OPTIMIZATION OF CULTURE PREPARATION AND CULTIVATION CONDITIONS FOR E. COLI PRODUCING GLYCOCONJUGATE VACCINE CANDIDATES AGAINST AVIAN INFLUENZA AND CAMPYLOBACTERIOSIS
The increased production of poultry meat in Indonesia poses a serious threat through the emergence of zoonotic diseases such as Avian Influenza and Campylobacteriosis, which present health risks to humans and significant economic losses to the poultry industry. As a solution, glycoconjugate vaccine...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/87244 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | The increased production of poultry meat in Indonesia poses a serious threat through the emergence of zoonotic diseases such as Avian Influenza and Campylobacteriosis, which present health risks to humans and significant economic losses to the poultry industry. As a solution, glycoconjugate vaccine candidate targeting the H5N1 virus and Campylobacter jejuni has been developed and produced in previous research. However, challenges in the production of these vaccine candidates include unstable culture growth following Isopropyl ?-d-1-thiogalactopyranoside (IPTG) induction and failure of protein glycosylation. This study aims to: (1) determine the optimal culture preparation method for vaccine candidate production, and (2) assess the variations in induction temperature, induction time, and E. coli strains for the production and glycosylation of the vaccine candidate. In this study, the culture preparation method utilizing serial dilution of glycerol stocks successfully isolated colonies with more stable growth. However, preliminary protein analysis using Western Blot revealed that the produced proteins were not glycosylated. Therefore, optimization tests of production cultures was conducted using various E. coli strains (E. coli BL21(DE3), Top10, Top10F’, and SCM6) under different induction conditions (induction temperatures of 30°C and 23°C and induction times of 4 and 16 hours) to produce glycosylated vaccine candidate proteins. A preliminary Dot Blot test was conducted to eliminate ineffective induction temperature variations for subsequent testing. Protein analysis of the production results using Western Blot indicated that the E. coli SCM6 strain from fresh transformant stock, with an induction temperature of 30°C and induction time of 4 hours, provided the highest glycosylation efficiency of 46%, compared to other strains with similar induction temperatures, which ranged between 8-38%. Thus, this study demonstrates that the production of glyconjugate vaccine candidate using E. coli SCM6 cultivation methods with: (1) serial dilution culture preparation from glycerol stocks, and (2) cultivation conditions of fresh transformant culture with an induction temperature of 30°C and an induction time of 4 hours, can consecutively provide stable culture growth conditions and optimal glycoprotein/glycoconjugate vaccine production.
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