The Primary Study Of Strategy For Enhancing The Expression Of Genetic Engineered Antibody

The genetic engineered antibody has been widely used in clinics recently, but the dosage for one period of treatment is considerably large. For example, about 1.2 g of anti-CD20 (Rituxan) antibody for the treatment of B-cell lymphoma and 0.88 g of anti-HER2 (Herceptin) antibody for breast cancer are required. The low yield is the biggest obstacle of large-scale application for the antibody in clinics. This study focused on how to improve the yield of engineered antibody. The results are shown as following:Constructing high efficiency expression vector containing a mutated neo gene to introduce anti-erbB2-scFv-Fc-IL-2 fusion protein and mutated neo genes into a hot spot of host cell genome.1. By site-directed mutagenesis the neo gene mutant was obtained by PCR. The dicistronic expression vector containing the anti-erbB2-scFv-Fc-EL-2 fusion protein and the mutated neo gene was constructed. The fusion protein and the neo genes were co-expressed in CHO cell.2. The transfectants containing the mutated neo gene grew considerably slower than those containing the normal neo gene in the presence of high concentrations of G418, however the former expressed strikingly higher level of fusion proteins than the later did, and the yield was about 3 u g / ml. In addition, the expression level was not changed with time and the cell growth remained very stable for the duration of the culture, which suggested that the targeted genes have been integrated into the hot spot of host cell genome. This project not only increased the expression level of the exogenous gene, but also drastically reduced the labor strength of screening positive clones.Establishing database of regulatory elements of the mouse antibody genes1. The information of expression regulatory elements of mouse antibody genes wascollected, sorted and analyzed based on the published articles and related otherdatabases. Web page database of expression regulatory elements of mouse antibody genes was established by the various tools of web pages and diagrams. 2. In this database, expression regulatory elements and trans-factors of closely-related mouse antibody genes can be found and their positive or negative effects on regulation of antibody genes learned. Explanation and references for the important regulatory elements and trans-factors are given in the database. Much more details can be enquired by linking to other databases on internet. The database will be very helpful to analyze the core sequences of promotors and enhancers harbored in the non-encoding regions of the antibody genes and trans-factors which bind to these sequences and positively regulate the antibody expression. Furthermore, it will be very useful to construct the novel and highly efficient eukaryotic expression vectors.