Produksi Antiserum Poliklonal Penyakit Citrus Vein Phloem Degeneration (CVPD): 1. Pemurnian Protein Antigenik Bakteri Liberobacter asiaticus

(Production of Polyclonal Antiserum of Huang Lung Bin Disease: 1. Purification of Bacterial Antigenic Protein)

Nurhadi1, Agus Muharam2, Yoyo Sulyo3, Arry Supriyanto4 M.E. Dwiastuti4
1Balai Penelitian Tanaman Buah, Solok
2Balai Pengkajian Teknologi Pertanian Jawa Barat
3Balai Penelitian Tanaman Hias
4Loka Penelitian Tanaman Jeruk Dan Hortikultura Subtropik, Batu

ABSTRAK
Citrus Vein Phloem Degeneration (CVPD) tergolong sebagai penyakit destruktif tanaman jeruk di berbagai daerah produksi jeruk di Indonesia. Penyakit ini disebabkan oleh bakteri “candidatus” Liberobacter asiaticus (b-Las). Strategi efektif untuk mengendalikan penyakit ditentukan oleh hasil diagnosis melalui “Polymerase Chain Reaction” (PCR) yang saat ini masih mengandalkan pada peralatan canggih, karenanya metoda serologi merupakan alternatif teknik diagnosis sederhana yang lebih murah. Penelitian bertujuan untuk memurnikan protein antigenik (PA) b-Las yang disiapkan sebagai antigen CVPD untuk memproduksi antiserum poliklonal. Penelitian dilakukan di laboratorium virologi Balai Penelitian Tanaman Buah pada Januari sampai Desember 2003. Dengan menggunakan isolat CVPD RIF/008 yang dipelihara pada tanaman periwinkle, tulang daun dipisahkan dari daun-daun yang bergejala dan di sterilkan permukaannya. Jaringan fluem dipisahkan setelah mendigesti tulang daun pada larutan enzim yang mengandung 100 ml air destilasi steril, 0.8 g cellulose, 0.4 g macerozyme, 1.0 mM CaCl2.H2O, 0.5 g PVP-40 dan 12.5 g mannitol pada suhu ruang (24-31°C) semalam. Jaringan fluem dicuci lima kali pada buffer ekstraksi (0.6 M mannitol; 25 mM Tris-HCl dan 5 mM magnesium acetate pH 7.5 dan dimacerated pada 5 ml buffer ekstraksi. Pellet diresuspensi pada 1 ml buffer ekstraksi setelah dua siklus sentrifugasi ( 29 x g, 5 menit dan 17,000 x g, 30 menit) the homogenate. 16 S rDNA organisme CVPD dideteksi melalaui PCR menggunakan suspensi tersebut. Suspensi diperlakukan dengan 4% glutaraldehyde pada buffer ekstraksi melalui dialisis semalam, kemudian didialisa pada buffer eksraksi sehari dan semalam. Supernatan dikumpulkan secara hati-hati setelah sentrifugasi pada kecepatan rendah menggunakan 20% sucrose cushion dan disentrifugasi pada 20% sucrose cushion pada kecepatan tinggi. Pellet diresuspensi dengan 500-1,000 µl larutan NaCl 0.85%. Prosedur pemurnian yang dilakukan selama pelaksanaan penelitian ini mampu mengoptimalkan rekoveri antigen CVPD, efektif meminimumkan kehilangan bakteri selama proses purifikasi dan kemungkinan kontaminasi produk akhir immunogen dengan komponen tanaman. Dengan materi awal50 gramjaringan daun periwinkle dari tunas semi dorman isolat CVPD RIFI008, dihasilkan 350 g PA b-Las sebagai antigen untuk imunisasi. Kesederhanaan prosedur mempunyai prospek untuk dikembangkan karena secara ekonomis dapat diterima dan secara teknis dapat diadopsikan karena antigen dapat disiapkan dengan peralatan dan bahan kimia yang minimum.
Kata kunci: Poliklonal antiserum, Monospesifik, CVPD

ABSTRACT
Citrus Vein Phloem Degeneration (CVPD) constitutes one of the most destructive diseases affecting citrus trees in most citrus production areas of Indonesia. The disease is caused by “candidatus” Liberobacter asiaticus (b-Las). Effective strategies for controlling CVPD rely on “Polymerase Chain Reaction” (PCR) diagnosis currently depend on the sophisticated equipments, thus serological methods may offer simple diagnoses that are more cost effective. Recent study aimed to purify the CVPD antigen and produced its polyclonal antibody. Study was carried out at microbiology laboratory of Research Institute for Fruits during January and December 2003. Using CVPD isolate designated RIFI008 maintained in periwinkle plants midribs were separated from leaves showing symptoms then were surfaces sterilized. Phloem tissues were separated after digesting the diseased midribs in an enzyme solution of 100 ml sterile distilled water, 0.8 g cellulose, 0.4 g macerozyme, 1.0 mM CaCI2-H2O, 0.5 g PVP-40, and 12.5 g mannitol at room temperature (24-31°C) overnight. The phloem tissues were washed five times in the extraction solution (0.6 M mannitol, 25 mM Tris-H’CI and 5 mM magnesium acetate, pH 7.5, and were macerated in 5 ml of the extraction solution. The pellet was resuspended in 1 ml of the extraction solution after one cycle of centrifugation (29 x g, 5 min and 17,000 x g, 30 min ) of the homogenate. The 16 S rDNA of CVPD pathogen was detected by PCR using the suspension. The suspension was treated with 4% glutaraldehyde in extracted solution by dialysis for one night, and then was dialyzed in the extraction solution for one day and night. The supernatant was carefully collected after centrifuging at a low speed using a 20% sucrose cushion, and was centrifuged on 20% sucrose cushion at high speed. The pellet was resuspended in 500-1,000 µl of the 0.85% NaCl solution. Purification procedure described in this study was able to optimally recover an antigen for CVPD, effective to minimize bacterium loss during purification processes and the possible contamination of the final immunogen with plant components. With a starting material consisted of 50-gram bark tissue of semi dormant flush from CVPD isolate RIF/T008, 350 g of eluted anti genic protein of b-Las as an antigen for immunization was obtained. The simplicity of the procedure makes it economically acceptable and technically adoptable because the antigen can be prepared with limited chemical and equipment.
Keywords ; PolycJonal antiserum, Monospesific, CVPD

 

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