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埃及斑蚊雄蚊副腺蛋白(MAG-317)之分離、純化、氨基酸N-端序列分析存在局部之探討
Journal Volume
中山醫學雜誌
Journal Issue
v16 n.2 p195-208
Date Issued
2010-08-06T04:00:59Z
Abstract
目的：本研究以SDS-PAGE，Coomassie brilliant blue染色檢視及純化埃及斑蚊雄蚊副腺蛋白(MAG-317)，探討MAG-317蛋白的特性及在交尾過程中的傅象。方法：大量收集MAG-317蛋白用以注射及追加注射澳洲白鼻兔使其生MAG-317多株抗體，再以西方點墨法檢測其抗體效價。同時取大量MAG-317蛋白進行胺基酸N-端序列分析，以FITC in situ localization方法探討MAG-317蛋白在雄埃及斑蚊副腺分布及在交尾過程中的傅象。結果：MAG-317蛋白氨基酸N-端序列黑腹果蠅(Drosophila melanogaster) CG6041基因物有79%的相似性，且埃及斑蚊3al cDNA的中腸消化蛋白酵素原氨基酸N-端序列有47%的相似性“在雄埃及斑蚊副腺蛋白質的MAG-317蛋白特性分析中證實MAG-317蛋白一可溶性蛋白質，在雄埃及斑蚊副腺分布及在交尾過程中的傅象，證實MAG-317蛋白可經由交尾過程中傅到雌蚊儲精囊的象。結論：雄埃及斑蚊可經由交尾過程中副腺蛋白質(MAG-317)傅輸到雌蚊的儲精囊，證實了交尾傅播的象。
Purpose: A male accessory gland 31.7 kDa protein (MAG-317) was purified and characterized from the mosquito Aedes aegypti (L.) by SDS-PAGE and Coomassie brilliant blue staining. Methods: The specificity of the polyclonal antibodies raised in rabbit to the purified MAG-317 protein was verified by immunoblot analysis of crude male accessory gland (MAG) extracts and the protein was separated by SDS-PAGE. Results: The N-terminal amino acid sequence of the purified MAG-317 has 79% identity to the derived amino terminal sequence of the CG6041 gene product of Drosophila melanogaster and 47% identity to the derived amino terminal sequence of a 3al cDNA of midgut of Aedes aegypti which encodes for a putative trypsinogen. MAG-317 was a soluble protein that was detected only in the supernatant of tissue homogenate. For in situ localization of the protein, confocal laser-scanning microscope was used to observe cross-sections of the MAG-317 was found to distribute in the whole accessory gland. Subsequently labeled the male mosquitoes with the fluorescence isothiocynate conjugated MAG-317 in vivo and allowed these males to copulate with virgin females showed that MAG-317 could be detected in female spermathecae. Conclusion: Our study concludes that MAG-317 can be transferred from male to the female mosquito during copulation.
Purpose: A male accessory gland 31.7 kDa protein (MAG-317) was purified and characterized from the mosquito Aedes aegypti (L.) by SDS-PAGE and Coomassie brilliant blue staining. Methods: The specificity of the polyclonal antibodies raised in rabbit to the purified MAG-317 protein was verified by immunoblot analysis of crude male accessory gland (MAG) extracts and the protein was separated by SDS-PAGE. Results: The N-terminal amino acid sequence of the purified MAG-317 has 79% identity to the derived amino terminal sequence of the CG6041 gene product of Drosophila melanogaster and 47% identity to the derived amino terminal sequence of a 3al cDNA of midgut of Aedes aegypti which encodes for a putative trypsinogen. MAG-317 was a soluble protein that was detected only in the supernatant of tissue homogenate. For in situ localization of the protein, confocal laser-scanning microscope was used to observe cross-sections of the MAG-317 was found to distribute in the whole accessory gland. Subsequently labeled the male mosquitoes with the fluorescence isothiocynate conjugated MAG-317 in vivo and allowed these males to copulate with virgin females showed that MAG-317 could be detected in female spermathecae. Conclusion: Our study concludes that MAG-317 can be transferred from male to the female mosquito during copulation.