at 27°C for 72 hours. Aspirate the culture medium in each well, add 100 μL of pNPP solution (1mg / mL pNPP, 0.06mol / L sodium acetate, 0.001mol / LEDTA, 0.2% Triton X-100, pH6.0) in sequence, and place at 37°C for 1h. Add 10 μL of 1mol / L NaOH to each well, mix well, and place at room temperature for 5-20 minutes. Read the absorbance value at 405nm on a BIO-TEK micr...

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Abstract

The invention provides a quantitative determination method for the titer of recombinant insect baculovirus. The method utilizes the fact that the recombinant insect baculovirus interferes in growth and duplication of host cells after the recombinant insect baculovirus infects insect cells, and then the method detects the number of the cells by using an acidic phosphatase method so as to realize quantitative determination of the titer of the recombinant insect baculovirus. The method has the advantages of high sensitivity, good accuracy, usage of cheap and easily available reagents, simple and convenient operation, easy mastery of technology, small consumption of time and higher efficiency.

Description

technical field [0001] The invention relates to virus titer measurement, in particular to a method for quantitatively measuring the titer of recombinant insect baculovirus. Background technique [0002] Since the discovery of the strong promoter characteristics of the polyhedrin gene (polh) of the baculoviridae nuclear polyhedrosis virus in the early 1980s, Smith and Summer [Smith GE, MD Summers and MJ Fraser. Production of human beta interferon in insect cells infected with a baculovirus expression vector. Mol Cell Biol.1983; 3(12): 2156-2165] first established a baculovirus expression vector system (Baculovirus Expression Vector System, BEVS). The baculovirus expression system has become one of the four major expression systems in the field of genetic engineering. Compared with E. coli and yeast mammalian cell expression systems, BEVS has special research value in the following four aspects: (1) as an ultra-efficient Eukaryotic gene expression system to produce useful tar...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/569G01N21/31
CPCG01N33/56911G01N2333/005G01N2333/43552
Inventor 周虎朱俊铭
Owner WATERSTONE PHARMA WUHAN
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