أختبار قابلية اللكتين المنقى جزئيا من بذور نبات
Bacillus subtilis ﻤﺤﻤﺩ ﻋﺒﺩ ﺍﷲ ،ﺒﺸﻴﺭ ﻋﺒﺩ ﺍﻟﺤﻤﺯﺓ ،ﻴﺎﺴﺭ ﺤﻴﺩﺭ ﺠﻠﻴل ﺍﻟﺨﻼﺼﺔ ﺠﺎﻤﻌﺔ ﺒﺎﺒل /ﻜﻠﻴﺔ ﺍﻟﻌﻠﻭﻡ basalwani@yahoo.com ﺘﻡ ﺍﻟﺘﺤﺭﻱ ﻋﻥ ﻓﻌﺎﻟﻴﺔ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﺴﻜﺭﻱ ﻓﻲ ﺒﺫﻭﺭ ﺴﺘﺔ ﺃﻨﻭﺍﻉ ﻤﻥ ﺍﻟﻨﺒﺎﺘﺎﺕ ﺍﻟﺒﻘﻭﻟﻴﺔ ﻭﻫﻲ :ﺍﻟﺒﺯﺍﻟﻴﺎ Pisum sativum L.ﻭﺍﻟﺤﻤﺹ Cicer arictinum L.ﻭﺍﻟﻤﺎﺵ Phaseolus aureus Roxb.ﻭﺍﻟﺒﺎﻗﻼﺀ Fecia vabaeﻭﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ Phaseolus vulgaris L.ﻭﺍﻟﻌﺩﺱ Lens culinarisﻭﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺃﻥ ﻤﺴﺘﺨﻠﺹ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ ﺍﻟﻤﺠﻔﻔﺔ Phaseolus vulgaris L. cv. Whiteﻴﻤﺘﻠﻙ ﺃﻋﻠﻰ ﻤﺴﺘﻭﻯ ﻓﻌﺎﻟﻴﺔ ﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻟﻤﺎ ﺃﺜﺒﺘﻪ ﻤﻥ ﻓﻌﺎﻟﻴﺔ ﺘﻼﺯﻨﻴﺔ ﺇﺘﺠﺎﻩ ﻜﺭﻴﺎﺕ ﺍﻟﺩﻡ ﺍﻟﺤﻤﺭﺍﺀ ﻭﻗﺩﺭﻫﺎ ٧ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻓﻀﻼ ﻋﻥ ﺍﻟﻤﺤﺘﻭﻯ ﺍﻟﺒﺭﻭﺘﻴﻨﻲ ﺍﻟﻌﺎﻟﻲ ،ﻟﺫﺍ ﺃﺨﺘﻴﺭﺕ ﺒﺫﻭﺭ ﻨﺒﺎﺕ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ ﻤﺼﺩﺭﺍ ﻟﻠﺩﺭﺍﺴﺔ ،ﻨﹸﻘﻲ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﺴﻜﺭﻱ ﺒﺴﻠﺴﻠﺔ ﻤﻥ ﺨﻁﻭﺍﺕ ﺍﻟﺘﻨﻘﻴﺔ ﺸﻤﻠﺕ :ﺍﻟﺘﺭﺴﻴﺏ ﺒﻜﺒﺭﻴﺘﺎﺕ ﺍﻷﻤﻭﻨﻴﻭﻡ ﺒﻨﺴﺒﺔ ﺇﺸﺒﺎﻉ %٧٠ﺤﻴﺙ ﺴﺠﻠﺕ ﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ﻗﺩﺭﻫﺎ ٣٧،٦١ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ،ﻭﺨﻁﻭﺓ ﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺘﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ ﺒﺎﺴﺘﻌﻤﺎل