Insect cell suspension culture method to prepare recombinant TPO protein

Abstract

The invention discloses an insect cell suspension culture method to prepare a recombinant TPO protein. At first, recombinant baculovirus is transected to insect cells in a logarithmic phase; after 96hours, the supernate is collected, P0 generation recombinant baculovirus liquid is harvested; primary recombinant baculovirus is subjected to cell passage cultivation; passage inoculated cells with adensity not less than 3-5*10<5> are inoculated into a triangular flask, when the cell density reaches 2-3*10<6>, subculture is carried out again; when the density reaches 2*10<6> cells / mL, P0 generation virus liquid is inoculated (MOI=0.1-0.5), the supernate of cell culture is collected and subjected to a centrifugal treatment to obtain P1 generation virus liquid; the P1 generation virus liquid iswrapped by a tin foil and is stored in the absence of light at a temperature of 4 DEG C; the method mentioned above is repeated to obtain P2 generation virus liquid, subculture is carried out, when the density reaches 2*10<6> cells / mL, P2 generation virus liquid is inoculated (MOI=1-5), after 3 to 5 days, the cell culture supernate is collected, after a centrifugal treatment, P3 generation virusliquid is obtained, and after titer detection, the recombinant TPO protein is obtained. The antigen specificity of prepared protein is good, the sensitivity and titer are high, and the sensitivity, precision, and stability of a kit are greatly improved.

Description

technical field [0001] The invention relates to biological detection technology, in particular to a method for preparing recombinant TPO protein by suspension culture insect cells. Background technique [0002] Baculovirus is a vector system widely used in recent years to efficiently express foreign proteins. The genome of baculovirus is a single closed circular double-stranded DNA molecule with a size of 80-160 kb. transcription. After DNA replication, it is assembled in the nucleocapsid of baculovirus, which has greater flexibility and can accommodate the insertion of larger fragments of foreign DNA, so it is an ideal carrier for expressing large fragments of DNA. Using baculovirus as a carrier can efficiently express foreign genes. Up to now, hundreds of genes including animals, plants, viruses, bacteria, and fungi have been efficiently expressed in insect cells or larvae. The main feature of this expression system is that it can obtain a large number of soluble recombi...

AI Technical Summary

Problems solved by technology

When Spinner is cultured, the culture volume is 1 / 2 of the bottle volume, but the bottle cap is not ventilated, so it is difficult to guarantee the DO of the cell growth process, which leads to the phenomenon of low titer; on the other hand, the Spinner bottle is a glass bottle , and there are 2 glass rotors, you need to be careful when using it, it is easy to break, and the price is more expensive. In addition, each Spinner device can only accommodate 4 bottle positions, and the number of bottle positions is small. You need to re-purchase equipment for enlargement, which is difficult to enlarge

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