Ekspresi dan Respon Imun Kandidat Vaksin DNA dari Gen gag-ca Virus Penyakit Jembrana

WIBAWA, Hendra, Dr.drh. Asmarani Kusumawati, MP

2005 | Tesis | S2 Bioteknologi

Penyakit Jembrana (PJ) adalah suatu penyakit yang spesifik menyerang sapi Bali. Agen penyebab penyakit ini adalah suatu virus dari famili Retroviridae dan subfamili Lentiviridae yang kemudian disebut Virus Penyakit Jembrana (VPJ). Pengembangan kandidat vaksin DNA untuk VPJ, pcDNA-CA, dibuat dengan memasukkan gen gag-ca yang menyandi antigen capsid (CA) virus ke dalam sebuah vektor ekspresi eukariot, pcDNA3.1. Tujuan penelitian ini adalah untuk mengetahui ekspresi gen gag-ca pada sel mamalia dan untuk mengetahui respon imun yang ditimbulkan oleh kandidat vaksin DNA. Ekspresi gen gag-ca dipelajari dengan melakukan transfeksi DNA ke dalam kultur sel mamalia (sel line Hela). Transfeksi DNA dilakukan dengan teknik kopresipitasi DNA-kalsium fosfat dalam 2X bufer HeBS. Ekspresi gen gag-ca dianalisis pada aras sintesis mRNA menggunakan teknik RT-PCR (Reverse Trancription Polymerase Chain Reaction) dan pada aras sintesis protein mengguna-kan teknik Western Immunoblotting. Respon imun yang menimbulkan antibodi spesifik diukur dengan teknik ELISA terhadap serum-serum dari mencit Balb/c yang diinjeksi plasmid DNA secara intramuskular dengan dosis 100 ug per ekor pada minggu ke-0, 3 dan 6. Serum dikoleksi pada dua dan empat minggu setelah vaksinasi terakhir. Hasil penelitian menunjukkan bahwa konstruksi kandidat vaksin DNA dapat mengekspresikan gen gag-ca pada kultur sel Hela baik dalam bentuk mRNA maupun protein CA. Hasil vaksinasi menunjukkan bahwa mencit-mencit yang divaksin dengan pcDNA-CA menunjukkan titer antibodi terhadap antigen CA.

Jembrana disease is a disease which specifically attack Bali cattle. The causative agent of disease is a virus from family of Retroviridae and subfamily of Lentiviridae which was called as Jembrana Disease Virus (JDV). The development of DNA vaccine candidate, pcDNA-CA, was produced by inserting the gag-ca gene which code the capsid (CA) antigen of the virus to an eukaryotic expression vector, pcDNA3.1. The objectives of this research were to study the expression of gag-ca gene in mammalian cells and to study the immune response that was elicited by DNA vaccine. The gag-ca gene expression was examined by using DNA transfection to mammalian cell culture (Hela cell line). DNA transfection was done by co-precipitation technique of DNA-calcium phosphate in 2X HeBS buffer. The gag-ca expressions were analyzed at the level of mRNA synthesis by RT-PCR (Reverse Transcription Polymerase Chain Reaction) and at the level of protein synthesis by Western Immunoblotting. The immune response which produced the specific antibody was measured by ELISA technique to Balb/c mice serums, which its were injected intramuscularly by the DNA plasmid with the dosage of 100 ug each at 0, 3 and 6 week. Serums were collected at two and four weeks after the last vaccination. The result showed that the construction of DNA vaccine candidate expressed gag-ca gene in Hela cell culture either mRNA or CA protein type. The result of vaccination showed that mice were vaccinated by pcDNA-CA produced antibodies against CA antigen.

Kata Kunci : Bioteknologi, Gen gag,ca, Vaksin DNA, Respon Imun, Jembrana disease virus, DNA vaccine, gag-ca gene, gene expression, and antibody response.