High Expression Of Complete Antibody In CHO Cells

The therapeutic complete antibody is potential in clinical treatment and business. However, the consumption of therapeutic complete antibody during every treatment period is very large. This makes the high expression of complete antibody to be the bottleneck of its widely clinical use . Now, most of the antibodies are expressed in CHO cells (Chinese hamster ovary cells), whether it has come to the market or in research. Expression vector is one of the most important factors to prepare high expression cell clone. So it's significant to study the effects of every element and its structure of the expression vector.At first, anti-HCC complete antibody (HAb25) was expressed in CHO cells by vectors pSWD3-hc and pDNHG-hc. As a result, we found the expression levels of anti-HCC complete antibody in CHO cell were very low. Then, we constructed two kinds of dicistronic expression vectors pIRESdhfr-hc and pDI1-4 via weakening the expression of dhfr gene. By comparison of the ability of producing cell clones of these expression vectors, we got the result that the dhfr gene had been weakened in every expression vector, decreasing from pDIl,pIRESdhfr-hc,pDI2,pDI3, pDI4. We think the expression vectors pDI2 and pIRESdhfr-hc are suitable for complete antibody to be highly expressed. So, we expressed anti-HCC complete antibody (HAb25) in CHO cells by vector pDI2. 84 monoclones were obtained, with 14 detected positively by sandwich ELISA. One of these clones expressed better than other clones with 6.15ng/105cell-3day under the selective condition (IMDM, 10% dialyzed serum without H and T). But the expression level dropped sharply during passages and culture. At last, the expression level can not be detected. We compared the transient expression level of anti-HCC complete antibody (HAb25) and a human-mouse chimeric antibody against humanbladder tumor in COS-7 cells under the same condition. The result showed that it is more difficult to express the anti-HCC complete antibody (HAb25) than to express chimeric antibody against human bladder tumor.In order to prove that certain antibodies could be expressed in these expression vectors constructed previously, we changed the anti-HCC complete antibody (HAb25) with chimeric antibody against human bladder tumor. Three expression vectors (pIRESdhfr-B, pIRESdhfr-BA and pDI-B2) were used in this research work. The result showed that chimeric antibody against human bladder tumor could be constantly expressed in CHO cells by any of these three expression vectors with a positive ratio of 100% under selective condition (IMDM, 10% dialyzed serum without H and T). The best clones were subjected to gene amplification in medium containing gradually increasing methotrexate(MTX, 1 x 10-8M, 2 10-8M, 5 10-8M ), the expressed chimeric antibody was quantitated by sandwich ELISA. The result showed that the antibody production could be significantly improved up to 50 times than the expression level under selective condition (IMDM, 10% dialyzed serum without H and T). One clone with antibody production of 13.6ug/106cell-3day was obtained (MTX, 5 10-8M).All these results showed that the dicistronic expression vectors (especially pDI-B2 and pIRESdhfr-B) constructed in this research work could be used to realize high expression of complete antibody.