ﺍﻟﻤﺒﺎﺩل ﺃﻷﻴﻭﻨﻲ DEAE- Celluloseﻭﺴﺠﻠﺕ ﻫﺫﻩ ﺍﻟﺨﻁﻭﺓ ﻓﻌﺎﻟﻴﺔ ﺘﻼﺯﻨﻴﺔ ﻗﺩﺭﻫﺎ ٤٥،٥ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻭﺨﻁﻭﺓ ﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ﺒﺎﺴﺘﻌﻤﺎل ﻫﻼﻡ Sephadex G-200ﻭﺴﺠﻠﺕ ﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ﻗﺩﺭﻫﺎ ٥٧،٠٧ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ،ﺒﻴﻨﺕ ﺍﻟﺩﺭﺍﺴﺔ ﺇﻤﻜﺎﻨﻴﺔ ﺘﻘﻴﻴﺩ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻨﻘﻰ ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﻤﺠﻔﻔﺔ ﺒﺎﺴﺘﻌﻤﺎل ﺍﻟﻜﻠﻭﺘﺭ ﺍﻟﺩﻴﻬﺎﻴﺩ ﺤﻴﺙ ﺃﻋﻁﻰ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﻓﻌﺎﻟﻴﺔ ﺘﻼﺯﻨﻴﺔ ﻨﻭﻋﻴﺔ ﻗﺩﺭﻫﺎ ٩٩،٥ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ. ﻜﻤﺎ ﺃﺨﺘﹸﺒﺭﺕ ﻓﻌﺎﻟﻴﺔ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻨﻘﻰ ﻭﺍﻟﻤﻘﻴﺩ ﻓﻲ ﺘﺜﺒﻴﻁ ﻨﻤﻭ ﺨﻼﻴﺎ ﺍﻷﺤﻴﺎﺀ ﺍﻟﺩﻗﻴﻘﺔ ﻭﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻓﻌﺎﻟﻴﺔ ﻋﺎﻟﻴﺔ ﻟﻠﻜﺘﻴﻥ ﻓﻲ ﺘﺜﺒﻴﻁ ﻨﻤﻭ ﺒﻜﺘﺭﻴﺎ .Bacillus subtilis ﺍﻟﻜﻠﻤﺎﺕ ﺍﻟﻤﻔﺘﺎﺤﻴﺔ :ﺍﻟﻜﺘﻴﻥ ﺍﻟﺴﻜﺭﻱ ،ﺒﺫﻭﺭ ﻨﺒﺎﺕ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ ،ﺒﻜﺘﺭﻴﺎ Bacillus subtilis Abstract The glycoprotein activity of lectine were survey in dried seeds for six plant species: Cicer arictinum L., Phaseolus aureus Roxb., Pisum sativum L., Phaseolus vulgaris L., Lens culinaris and Fecia vabae. the extract of crud protein from dried seeds of Phaseolus vulgaris L. possess highest protein specific activity