Construction Of Antibody Library And Screening Of Antifungal β - Mannose Antibody On Glycine - Based Yeast

At present, the biological drug is a of the most popular drugs in the market, and large proportion of these drugs are antibody drugs. Most of the drugs are used mammalian cell expression system, because the protein’s modify of the mammalian cell expression system is familiar with the native structure. However, because of the high cost and low yield of the mammalian cell expression system, it has seriously hindered the development of biological drugs. Yeast, as a kind of eukaryotic organism,has many advantages, such as high growth rate, high expression and low cost, and can be used in the expression of various recombinant proteins. By using glycosylation engineering yeast, the protein glycosylation modification for complex glycosuria instead of yeasts are generally of high mannose glycosuria, which is similar with the native structure. So in this research, we use glycosylation engineering yeast to express antibody.Surface display technology is a new gene manipulation technique. This technology can expressing the protein on cell surface, and the protein that displayed on the cell surface and can keeping the protein’s original structure and the biological activity. The protein that Glycosylation engineering yeast is familiar with the eukaryotic cells, and the yield is higher than mammalian cells. Therefore, the yeast surface displayed technology is a good way to study eukaryotes expression system,especially the protein expression. The technology that based on screening antibody library is simply and convenience, has become one of the most important methods to study and prepare of antibody, and become an important way in the development of new monoclonal antibody drugs.We use the Sed protein as the anchoring protein. The antibody library that I constructed, can be done in the same yeast cell by surfacing display and expressing structural integrity of the monoclonal antibody. Most kinds of the antibody that screening from the library are not native structure, lots of them are single chain antibodies. Therefore, it is necessary to construct complete antibody. But through the Sed anchoring protein, it can screen and express complete antibody into the same yeast without other steps. Here, we use this method, named dual mode, to design,select and express of complete monoclonal antibody through the glycosylation engineering yeast.First of all, we construct the immune antibody library by using glycosylation engineering yeast. We construct the Fc-Sed sequence and transform into the yeast cells. After using HA7 as antigen to immune mice, we take the mouse’s spleen cells,extract the RNA from the spleen cells, amplify the heavy chain and light chain variable region gene of the cell, and construct the antibody plasmid. Finally, we construct the antibody library by using this plasmid. We screen the antibody library by using carboxyl magnetic particles. It can crosslinking the HA7 and then screening the antibody library which can combine with HA7.Then, in order to determine the binding capacity of antibody and HA7 that magnetic beads combined, we use ELISA double sandwich method. The obtained antibody was detected by this method, and the expression of the most powerful antibody was obtained. Then, we analysis the structure and function of this antibody.First, in order to determine the binding position of this antibody to HA7, we use the PNGase F enzyme, this enzyme can incise the carbohydrate chain. Western blot results showed that, after cutting HA7 carbohydrate chain, antibody were no longer binding HA7, so the antibody may binding the HA7 carbohydrate chain. Further analysis of the structure for this antibody, we use β-mannosidase from Cellulomonas fimi to cut the HA7 carbohydrate chain. The β-mannosidase from Cellulomonas fimi can only cut the β-mannon. Western results showed that, after cutting the β-mannon,the HA7 is no longer binding the antibody. So it shows that the antibody is binding the β-mannon. After determining the structure of the antibody that binding, we analyzed the function of the antibody in vivo.In order to determine the function of the antibody, we used Bal B/C mice as experimental animals. First we dilute the C.albicans with physiological saline dilution,configurate to different concentrations of C.albicans liquid, mice were divided into different groups of bacteria liquid injection concentration, bacteria liquid inject by intraperitoneal injection. Then observe and record the changing of the weight of the mice. We can know from the record that the weight changing of the mice obviously.From the experimental result, we determine the injection concentration for the next experiment. The new Bal B/C mice were then divided, in addition to the normal saline group and the Candida albicans group, with different amounts of antibody and Candida albicans premixed, and then injected into the mice.Experimental results show that, the weight of mice with normal saline group increasing all the time, other groups of mice weight at the beginning of the injection decreased but the largest decrease is different amount of antibody decreased the amplitude is relatively small, not antibody injection group fell the most, but the difference is not so obvious. Compared with the mice weight recovery time is obviously different, the largest group of antibody injection group rise significantly earlier in other groups, and the highest recovery speed, we can know from the result that the antibody has neutralizing activity.