Sub kloning Gen Hemaglutinin 32 kDa Sub Unit Pili Pasteurella multocida Serotipe B:2 pada vektor ekspresi Eukariot

SUYANTO, Dr. Widya Asmara, SU.,Ph.D

2004 | Tesis | S2 Bioteknologi

Pasteurella multocida serotipe B:2 merupakan bakteri penyebab penyakit septikemi hemoragika yang menyerang kerbau dan sapi. Usaha pengendalian dilakukan dengan pencegahan masuknya bakteri pada inang. Vaksin DNA yang menyandi gen hemaglutinin sub unit pili P. multocida diharapkan mampu memenuhi tujuan tersebut. Gen hemaglutinin 32 kDa sub unit pili P. multocida terdapat pada pKH405 sebesar 786 bp diapit oleh EcoR I dan Hind III. Untuk memperoleh kandidat vaksin, maka gen ini disubklon pada vektor ekspresi eukariot. Penelitian ini bertujuan memperoleh DNA rekombinan gen hemaglutinin pada pCMV-Script, memasukkan DNA rekombinan pada mencit dan mengetahui ekspresi rekombinan DNA tersebut. Gen hemagutinin diisolasi dari pKH405 melalui digesti plasmid dengan enzim endonuklease restriksi EcoR I dan Hind III diikuti dengan elektroelusi fragmen gen. Fragmen gen hemaglutinin diligasi dengan pCMV-Script yang sebelumnya sudah dilinearisasi dengan EcoR I dan Hind III dan kemudian ditransformasikan ke E. coli DH5α. Molekul rekombinan pKH408 kemudian disuntikkan intra muscular ke mencit Balb/c dengan dosis 100 μg dan 150 μg. Deteksi ekspresi gen hemaglutinin dilakukan secara tidak langsung dengan jalan mengukur titer IgG antihemaglutinin dengan teknik ELISA. Hasil penelitian yaitu diperolehnya DNA rekombinan antara gen hemaglutinin 32 kDa pada pCMV-Script (pKH408). Molekul DNA rekombinan tidak menimbulkan reaksi negatif pada mencit pasca imunisasi. Molekul DNA rekombinan yang diperoleh tersebut dapat terekspresi dalam sistem ekspresi mamalia. Dosis 100 μg pKH408, memberikan titer IgG 1:50, 1:50, 1:400, 1:400 pada waktu 2, 4, 6, dan 8 minggu setelah vaksinasi. Dosis 150 μg pKH408, memberikan titer IgG 1:100, 1:800, 1:400, 1:400 pada selang waktu pengamatan yang sama.

Pasteurella multocida serotypes B:2 is the agent of hemorrhagic septicemia in cattle and buffalo. Controls of the disease were carried out by protecting the bacteria to enter the host. DNA vaccine which cotain of hemagglutinin sub unit pili gene from P.multocida would be expected to induce a good protection. That gene was obtained from pKH405 by digesting the plasmid with EcoR I and Hind III (Asmara, 2002). To have the expression of the gene in animal, the hemagglutinin gene should be cloned into an eukaryotic expression vector. This research had conducted by series activity. Preparation hemagglutinin gene and linear vector were done by digestion pKH405 and pCMV-Script vector used restriction enzyme EcoR I and Hind III, then was been electroeluted. Result of the ligation between the vector and the insert were transformed into competent cells E.coliDH5α with heat shock methods. Isolation of the plasmid used alkaline lysis Sambrook et al., (1989). The recombinant DNA then was introduced into mice Balb/c with 100 μg and 150 μg doses. Negative controls used the mouse that was injected by PBS. The Detection of expression recombinant DNA passed through measurement of IgG titers antihemagglutinin by ELISA. Titers were defined as resiprocal the highest dilution that had given positive value. The cut of point value was two time number of OD405 ELISA reader from negative controls (serum from PBS imunization) (Al-Mariri, 2001). The result of this research was gained recombinant DNA between gene hemagglutinin 32 kDa on pCMV-Script vector. Introduction recombinant DNA (pKH408) in mouse was not negative reaction after immunization. The recombinant DNA could been detected about its expressions in the expression system of the mice.

Kata Kunci : Bioteknologi,Bakteri Septikemihemoragika,Gen Hemaglutinin 32 kDa Sub Unit Pili, The gene hemagglutinin, P.multocida, Sub Cloning, Expression, Eukaryotic, E.coliDH5α