observed were 7 A.U./mg, therefore, it was selected as a lectine source for studying this glycoprotein. the lectine extracted from Phaseolus vulgaris L. was purified by several steps, including precipitation with ammonium sulfate 70%, ion exchange chromatography using DEAE-cellulose and gel filtration on sephadex G-200 Colum, this study revealed that lectine was immobilized by covalent bonding gluteraldehyde, showed specific activity 95.5 A.U./mg. The results of the study showed the ability to use immobilized lectine to use it as inhibitors to inhibition the growth of Salmonella typhi. key words: : Ketan diabetes, plant the seeds of white beans, the bacterium Bacillus subtilis ﺍﻟﻤﻘﺩﻤﺔ ﺍﻟﻠﻜﺘﻴﻨﺎﺕ :ﻫﻲ ﺒﺭﻭﺘﻴﻨﺎﺕ ﺴﻜﺭﻴﺔ ،ﻏﻴﺭ ﻤﻨﺎﻋﻴﺔ ﺍﻷﺼل ﻭﻏﻴﺭ ﺍﻨﺯﻴﻤﻴﺔ ﻤﻥ ﻨﺎﺤﻴﺔ ﺍﻟﻔﻌﺎﻟﻴﺔ ﻭﻟﻬﺎ ﻗﺩﺭﺓ ﺘﺨﺼﺼﻴﺔ ﻋﺎﻟﻴﺔ ﻋﻠﻰ ﺍﻷﺭﺘﺒﺎﻁ ﺒﺎﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺕ )ﺍﻟﺴﻜﺭﻴﺎﺕ( ﻭﺘﺭﺴﻴﺏ ﺍﻟﺨﻼﻴﺎ ﺤﻴﺙﹸ ﺍﻥ ﻗﺎﺒﻠﻴﺘﻬﺎ ﺍﻟﻔﺭﻴﺩﺓ ﻋﻠﻰ ﺍﻟﺘﻤﻴﻴﺯ ﻭﺍﻻﺭﺘﺒﺎﻁ ﻭﺒﺸﻜل ﻋﻜﺴﻲ ﻤﻊ ﻜﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﻤﻌﻴﻨﺔ ﻭﺒﺩﻭﻥ ﺍﻱ ﺘﺤﻭﻴﺭ ﻜﻴﻤﻴﺎﺌﻲ ﻴﺠﻌﻠﹸﻬﺎ ﺃﺩﻭﺍﺕ ﻗﻴﻤﺔ ﻓﻲ ﺍﻟﻤﺠﺎل ﺍﻟﻁﺒﻲ ﺍﻟﺤﻴﻭﻱ ﻤﻥ ﻨﺎﺤﻴﺔ ﻗﺩﺭﺘﻬﺎ ﻋﻠﻰ ﺍﻹﺭﺘﺒﺎﻁ ﺒﺎﻟﻤﺴﺘﻘﺒﻼﺕ ﺍﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺘﻴﺔ ﻋﻠﻰ ﺴﻁﻭﺡ ﺍﻟﺨﻼﻴﺎ ﻭﺒﺎﻟﺘﺎﻟﻲ ﺘﻠﺯﻴﻨﻬﺎ ) .