Method for culturing insect cells and preparing HPV (human papillomavirus) 16/18 L1 proteins by applying biological reactor

An HPV16L1 and bioreactor technology, which is applied in the field of preparing exogenous proteins such as human papillomavirus 16 and 18 L1 proteins, can solve problems such as cell damage and death, and achieve the effects of optimizing conditions and methods and increasing the scale of culture.

Inactive Publication Date: 2011-09-14
DALIAN ALEPH BIOMEDICAL
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  • Summary
  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

However, the oxygen demand of insect cells is very high during the culture process, which makes it necessary to carry out a large number of oxygen delivery operations such as stirring, air injection, etc., but the shear force generated by these operations often causes cell damage and death. Therefore, if you want to To further increase the concentration of insect cells in suspension culture, it is necessary to consider many aspects, such as the design of the bioreactor, the smoothness of mass transfer and oxygen transfer, the strength of shear force, the level of cell inoculum concentration, the medium group, etc. score selection etc.

Method used

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  • Method for culturing insect cells and preparing HPV (human papillomavirus) 16/18 L1 proteins by applying biological reactor
  • Method for culturing insect cells and preparing HPV (human papillomavirus) 16/18 L1 proteins by applying biological reactor
  • Method for culturing insect cells and preparing HPV (human papillomavirus) 16/18 L1 proteins by applying biological reactor

Examples

Experimental program
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Effect test

Embodiment 1

[0031] Embodiment 1. utilizes the condition of bioreactor to cultivate Sf-9 cell

[0032] The frozen insect cell Sf-9 (Invitrogen Company) was taken out from liquid nitrogen, quickly placed in a 27°C water bath for thawing, and then centrifuged at 1000rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in 0.5ml Grace growth solution (Grace (Gibco company) 4.572%, NaHCO 30.035%, hydrolyzed milk protein (Oxid company) 0.33%, yeast extract (Oxid company) 0.33%, fetal bovine serum (Hyclone company) 10%, penicillin 4U, streptomycin 10U, pH 6.5), and then transfer Transfer to a T25 culture flask containing 6ml of Grace Growth Medium and grow in an incubator at 27°C for 3 days. After the Sf-9 cells confluent into a monolayer, the culture supernatant was discarded, and 10 ml of fresh Grace growth solution was added. The adherent cells were gently blown off, then transferred to a sterile Erlenmeyer flask (sealed with a gas-permeable film), and the ...

Embodiment 2

[0035] Example 2. Determination of Proliferation of Recombinant Baculovirus

[0036] When the Sf-9 cells proliferated in the bioreactor to the end of the logarithm or the early stage of the plateau, a recombinant baculovirus carrying the HPV16 L1 gene (GeneBank accession number 1489082) was added (the baculovirus was derived from a kit from BD, USA, BD Baculo Gold TM , 560129) or the recombinant baculovirus carrying the HPV18 L1 gene (GeneBank accession number ADC35715), continue to culture for 4 days. Take 1ml of cell culture medium every 12 hours, and measure the titer of recombinant baculovirus therein. Briefly, 1ml of the infected cell culture solution was taken, centrifuged at 1000rpm for 5 minutes, and the supernatant and cell pellet were collected respectively. Use Grace diluent (Grace (Gibco company) 4.572%, NaHCO 3 0.035%, hydrolyzed milk protein (Oxid company) 0.33%, yeast extract (Oxid company) 0.33%, penicillin 4U, streptomycin 10U, pH 6.5) carry out 10-fold ...

Embodiment 3

[0038] Example 3. Determination of HPV16 / 18 L1 protein expressed by insect cells

[0039] After infection of Sf-9 cells cultured in a bioreactor with recombinant baculovirus, HPV16 / 18 L1 protein expressed by insect cells was measured every 12 hours. Briefly, the cell culture fluid was taken and the cell pellet was separated. to every 10 6 100 μl of lysate (50 mmol / L Tris-HCl (pH 8.0); 15 mmol / L NaCl; 0.1 mmol / L PMSF; 0.1% NP40; 5 mmol / L EDTA) was added to each cell and ultrasonically disrupted to lyse Sf-9 cells. After lysis, centrifuge at 10,000 g for 15 min at 4°C to remove cell debris. 100 μl of supernatant was added to a 96-well plate coated with anti-HPV16 L1 or anti-HPV18 L1 polyclonal antibody (SANTA CRUZ BIOTECHNOLOGY Company). At the same time, a predetermined amount of purified HPV16 / 18 L1 protein was used as a control to prepare a standard curve for quantitative protein. Plates were then incubated for 1 hour at 37°C. After incubation, the plate was washed thr...

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Abstract

The invention provides a method for preparing extrogenous proteins, such as human papillomavirus 16 type and 18 type L1 proteins by adopting a biological reactor for large-scale culture of insect cells. Particularly, in the invention, the conditions for culturing the insect cells by using the biological reactor are optimized, and the conditions of using the insect cells infected with recombinant rhabdovirus to express the extrogenous proteins, such as the HPV (human papillomavirus) 16 / 18 L1 proteins are further optimized, thereby getting the method for high-level expression of the extrogenous proteins, such as the HPV 16 type and 18 type L1 proteins.

Description

technical field [0001] The present invention relates to the field of biotechnology. In particular, the present invention provides a method for producing foreign proteins such as human papillomavirus type 16 and 18 L1 proteins by culturing insect cells on a large scale using a bioreactor. In particular, the present invention optimizes the conditions for culturing insect cells using a bioreactor, and for infecting insect cells with recombinant baculoviruses to express foreign proteins such as HPV16 / 18 L1 protein, thereby obtaining high-level expression of foreign proteins such as Method for HPV16 / 18 L1 protein. Background technique [0002] The insect cell-baculovirus expression system has unique advantages in high-efficiency expression of foreign proteins, and thus has been widely used. The expression system has the following advantages: 1) high safety: baculovirus only replicates in invertebrate insects or insect cells, but is not infectious to humans, mammals, other inver...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/09C12N5/07C12N5/10
Inventor 王敏文王玉峰周长民闫昆明李春明张千王飞宇聂飞周蕾郭文军高伟星段广宇李卫东
Owner DALIAN ALEPH BIOMEDICAL
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