CONSTRUCTION OF GENE EXPRESSION VECTOR PLASMODIUM FALCIPARUM ERYTHROCYTE BINDING ANTIGEN 140 (PFEBA140) REGION III-V IN PICHIA PASTORIS

• Main Author: FARADILA (NIM:10514038), NURUL
• Format: Final Project
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/29808
• Institution: Institut Teknologi Bandung

Malaria is an infectious disease caused by the Plasmodium parasite transmitted through the bite of a <br /> <br /> <br /> <br /> <br /> <br /> female Anopheles mosquito. Malaria caused by Plasmodium falciparum is one of the causes of high <br /> <br /> <br /> <br /> <br /> <br /> morbidity and mortality in the world, so that a policy of handling is needed, one of them is through <br /> <br /> <br /> <br /> <br /> <br /> the development of a malaria vaccine. PfEBA-140 is a ligand protein used in invasion process at the <br /> <br /> <br /> <br /> <br /> <br /> level of erythrocytes in the blood phase. PfEBA-140 consists of region VI and the results show that <br /> <br /> <br /> <br /> <br /> <br /> region III s.d. V is able to provide a high immune response through the amount of IgG3 which <br /> <br /> <br /> <br /> <br /> <br /> indicates long-term infection or chronic disease in the body. Therefore, this research will clone the <br /> <br /> <br /> <br /> <br /> <br /> PfEBA-140 region III to V gene. PfEBA140 region III-V is amplified by PCR method at annealing <br /> <br /> <br /> <br /> <br /> <br /> temperature of 56 oC using a primer consisting of primer forward 5'- <br /> <br /> <br /> <br /> <br /> <br /> GAATTCGGTGATGGAACGCCAATAAG-3 'and reverse 5'- <br /> <br /> <br /> <br /> <br /> <br /> GTCGACCCTTTCGTCAGAATAGGT-3' then ligated to the pGEM-T vector cloning. The results of <br /> <br /> <br /> <br /> <br /> <br /> ligation was transformed into Escherichia coli TOP10F 'by heat shock method. Recombinant plasmid <br /> <br /> <br /> <br /> <br /> <br /> pGEM-T-PfEBA140-RIII-V. The recombinant plasmid then determines the order of its nucleotide by <br /> <br /> <br /> <br /> <br /> <br /> sequencing using the Sanger method. Sequencing results showed that the pGEM-T-PfEBA140-RIII-V <br /> <br /> <br /> <br /> <br /> <br /> recombinant plasmid construction was successfully carried out and a 100% similarity level was <br /> <br /> <br /> <br /> <br /> <br /> available with PfEBA-140-RIII-V. Then the PfEBA-140-RIII-V which was originally on the pGEM-T <br /> <br /> <br /> <br /> <br /> <br /> cloning vector was subcloned to the linear pPICZ&#945;A expression vector by double duplicating using <br /> <br /> <br /> <br /> <br /> <br /> EcoRI and SalI restriction enzymes. Both of PfEBA-140-RIII-V and linear pPICZ&#945;A were ligated and <br /> <br /> <br /> <br /> <br /> <br /> transformed to Escherichia coli TOP10F '.