(Goldstein et al., 1980ﻭﺒﺴﺒﺏ ﺍﻟﺘﺨﺼﺼﻴﺔ ﺍﻟﻌﺎﻟﻴﺔ ﻟﻠﻜﺘﻴﻨﺎﺕ ﻟﻼﺭﺘﺒﺎﻁ ﺒﺎﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﺍﻟﺒﺴﻴﻁﺔ ﻭﺍﻟﺜﻨﺎﺌﻴﺔ ﻭﺍﻟﻤﺘﻌﺩﺩﺓ ﻤﻨﻬﺎ ﻭﺍﻟﺘﻲ ﺘﺘﻭﺍﺠﺩ ﻓﻲ ﺘﺭﻜﻴﺏ ﻤﻌﻅﻡ ﻤﺴﺘﻘﺒﻼﺕ ﺴﻁﻭﺡ ﺨﻼﻴﺎ ﺍﻻﺤﻴﺎﺀ ﺍﻟﻤﺠﻬﺭﻴﺔ ﺍﻟﻤﺭﻀﻴﺔ ﻭﺍﻟﺨﻼﻴﺎ ﺍﻟﺴﺭﻁﺎﻨﻴﺔ ﻓﺈﻥ ﻫﺫﺍ ﻴﺠﻌل ﻤﻥ ﺍﻟﻠﻜﺘﻴﻨﺎﺕ ﺘﺴﺘﻌﻤل ﺒﺸﻜل ﻭﺍﺴﻊ ﻜﻤﻀﺎﺩﺍﺕ ﻟﻨﻤﻭ ﺍﻻﺤﻴﺎﺀ ﺍﻟﻤﺠﻬﺭﻴﺔ ،ﺤﻴﺙ ﺍﺴﺘﻌﻤل ﺍﻟﻠﻜﺘﻴﻥ ﻜﻤﻀﺎﺩ ﻓﻁﺭﻱ ﻭ ﻤﻀﺎﺩ ﻓﻴﺭﻭﺴﻲ ﻭﻤﻀﺎﺩ ﻟﻠﻁﻔﻴﻠﻴﺎﺕ ﻭﻤﻀﺎﺩ ﻟﻠﺤﺸﺭﺍﺕ ﻓﻀﻼ ﻋﻥ ﺇﺴﺘﻌﻤﺎﻟﻪ ﻜﻤﻀﺎﺩ ﺤﻴﻭﻱ ﻟﺘﺜﺒﻴﻁ ﻨﻤﻭ اﻟﺒﻜﺘﺮﯾﺎ ).(Yufang et al., 2010 2104 ١ﺍﻟﻤﻭﺍﺩ ﻭﻁﺭﺍﺌﻕ ﺍﻟﻌﻤل ١-١ ﺍﻟﻤﻭﺍﺩ ﺒﺫﻭﺭ ﺍﻟﻨﺒﺎﺘﺎﺕ :ﺍﻟﻤﺎﺵ ) ، ( Phaseolus aureus Roxb.ﺍﻟﺒﺯﺍﻟﻴﺎ )، (Pisum sativum L. ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ ) ، (Phaseolus vulgaris L.ﺍﻟﺤﻤﺹ ) ،(Cicer arictinum L.ﺍﻟﻌﺩﺱ ) Lens (culinarisﻭ ﺍﻟﺒﺎﻗﻼﺀ ) ،(Fecia vabaeﺘﻡ ﺠﻤﻊ ﺍﻟﺒﺫﻭﺭ ﻤﻥ ﺍﻟﺴﻭﻕ ﺍﻟﻤﺤﻠﻴﺔ ﻓﻲ ﻤﺤﺎﻓﻅﺔ ﺒﺎﺒل ،ﻤﺤﻠﻭل ﻋﺎﻟﻕ ﻜﺭﻴﺎﺕ ﺍﻟﺩﻡ ﺍﻟﺤﻤﺭﺍﺀ ،ABO blood group ،%٢ﻤﺤﻠﻭل NaClﺒﺎﻟﺘﺭﺍﻜﻴﺯ )،(%٢٠ ،%١٥ ،%١٠ ،%٥ Phenol ، Tris buffer 0.02M ،BSA (Bovin serum albumin) ،Commassie blue G-250 ،Iodine ،blueﻛﺤﻮل.Gluteraldehyde ،Brain-Heart infusion ،Safranine ،%95 ٢-١ﻁﺭﺍﺌﻕ ﺍﻟﻌﻤل ١-٢-١ﺘﺤﻀﻴﺭ ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻟﺒﺭﻭﺘﻴﻨﻲ ﺍﻟﺨﺎﻡ ﻁﹸﺤﻨﹶﺕ ﺍﻟﺒﺫﻭﺭ ﺍﻟﺘﻲ ﺠﻤﻌﺕ ﺒﺸﻜل ﻤﺴﺤﻭﻕ ﻭﺘﻡ ﻨﺨﻠﻬﺎ ﺒﻘﻁﻌﺔ ﻤﻥ ﻗﻤﺎﺵ ﺍﻟﺸﺎﺵ ﻟﻠﺘﺨﻠﺹ ﻤﻥ ﺍﻟﺸﻭﺍﺌﺏ ﻜﺎﻟﻘﺸﺭﺓ ﺃﻭ ﺍﻟﻐﻼﻑ ﺍﻟﺨﺎﺭﺠﻲ ﻟﻠﺒﺫﻭﺭ ،ﻭﻤﻥ ﺜﻡ ﺃُﻀﻴﻑ ﺍﻟﻤﺴﺤﻭﻕ ﺍﻟﻰ ﻤﺤﻠﻭل NaClﺒﺘﺭﺍﻜﻴﺯﻩ ﺍﻟﻤﺨﺘﻠﻔﺔ )،%٥ (%٢٠ ،%١٥ ،%١٠ﻭﻤﺯﺠﻪ ﺠﻴﺩﺍ ،ﺘﹸﺭﻙ ﺍﻟﻤﺯﻴﺞ ﻟﻤﺩﺓ ٤٨ﺴﺎﻋﺔ ﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ °٤ﺩﺭﺠﺔ ﻤﺅﻴﺔ ،ﺜﻡ ﺭﺸﺤﺕ ﺍﻟﻌﻴﻨﺔ ﺃﻭ ﺍﻟﻤﺯﻴﺞ ﺒﻌﺩ ﺫﻟﻙ ﺒﻘﻁﻌﺔ ﻤﻥ ﺍﻟﺸﺎﺵ ﻭﻨﺒﺫﺕ ﺒﺎﻟﻁﺭﺩ ﺍﻟﻤﺭﻜﺯﻱ ﺒﻘﻭﺓ Xg 6000ﻟﻤﺩﺓ ٣٠ﺩﻗﻴﻘﺔ ﺃﻫﻤـل ﺍﻟﺭﺍﺴﺏ ﻭﺃﺨﺫ ﺍﻟﺭﺍﺌﻕ ﺍﻟﺫﻱ ﻴﻤﺜل ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻻﻨﺯﻴﻤﻲ ﺍﻟﺨﺎﻡ ) .