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A kind of insect cell line and its application of serum-free suspension culture

An insect cell line and cell line technology, which is applied in the fields of agriculture and biology, can solve the problems of difficulty in reaching the highest cell density and production yield, low expression of virus polyhedron recombinant protein, and easy cluster growth of cells, so as to achieve virus production. And the effect of high protein expression level, high cell density and fast growth rate

Active Publication Date: 2021-10-01
QINGDAO AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At the same time, most insect cell lines grow adherently. Due to the limitation of the adherent area, it is difficult to achieve the highest cell density and production yield, and large-scale production is limited.
[0003] At present, the most widely used insect cell lines in the world in terms of baculovirus production and exogenous recombinant protein expression are Sf-21 of Spodoptera frugiperda or its clone Sf-9 and BTI- of Trichoplusia ni. Tn5B1-4 (High Five), Sf-9 and BTI-Tn5B1-4 cell lines have their own advantages and disadvantages in practical applications. The growth speed of the two cell lines is fast under adherent conditions, but the cells are easy to clump in suspension culture. affect its growth
The titer of insect baculovirus forming budded virus (BV) in Sf-9 cells was higher, but the production of viral polyhedron and the expression of recombinant protein were lower than those in BTI-Tn5B1-4 cells

Method used

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  • A kind of insect cell line and its application of serum-free suspension culture
  • A kind of insect cell line and its application of serum-free suspension culture
  • A kind of insect cell line and its application of serum-free suspension culture

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The establishment and screening and biological characteristics of embodiment 1 cell lines

[0023] 1.1 Establishment of cell lines

[0024] Collect about 100 newly-produced (less than 24 hours) eggs of Trichoplusia ni, disinfect the surface of the eggs with 10% sodium hypochlorite and 70% alcohol in an ultra-clean workbench for 5 minutes, and rinse with sterile water for 3 minutes. time, transfer to a fine sieve with a pore size of 100 μm, add 5 mL of Sf-900 TM ⅢSFM medium, grind eggs with sterile rubber tip, pipette cell filtrate into 25cm 2Cell culture flasks were cultured in a 28°C incubator. Regularly replace a certain proportion of fresh medium according to the growth state of the cells. After the primary cultured cells grow to cover the bottom of the culture bottle, blow the cells in the ultra-clean workbench to prepare a cell suspension, and carry out cell passage according to a certain proportion. After about 10 passages, when the cell growth state is stable,...

Embodiment 2

[0037] The serum-free suspension culture of embodiment 2 cell lines

[0038] Put QAU-Tn-E-7 cells in 75cm 2 Cultured in cell culture flasks (T-flask), 15 mL per bottle, cell suspension was taken during the logarithmic growth phase, inoculated into 125 mL suspension culture flasks (Spinner flask, Corning Company), and each bottle was inserted into 80 mL at a concentration of 2.0 × 10 5 cells / mL of QAU-Tn-E-7 cells, the medium is Sf-900 TM ⅢSFM serum-free medium, placed in an incubator at 28°C, cultured cells in suspension at speeds of 80r / min, 100r / min and 120r / min respectively, treated 3 bottles of cells at each speed, and removed from each bottle every 24h Take 1 mL of cell suspension each, and count with a hemocytometer to determine the cell concentration; at the same time, mix the same amount of cell suspension with 0.4% trypan blue to measure the survival rate of cells with different treatments.

[0039] The screening results of suspension culture at different rotation ...

Embodiment 3

[0040] The virus output of embodiment 3 cell line under the serum-free suspension culture

[0041] The QAU-Tn-E-7 cells in the logarithmic growth phase were divided into 2.0×10 5 The concentration of cells / mL was inoculated in a 125mL suspension culture bottle (Spinner flask, Corning Company), each bottle was inserted into 80mL, and the medium was Sf-900 TM ⅢSFM serum-free medium was placed in a 28°C incubator, and the cells were cultured in suspension at a speed of 100r / min. After culturing for 3 days, samples were taken to detect the cell concentration, and the cells were inoculated with Autographa californica nuclear polyhedrosis virus (AcMNPV) at a multiplicity of infection (MOI) of 10 according to the cell concentration, and repeated three times. On the 4th day of virus infection, the cell suspension was taken to check the virus infection rate under a microscope, and the cell suspension was collected and lysed by ultrasonic waves, so that the cells were lysed to release ...

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Abstract

The present invention develops an insect cell line, which is derived from the embryonic cell line QAU‑Tn‑E‑7 of Trichoplusia spp., and the preservation number of the cell line is CCTCC NO: C201865. The cell line of the present invention is suitable for serum-free suspension culture, does not contain TNCL virus, has fast growth rate, high cell density, high virus yield and protein expression level under the condition of serum-free suspension culture, and is a large-scale production of insect virus killing agent. Ideal cell line for pesticides and foreign recombinant proteins.

Description

technical field [0001] The invention belongs to the fields of agriculture and biotechnology, and in particular relates to a serum-free suspension culture insect cell line and its application, i.e. Trichoplusiae embryonic cell line QAU-Tn-E-7, which is suitable for insect virus insecticides and external Large-scale production of source recombinant proteins. Background technique [0002] Insect cell lines play a very important role in the production of baculovirus and the expression of recombinant proteins. Insect cell culture medium generally contains a certain proportion of animal serum to support the growth and proliferation of cells. However, due to the high cost of serum and the composition It is complicated, and it will cause difficulties in the separation, purification and detection of later culture products, which limits its application. Research and development in the fields of cell engineering, genetic engineering, protein engineering, and medical biology urgently n...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/07C12N7/00C12N9/38C12N9/16C12R1/93
CPCC12N5/0601C12N7/00C12N9/16C12N9/2471C12N2710/14151C12Y301/03001C12Y302/01023
Inventor 李长友郑桂玲陈英健孙雨
Owner QINGDAO AGRI UNIV
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