The Modification Of CHO-dhfr～- Cells For Biopharmaceutical Production

CHO-dhfr~- cell is the most important host cell for biopharmaceutical protein, however, there are some problems still existing in CHO-dhfr~- cell culturing.In order to make CHO-dhfr~- cell more suitable for culturing on a large scale, we modified the cell by steady integrating the anti-apoptosis gene Bcl-2 in genome of CHO-dhfr~- used the Flp target integrating system.Firstly, human anti-apoptosis gene Bcl-2 was target integrated into CHO-dhfr~- cell and overexpressed. The viability of the reconstructing cell increased to face the inductive apoptosis factors such as culturing in serum-free and in high-concentration ammonia ion.As accumulation of Lactic acid which is a kind of side products can obviously influence the growth of the cell. In order to reduce lactate formation in the cell culture by genetically manipulation of the pathway of lactate synthesis, 4 RNAi targets were selected in the LDH-A gene of CHO-dhfr~-cell. Four DNA sequence to produce corresponding shRNA is designed and cloned in the lower position of the expressing vector pAV/U6. shRNA toward LDH-A gene of CHO-dhfr~- can be expressed continuously by steady integrating expressing vector within genome of CHO-dhfr~- cell. One cell line was screened out from the 4 reconstructing cells lines, the result of analysis showed that the LDH-A mRNA , the accumulation of lactate , cell apoptosis in medium all decreased, the density of cell culturing clinging to walls increased by 11. 4%.The third part of the job was to control the cell proliferation bioprocesses. Because the over proliferation of the cell in culturing process was unfavorable for the heterologous protein production, growth stagnation is...