(Sharma et al., 2009 ٢-٢-١ﻗﻴﺎﺱ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﺃﻀﻴﻑ ٢٥ﻤﺎﻴﻜﺭﻭﻟﻴﺘﺭ ﻤﻥ ﻤﺤﻠﻭل ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻟﺨﺎﻡ ﺍﻟﻰ ﻨﻔﺱ ﺍﻟﺤﺠﻡ )٢٥ﻤﺎﻴﻜﺭﻭﻟﻴﺘﺭ( ﻤﻥ ﻋﺎﻟﻕ ﺍﻟﺩﻡ %٢ﻓﻲ ﺼﻔﻴﺤﺔ ﺒﻼﺴﺘﻴﻜﻴﺔ ﺘﺤﻭﻱ ﺤﻔﺭﺍﹰ ﺒﺸﻜل ﺤﺭﻑ (V Well plate) Vﺘﻤﺯﺝ ﺍﻟﻌﻴﻨﺔ ﺠﻴﺩﺍ ﻭﺘﺘـﺭﻙ ﻟﻤـﺩﺓ ﻨﺼﻑ ﺴﺎﻋﺔ ﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ﺍﻟﻐﺭﻓﺔ ،ﻓﻌﻨﺩ ﺘﺭﺴﺏ ﻜل ﻋﻴﻨﺔ ﺍﻟﺩﻡ ﻓﻲ ﻗﻌﺭ ﺍﻟﺤﻔﺭﺓ ﻭﻅﻬﻭﺭﻫﺎ ﺒﺸﻜل ﻨﻘﻁـﺔ ﺤـﺎﺩﺓ ﺍﻟﺤﻭﺍﻑ ﻓﺄﻨﻬﺎ ﺩﻟﻴل ﺍﻟﻨﺘﻴﺠﺔ ﺍﻟﺴﺎﻟﺒﺔ ﻭﻫﻲ ﺘﺭﺴﺏ ﻜﺭﻴﺎﺕ ﺍﻟﺩﻡ ﺍﻟﺤﻤﺭﺍﺀ ﺩﻭﻥ ﺘﻠﺯﻨﻬﺎ ،ﺃﻤﺎ ﺍﻟﻨﺘﻴﺠﺔ ﺍﻟﻤﻭﺠﺒـﺔ ﻓﻬـﻲ ﺘﺠﻤﻊ ﻜﺭﻴﺎﺕ ﺍﻟﺩﻡ ﺍﻟﺤﻤﺭﺍﺀ ﺩﻭﻥ ﺃﻥ ﺘﺘﺭﺴﺏ ﻭﺘﻅﻬﺭ ﺒﺸﻜل ﺒﻘﻌﺔ ﻏﻴﺭ ﻤﻨﺘﻅﻤﺔ ﺍﻟﺤﻭﺍﻑ ﻭﻫﻲ ﺩﻟﻴل ﺘﻠﺯﻥ ﻜﺭﻴﺎﺕ ﺍﻟﺩﻡ ﺍ ﻟﺤﻤﺭﺍﺀ ﺒﻔﻌل ﺍﻟﻠﻜﺘﻴﻥ ﻭﺍﻟﺘﺼﺎﻗﻬﺎ ﺒﺒﻌﻀﻬﺎ) ،(Yufang et al., 2010ﺘﹸﻤﺜل ﻋﺩﺩ ﺍﻟﻭﺤﺩﺍﺕ ﺍﻟﺘﻼﺯﻨﻴﺔ ﻋـﺩﺩ ﺍﻟﺤﻔﺭ ﺍﻟﺘﻲ ﺃﻋﻁﺕ ﻨﺘﻴﺠﺔ ﺇﻴﺠﺎﺒﻴﺔ ﻭﺍﻀﺤﺔ ،ﻭﺘﻘﺎﺱ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﺍﻟﻨﻭﻋﻴﺔ ﺑﺎﻟ ـ HU/mgﺍﻱ ﻋﺩﺩ ﺍﻟﻭﺤـﺩﺍﺕ ﺍﻟﺘﻼﺯﻨﻴﺔ ﻟﻜل ﻭﺤﺩﺓ ﺘﺭﻜﻴﺯ ﺒﺭﻭﺘﻴﻥ ).(Sharon and Lis, 1990 ٣-٢-١ﺘﻘﺩﻴﺭ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺃﻀﻴﻑ ﺍﻟﻰ ﺃﻨﺒﻭﺒﺔ ﺍﺨﺘﺒﺎﺭ ﻨﻅﻴﻔﺔ ٠،٠٥ﻤﻠﻴﻠﺘﺭ ﻤﻥ ﻋﻴﻨﺔ ﻤﺤﻠﻭل ﺍﻟﺒﺭﻭﺘﻴﻥ ٠،٤٥ ،ﻤﻠﻴﻠﺘﺭ ﻤـﻥ ﺩﺍﺭﺉ ﺍﻟﻔﻭﺴﻔﺎﺕ ﻭ ٢،٥ﻤﻠﻴﻠﺘﺭ ﻤﻥ ﺼﺒﻐﺔ ﺍﻟﻜﻭﻤﺎﺴﻲ ﺍﻟﺯﺭﻗﺎﺀ ، G-250ﺜﻡ ﻴﻤﺯﺝ ﺍﻟﺨﻠﻴﻁ ﺠﻴﺩﺍ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺠﻬﺎﺯ ﺍﻟﺭﺝ، ﻭﻴﺘﺭﻙ ﻟﻤﺩﺓ ﺩﻗﻴﻘﺘﻴﻥ ﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ﺍﻟﻐﺭﻓﺔ ﺜﻡ ﺘﹸﻘﺎﺱ ﺍﻻﻤﺘﺼﺎﺼـﻴﺔ ﻋﻠـﻰ ﺍﻟﻁـﻭل ﺍﻟﻤـﻭﺠﻲ ٥٩٥ﻨـﺎﻨﻭﻤﺘﺭ ) .