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Abstract
A major defense mechanism in insects is the melanotic encapsulation of foreign bodies. Animportant enzyme that participates in the melanization process is the pro- phenol oxidase (PPO). The PPO is released by hemocytes in the hemolymph and fat body and activated to phenol oxidase (PO) which in tum produces melanin pigments.
The melanin pigments are deposited on intruding foreign body as part of the immune response. The aim of our study was to detect the phenol oxidase in Anopheles maculatus, a species known to be refractory to Plasmodium, the causal agent of . malaria.
We obtained antibodies raised against Manduca sexta prophenol oxidase (PPO) from Dr Michael Kanost, Kansas State University, Manhattan, Kansas, USA. These antibodies were used to in Western blotting experiments to detect PO in Anopheles maculatus, Anopheles sinesis, Drosophila melanogaster and Bactrocera papayae.
The latter three insect species were used as controls. Results of the immunoblots showed that the anti-PPO antiboflies detected three major protein bands of 62 kD, 59 kD and 43 kD in both adult male and female An. maculatus. None of these protein bands were detected in An. maculatus larvae.
We also designed two sets of degenerate PCR primers based on the published eDNA sequence of the Manduca sexta PPO gene. Both sets of primers were used at several annealing temperatures but no amplification product was obtained from the PCR experiments and thus no DNA was a ailable for cloning.
Abstrak
Satu mekanisme perlindungan yang penting pada serangga ialah penkapsulan melanin terhadap jasad asing. Enzim penting yang terlibat dalam proses ini ialah pro-fenol oksidase (PPO). PPO dirembeskan oleh hemosit yang terdapat dalam hemolimfa dan pada jasad lemak and diaktitkan kepada fenol oksidase (PO) yang kemudiannya
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menghasilkan pigmen melanin. Pigmen melanin ini pula akan diendapkan pada jasad asing yang memasukki organism. Tujuan kajian ini ialah untuk mengesan fenol oksidase padaAnopheles maculatus, spesies nyamuk yang rintang terhadap jangkitan Plasmodium, agen malaria
Antibodi yang dijana terhadap profenol oksidase Manduca sexta telah di perolehi daripada Dr Michael Kanost, Kansas State University, Manhattan, Kansas, USA.
Autibodi tersebut digunakan dalam eksperimen pemblotan Western untuk mengesan PO pada Anopheles maculatus, Anopheles sinesis, Drosophila melanogaster and Bactrocera papayae. Tiga serangga yang terakhir itu dijadikan kawalan. Keputusan immunoblot menunjukkan antibody yang digunakan itu berjaya mengesan tiga jalur protein utama yang bersaiz 62 kD, 59 kD dan 43 kD pada An. maculatus dewasa jantan dan betina. Walau bagiamanpun, antibody tersebut tidak mengesan apa-apa
protein pada larva An. maculatus.
Berdasarkan jujukan cDNA PPO Manduca sexta yang diterbitkan, dua set primer PCR telah direkabentuk. Kesemua primer tersebut telah digunakan dalain eksperimen PCR dengan beberapa suhu penxypuhlindapan. Tiada produk DNA yang berjaya diamplifikasikall dan pellgklollan tidak dapat dijalankall.
