OPTIMASI EKSPRESI PROTEIN FUSI HBSAG-HBCAG MENGGUNAKAN PLASMID PET32B (+) PADA INANG ESCHERICHIA COLI BL21 (DE3) SEBAGAI KANDIDAT VAKSIN HEPATITIS B
Hepatitis B is an infectious disease caused by hepatitis B virus (HBV) infection. WHO estimates currently 257 million people suffer chronic hepatitis B. Vaccination using HBsAg subunit protein remains the most effective way to prevent the spread of hepatitis B. Although HBsAg immunogenicity is alrea...
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| Format: | Final Project |
| Language: | Indonesia |
| Online Access: | https://digilib.itb.ac.id/gdl/view/48718 |
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| Institution: | Institut Teknologi Bandung |
| Language: | Indonesia |
| Summary: | Hepatitis B is an infectious disease caused by hepatitis B virus (HBV) infection. WHO estimates currently 257 million people suffer chronic hepatitis B. Vaccination using HBsAg subunit protein remains the most effective way to prevent the spread of hepatitis B. Although HBsAg immunogenicity is already proven, some people show no immune response against HBsAg vaccination. HBsAg-HBcAg fusion protein developed by Utami (2013) is an alternative vaccine candidate expected to have better immunogenicity. This research aims to optimize HBsAg-HBcAg fusion protein expression using pET32b (+) as expression vector and Escherichia coli BL21 (DE3) as expression host and to optimize protein purification using Ni-NTA purification column. Protein expression was performed using LB medium with 100 ?g/ml ampicillin with various conditions as follows; 4 hours of induction with 1 mM IPTG, 12 hours of induction with 1mM IPTG, and 4 hours induction with 1, 1.5, and 2 mM IPTG. Total protein of Escherichia coli BL21 (DE3) transformed with pET32b (+) inserted with HBsAg-HBcAg fusion protein gene was analysed using SDS-PAGE. SDS-PAGE electrophoregram showed no sign of HBsAg-HBcAg fusion protein expression. Promoter sequence analysis using PhagePromoter and EMBOSS-WATER was performed to analyse promoter sequence regulating HBsAg-HBcAg fusion gene expression. It was shown there is no mutation with T7 promoter sequence that could alter gene expression. We suggest to perform terminator sequence analysis, protein expression analysis using western blotting, and reconstruction of pET32b (+) HBV plasmid.
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