Overexpression Of Heat Shock Protein 70 Enhance The Ability Of Cho Cells Expressing Antibodies To

In the long process of biological evolution, heat shock protein is preserved organisms can be used against external damage and can be an important mechanism of self-protection. Heat shock proteins are elevated in temperature or other stress conditions (hypoxia, low temperature or radiation) through the heat shock gene coding for a class of biological evolution in the most conservative with cellular proteins. Family of heat shock protein 70 most species, a total of 21 proteins and, therefore, become a heat shock protein family in the most studied, although they are mainly involved in protein folding, subunit composition and the degradation of the corresponding useless Protein and other processes, but also able to adjust and stabilize the activity of target cells and function, but they are not involved in the target protein (target protein) composition, so the important role of heat shock proteins are "molecular chaperone"role. Mainly in the promotion of the role of protein folding, transport and assembly; protection and stable cell structure; presenting antigens to help cellular immunity and tumor immunity, etc., but also to restore and accelerate the removal of intracellular proteins have been modified, enabling cells to produce tolerance to That it can adapt to environmental changes. The study found that over the years HSP70 can inhibit apoptosis (of course, some studies proved that HSP70 can promote cell apoptosis) and promote cell proliferation, to obtain heat-resistant organisms or resistance to achieve the protection of cells, so whether the study analyzed the cells by increasing the expression of HSP70 protein in the endocrine protein that promotes cell antibody production.This research is organized by Chinese hamster ovary cells over-expression of heat shock protein 70 antibody in order to improve the ability of its expression. The main technical route:first, by conventional PCR from Chinese hamster ovary cells in the expansion of the genomic DNA removed HSP70 gene; then taken by the expansion of HSP70 gene connected to the cloning vector pEASY-B to construct cloned into plasmid pEASY-HSP70; then this cloned in E. coli DH5a competent cells in the transformation of a number of monoclonal picked through the night after rolling two-plasmid strains mentioned restriction analysis and sequencing will identify the correct plasmid by restriction enzyme digestion to purified HSP70 gene ligated into the eukaryotic expression vector pcDNA3.1, the constructed expression plasmid pcDNA3.1-HSP70; then mediated by liposomes, stable transfected into CHO/dhfr- cells; by RT-qPCR detection of gene over-expression of HSP70; G418 pressure, screened over 96 heat shock protein 70 expression in stable cell lines were expanded and cultured for its follow-up experiments. Overexpression of HSP70 in the CHO/dhfr- cells transfected with empty vector group and the CHO/dhfr- pcDNA3.1 were transfected cell group in the expression of anti-HBs plasmid, ELISA detection of two cells of the ability of anti-HBs, and comparing the two groups of cells the ability of anti-HBs. Currently, people study the function of HSP70 has been very comprehensive. The establishment of a stable over-expression of heat shock protein 70 CHO/dhfr- cell lines, the study can be used to enhance the expression of target antibody.Through the above experiments the following conclusions:RT-qPCR results showed that the expression of transfected plasmid pcDNA3.1-HSP70 cells in the experimental group CHO/dhfr- HSP70 mRNA levels were significantly higher than those transfected with empty vector pcDNA3.1 in the CHO/dhfr-cells; ELISA test results showed that overexpression of HSP70 in CHO cells, the expression of anti-HBs higher than the CHO/dhfr- cells (P<0.05). Studies have shown that HSP70 can effectively promote cell secretory protein.