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Sf9 cell line free from Sf-RV pollution as well as screening method and application of Sf9 cell line

A screening method and cell line technology, applied in the field of insect cell lines, can solve problems such as concerns about the safety of biological preparations, and achieve the effect of ensuring safety

Pending Publication Date: 2019-11-08
CHANGCHUN ZHUOYI BIOLOGICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the discovery of a new rhabdovirus (Sf-RV) in the Sf cell line raises concerns about the safety of biologics produced by the insect cell-baculovirus expression vector system

Method used

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  • Sf9 cell line free from Sf-RV pollution as well as screening method and application of Sf9 cell line
  • Sf9 cell line free from Sf-RV pollution as well as screening method and application of Sf9 cell line
  • Sf9 cell line free from Sf-RV pollution as well as screening method and application of Sf9 cell line

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] [Example 1] Screening method of Sf9-ZY cell line and detection of Sf-RV virus

[0036] 1. Screening method of Sf9-ZY cell line

[0037] 1) Recovery of Sf9 cells

[0038] Take out the frozen cells from the liquid nitrogen tank, shake them quickly in a 37°C water bath until the ice cubes in the cryovial are nearly melted, add 2ml to the T25 culture flask, and transfer the cells in the cryovial to the Gently shake and mix well in the culture flask. At room temperature, place it on a horizontal tabletop for 5 minutes to adhere to the wall. Aspirate the unattached cells and add 3-5ml of Gibco Sf-900ⅢSFM medium. Place the T25 culture flask Static cultivation in a constant temperature incubator at 27°C;

[0039] 2) Sf9 cells passaged for 1 generation

[0040] When the Sf9 cells recovered by the above method are cultured for 2-3 days, or when the cell confluence is greater than 95%, the cells are subcultured: remove the excess medium in the culture bottle, add 5ml of new Gib...

Embodiment 2

[0081] [Example 2] Sf9-ZY cell proliferation characteristics

[0082] Culture Sf9-ZY cells and Sf9 cells separately, with 6.0×10 5 / ml cell density, 20ml cell suspension was inoculated in a 125ml triangular shaking culture flask, placed on a constant temperature shaking culture device, the speed was adjusted to 120 rpm, the culture temperature was 27±1°C, and samples were taken every 24 hours until the cells died , using an automatic cell counter to measure the number of viable cells and cell diameter in the sample, the cell doubling time calculation formula is as follows: Td=T×Log2 / Log(Q2 / Q1), Td=doubling time, T=from the last passage time, Q1 = cell seeding density, Q2 = number of viable cells.

[0083] Result: from Figure 4 It can be seen that the two cell lines Sf9-ZY and Sf9 were in the logarithmic growth phase 4 days before the culture, the cell doubling time was between 23 and 28 hours, and the cell diameter was 14-16 μm; and the Sf9-ZY cell The doubling time (23-26...

Embodiment 3

[0085] [Example 3] Detection of baculovirus proliferation level in Sf9-ZY cell line

[0086] Take Sf9 cells and Sf9-ZY cells respectively, inoculate 6-well plates, culture 18 groups each, cell inoculation density 8.0×105 cells / ml, 2ml per well, let stand for 10 minutes until the cells adhere to the wall, and inoculate recombinant rods according to MOI0.1 Viruses (HPV16L1 and HPV58L1), the 6-well plate was placed at 27±1°C for static culture for 3 to 4 days, the cells were collected, and the virulence was detected by immunofluorescence method, the steps were as follows:

[0087] 1) Aspirate SF-900ⅢSFM culture based on 96-well cell culture plate, 200 μl per well, add 50 μl of the test substance, mix well and then aspirate 50 μl into the second well, and so on for 5-fold dilution, aspirate 50 μl from the 11th well and discard , set a blank for each test;

[0088] 2) Add 50 μl of Sf9 cells at a concentration of 8×105 cells / ml to each well, and incubate at 27°C (±1°C) for 24±2 hou...

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Abstract

The invention relates to an insect cell line and discloses a Sf9 cell line free from Sf-RV pollution as well as a screening method and application of the Sf9 cell line. The cell line is screened fromcommercial Sf9 cells, named as an Sf9-ZY cell line and has the biological collection number of CCTCC NO:C201952. Detection of the proliferation level and protein expression quantity of baculovirus inthe Sf9-ZY cell line indicates that the Sf9-ZY cell line can replace Sf9 cells to serve as host cells of an insect cell-baculovirus expression vector system (BICS) to be applied to expression of recombinant protein for preparing biological products. The Sf9-ZY cell line provided by the invention solves the problem that host cells of the insect cell-baculovirus expression vector system (BICS) are polluted by Sf-RV and guarantees safety of the biological products.

Description

technical field [0001] The invention relates to an insect cell line, in particular to a Sf9 cell line without Sf-RV pollution, a screening method and application thereof. Background technique [0002] In 1969, Vaughn et al. established an insect cell line, which was isolated from the ovary tissue of the pupae of female Spodoptera frugiperda. At that time, 2.6% heat-inactivated insect blood was added to the culture medium. After the establishment, this cell line was provided to Oxford, England. The University's NERC Invertebrate Virology Unit, where this cell line was adapted to medium supplemented with newborn bovine serum instead of medium supplemented with insect blood, at this time the cell line was called IPLB-Sf-21 -AE was later provided to Texas A&M University in the United States, where it was cloned and applied to the protein expression system. The Sf9 cell line was cloned from IPLB-Sf-21-AE by G.E.Smith and C.L.Cherry. It has a more uniform cell shape, is sensitive...

Claims

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Application Information

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IPC IPC(8): C12N5/10C12N15/866
CPCC12N15/86C12N5/0601C12N2710/14043C12N2510/00
Inventor 杨屹苗丽侯玉婷刘发明吴海华朱蕾宋立强
Owner CHANGCHUN ZHUOYI BIOLOGICAL CO LTD
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