(Bradford, 1976 ٤-٢-١ﺘﺤﺩﻴﺩ ﺍﻓﻀل ﻤﺤﻠﻭل ﻤﻠﺤﻲ ﻷﺴﺘﺨﻼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﺘﻡ ﺍﺴﺘﺨﻼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﻓﻲ ﻜل ﻤﻥ ﻤﺤﺎﻟﻴل NaClﺒﺘﺭﺍﻜﻴﺯ %٥ﻭ %١٠ﻭ %١٥ﻭ %٢٠ﻭ ﺍﻟﻤـﺎﺀ ﺍﻟﻤﻘﻁﺭ ﻭﻤﺤﻠﻭل ﺍﻟﻤﻠﺢ ﺍﻟﻔﺴﻠﺠﻲ ،ﻭﻗﺩﺭﺕ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﻭﻗﹸﺩﺭ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ﻟﻜل ﻤﺤﻠﻭل ﺘﻡ ﺍﺴﺘﺨﺩﺍﻤﻪ. 2105 ٥-٢-١ﺘﺨﺼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﻟﺯﻤﺭ ﺍﻟﺩﻡ ﺘﻡ ﺩﺭﺍﺴﺔ ﺘﺨﺼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﺇﺘﺠﺎﻩ ﺯﻤﺭ ﺍﻟﺩﻡ ﻋﻥ ﻁﺭﻴﻕ ﺘﺤﻀﻴﺭ ﻋﻭﺍﻟﻕ ﺍﻟﺩﻡ ﻟﺠﻤﻴﻊ ﺍﻟﺯﻤﺭ ﻭﺒﺘﺭﻜﻴـﺯ %٢ﻭﻴﻤﺯﺝ ٢٥ﻤﺎﻴﻜﺭﻭﻟﻴﺘﺭ ﻤﻥ ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻟﻠﻜﺘﻴﻨﻲ ﺍﻟﻰ ﻨﻔﺱ ﺍﻟﺤﺠﻡ ﻤﻥ ﻋﺎﻟﻕ ﺍﻟﺩﻡ ﻭﻴﺘﺭﻙ ﺒﺩﺭﺠـﺔ ﺤـﺭﺍﺭﺓ ﺍﻟﻐﺭﻓﺔ ﻟﻤﺩﺓ ﻨﺼﻑ ﺴﺎﻋﺔ ﻭﺘﻘﺭﺃ ﺍﻟﻨﺘﺎﺌﺞ ﻤﻥ ﺨﻼل ﺭﺅﻴﺔ ﺍﻟﺘﻠﺯﻥ ﻟﻜﺭﻴﺎﺕ ﺍﻟﺩﻡ ﺍﻟﺤﻤﺭﺍﺀ ﻭﺍﻟﺘﻲ ﺘﻤﺜل ﺍﻟﻨﺘﻴﺠﺔ ﺍﻟﻤﻭﺠﺒﺔ ﻭﻫﻲ ﺍﻟﻔﺔ ﺍﻟﻠﻜﺘﻴﻥ ﻟﺯﻤﺭﺓ ﺍﻟﺩﻡ ).(Kuku et al., 2004 ٦-٢-١ﺍﻟﺘﻨﻘﻴﺔ ﺘﻡ ﺘﻨﻘﻴﺔ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺒﺎﻻﻋﺘﻤﺎﺩ ﻋﻠﻰ ﻋﺩﺓ ﺨﻁﻭﺍﺕ ﺍﻭﺘﻘﻨﻴﺎﺕ ﻭﻫﻲ ﺍﻟﺘﺭﺴﻴﺏ ﺒﻜﺒﺭﻴﺘﺎﺕ ﺍﻻﻤﻭﻨﻴﻭﻡ ﺒﻌﺩﺓ ﺘﺭﺍﻜﻴﺯ ) (%٩٠-%٢٠ﻭﺍﻟﺘﻨﻘﻴﺔ ﺒﺘﻘﻨﻴﺔ ﺍﻟﺘﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻤﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ DEAE-celluloseﻭﺍﻟﺘﻨﻘﻴﺔ ﺒﺎﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻫﻼﻡ .Sephadex G-200ﺤﻴﺙ ﺘﻀﻤﻨﺕ ﻋﻤﻠﻴﺔ ﺍﻟﺘﺭﺴﻴﺏ ﺒﻜﺒﺭﻴﺘﺎﺕ ﺍﻻﻤﻭﻨﻴـﻭﻡ ﺃﻀﺎﻓﺔ ﺃﻭﺯﺍﻥ ﻤﻌﻴﻨﺔ ﻤﻥ ﻜﺒﺭﻴﺘﺎﺕ ﺍﻷﻤﻭﻨﻴﻭﻡ ﺘﺩﺭﻴﺠﻴﺎ ﻋﻠﻰ ﺸﻜل ﻤﺭﺍﺤل ﺍﻟﻰ ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻟﺒﺭﻭﺘﻴﻨﻲ ﺍﻟﺨﺎﻡ ﺒﺩﺭﺠﺔ º٤ﻡ ﻤﻊ ﺍﻟﺘﺤﺭﻴﻙ ﺍﻟﻤﺴﺘﻤﺭ ﻟﺒﻠﻭﻍ ﻨﺴﺏ ﺇﺸﺒﺎﻉ ﺘﺭﺍﻭﺤﺕ ﺒﻴﻥ ) %(٩٠-٢٠ﻭﻨﺒﺫ ﺍﻟﻤﺤﻠﻭل ﺒﻌﺩ ﻜل ﻤﺭﺤﻠـﺔ ﻤـﻥ ﻤﺭﺍﺤل ﺍﻹﻀﺎﻓﺔ ﻋﻠﻰ ﺴﺭﻋﺔ Xg ٦٠٠٠ﻟﻤﺩﺓ ٢٥ﺩﻗﻴﻘﺔ ﺒﺩﺭﺠﺔ º٤ﻡ ﺜﹸﻡ ﻓﺼل ﺍﻟﺭﺍﺌﻕ ﻭﺫﻭﺏ ﺍﻟﺭﺍﺴﺏ ﻓﻲ ﺃﻗل ﻜﻤﻴﺔ ﻤﻥ ﻤﺤﻠﻭل NaClﺒﺭﻗﻡ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ ٨ﻭﺘﻤﺕ ﺩﻴﻠﺯﺘﻪ ﻤﻘﺎﺒل ﺍﻟﻤﺎﺀ ﺍﻟﻤﻘﻁﺭ ﺜﻡ ﻗﺩﺭﺕ ﻓﻴﻪ ﺘﺭﻜﻴﺯ ﺍﻟﺒـﺭﻭﺘﻴﻥ ﻭﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ) .(Dennison, 2002ﻓﻲ ﺤﻴﻥ ﺘﻀﻤﻨﺕ ﺍﻟﺘﻨﻘﻴﺔ ﺒﺎﻟﻤﺒﺎﺩل ﺍﻻﻴـﻭﻨﻲ DEAE-cellulose ﺍﻤﺭﺍﺭ ﺍﻟﻌﻴﻨﺔ ﺍﻟﺒﺭﻭﺘﻴﻨﻴﺔ ﺒﻌﻤﻭﺩ ﺍﻟﺘﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ ﺒﺎﺒﻌﺎﺩ ٢,٥٠×٥٠ﺴﻨﺘﻤﺘﺭ ﻭﺒﺄﺴﺘﺨﺩﺍﻡ ﺍﻟﺘﺩﺭﺝ ﺍﻟﻤﻠﺤﻲ ﻟﻤﺤﻠـﻭل NaClﻷﺠل ﺨﻁﻭﺓ ﺍﻻﺴﺘﺭﺩﺍﺩ ﺍﻟﻤﻠﺤﻲ ﻭﺠﻤﻌﺕ ﺍﻻﺠﺯﺍﺀ ﺍﻟﻤﻨﻔﺼﻠﺔ ﺒﻭﺍﻗﻊ ٢٠ﻤﻠﻴﻠﺘﺭ ﺒﺎﻟﺴﺎﻋﺔ ،ﻓﻲ ﺤﻴﻥ ﺘﻀﻤﻨﺔ ﺨﻁﻭﺓ ﺍﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ﺍﺴﺘﺨﺩﺍﻡ ﻋﻤﻭﺩ ﻫﻼﻡ Sephadex G-200ﺒﺄﺒﻌﺎﺩ ١,٢٥×٥٠ﺴـﻨﺘﻤﺘﺭ ﻭﺒﻭﺍﻗـﻊ ٢٥ ﻤﻠﻴﻠﺘﺭ ﺒﺎﻟﺴﺎﻋﺔ ﻟﻼﺠﺯﺍﺀ ﺍﻟﻤﻔﺼﻭﻟﺔ. ٧-٢-١ﺍﻟﺘﻘﻴﻴﺩ ﺒﺎﻟﻜﻠﻭﺘﺭ ﺍﻟﺩﻴﻬﺎﻴﺩ ﺘﻡ ﺘﻘﻴﻴﺩ ﺍﻟﻠﻜﺘﻴﻥ ﺒﺎﻀﺎﻓﺔ ﺜﻼﺜﺔ ﺃ ﺤﺠﺎﻡ ﻤﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻨﻘﻰ ﺍﻟﻰ ﺤﺠﻡ ﻭﺍﺤﺩ ﻤﻥ ﺍﻟﻜﻠﻭﺘﺭﺍﻟﺩﻴﻬﺎﻴـﺩ ﻴﻤـﺯﺝ ﺍﻟﻤﺤﻠﻭل ﺠﻴﺩﺍﹰ ﻭﺒﺤﺫﺭ ﻴﺭﺝ ﻭﻋﺎﺀ ﺍﻷﺨﺘﺒﺎﺭ ﺒﺸﻜل ﺩﺍﺌﺭﻱ ﺜﻡ ﻴﺘﺭﻙ ﺍﻟﻤﺤﻠﻭل ﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ﺍﻟﻐﺭﻓﺔ ﻟﻤﺩﺓ ٢٤ﺴﺎﻋﺔ، ﻴﺘﻜﻭﻥ ﺒﻌﺩﺌﺫ ﺒﻭﻟﻴﻤﺭ ﺒﺸﻜل ﺤﺒﻴﺒﺎﺕ ﺼﻐﻴﺭﺓ ﺠﺩﺍﹰ ﻏﻴﺭ ﺫﺍﺌﺒﺔ ،ﺘﹸﻔﺼل ﻭﺘﹸﻐﺴل ﻋﺩﺓ ﻤﺭﺍﺕ ﺒﺎﻟﻨﺒﺫ ﺒﻘﻭﺓ Xg ٦٠٠٠ ﻟﻤﺩﺓ ٥ﺩﻗﺎﺌﻕ ﻟﺤﻴﻥ ﻭﺼﻭل ﺍﻟﻘﺭﺍﺀﺓ ﻋﻠﻰ ﻁﻭل ﻤﻭﺠﻲ ٢٨٠ﻨﺎﻨﻭﻤﻴﺘﺭ ﺒﺠﻬﺎﺯ ﺍﻟﻤﻁﻴﺎﻑ ﻟﻠﺭﺍﺸﺢ ﺍﻟﻨﺎﺘﺞ ﻤﻥ ﺍﻟﻐﺴل ﺃﻗل ﻤﻥ ، ٠،٠١ﻴﻌﻠﹼﻕ ﺒﻌﺩ ﺫﻟﻙ ﺍﻟﺒﻭﻟﻴﻤﺭ ﺒﺩﺍﺭﺉ ﺍﻟﻔﻭﺴﻔﺎﺕ ﺍﻟﻤﻠﺤﻲ ﻭﺒﺭﻗﻡ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ ٧،٢ ) .(Pop, et al. 2006 ٨-٢-١ﺘﺤﻀﻴﺭ ﺍﻟﻌﺎﻟﻕ ﺍﻟﺒﻜﺘﻴﺭﻱ ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻰ ﺍﻟﻌﺯﻟﺔ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ Bacillus subtilisﺍﻟﻤﺸﺨﺼﺔ ﻭﺍﻟﻨﻘﻴﺔ ﻤﻥ ﻤﺨﺘﺒﺭﺍﺕ ﻗﺴﻡ ﻋﻠـﻭﻡ ﺍﻟﺤﻴﺎﺓ /ﺠﺎﻤﻌﺔ ﺒﺎﺒل ﻭﺘﺯﺭﻉ ﻋﻠﻰ ﻤﺭﻕ ﺨﻼﺼﺔ ﺍﻟﺩﻤﺎﻍ ﻭﺍﻟﻘﻠﺏ ﻭﺘﺤﻀﻥ ﻓﻲ ﺍﻟﺤﺎﻀﻨﺔ ﻟﻤﺩﺓ ٢٤ﺴﺎﻋﺔ ﻭﺒﺩﺭﺠﺔ ﺤﺭﺍﺭﺓ ٣٧ﻡ °ﺜﻡ ﺘﻭﻀﻊ ﻓﻲ ﺍﻟﺜﻼﺠﺔ ﻟﻐﺭﺽ ﺍﻟﺤﻔﻅ ﻭﺇﻴﻘﺎﻑ ﺍﻟﺘﻔﺎﻋﻼﺕ ﺍﻟﺤﻴﻭﻴﺔ ﻟﻠﺒﻜﺘﺭﻴـﺎ ،ﻭﺤﺴـﺏ ﺍﻟﻁﺭﻴﻘـﺔ ﺍﻟﻤﻭﺼﻭﻓﺔ ﻤﻥ ﻗﺒل ﺍﻟﺒﺎﺤﺙ Popﻭﺠﻤﺎﻋﺘﻪ (2006 ) ﺤﻴﺙ ﺃُﻀﻴﻑ ١،٥ﻤﻠﻴﻠﻴﺘﺭ ﻤﻥ ﻋﺎﻟﻕ ﺍﻟﺒﻜﺘﺭﻴﺎ ﺍﻟﻤﻨﻤﺎﺓ ﻋﻠﻰ ﻤﺭﻕ ﺨﻼﺼﺔ ﺍﻟﺩﻤﺎﻍ ﻭﺍﻟﻘﻠﺏ ﺍﻟﻰ ٠،٥ﻤﻠﻴﻠﻴﺘﺭ ﻤﻥ ﻋﺎﻟﻕ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﻓﻲ ﺃﻨﺎﺒﻴﺏ ﺍﻟﺘﻨﻤﻴﺔ ﻭﺘﹸﺤﻀﺭ ٣ﻤﻜﺭﺭﺍﺕ ﻤﻥ ﻋﺎﻟﻕ ﻟﻜﺘﻴﻥ )ﻤﻘﻴﺩ( -ﺒﻜﺘﺭﻴﺎ ﻟﻜل ﻋﺯﻟﺔ ﺒﻜﺘﻴﺭﻴﺔ ﻭﻴﻘﺭﺃ ﺍﻟﻁﻭل ﺍﻟﻤﻭﺠﻲ ﺁﻨﻴﺎﹰ ﻟﺘﹸﺴﺠل ﺍﻻﻤﺘﺼﺎﺼﻴﺔ ﺍﻟﻀـﻭﺌﻴﺔ 2106 ﻋﻠﻰ ﺍﻟﻁﻭل ﺍﻟﻤﻭﺠﻲ ٦٦٠ﻨﺎﻨﻭﻤﻴﺘﺭ ﻋﻨﺩ ﺍﻟﺯﻤﻥ ﺼﻔﺭ ) (t0ﺜﻡ ﺘﹸﺤﻀﻥ ﺍﻷﻨﺎﺒﻴﺏ ﻓﻲ ﺍﻟﺤﺎﻀﻨﺔ ﺒﺩﺭﺠـﺔ ﺤـﺭﺍﺭﺓ ٣٧ﻡ °ﻟﻤﺩﺓ ﻋﺸﺭ ﺩﻗﺎﺌﻕ ﻭﺘﹸﻘﺭﺃ ﺍﻻﻤﺘﺼﺎﺼﻴﺔ ﻋﻠﻰ ﻁﻭل ﻤﻭﺠﻲ ٦٦٠ﻨﺎﻨﻭﻤﻴﺘﺭ ﻋﻨﺩ ﺍﻟﺯﻤﻥ ) (t10ﺃﻱ ﺒﻌﺩ ﻤﺭﻭﺭ ﻋﺸﺭ ﺩﻗﺎﺌﻕ ﻟﻠﺴﻤﺎﺡ ﺒﺎﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﺒﺎﻷﺭﺘﺒﺎﻁ ﺒﻜﺭﺒﻭﻫﻴﺩﺭ ﺍﺕ ﺃﺴﻁﺢ ﺍﻟﺨﻼﻴﺎ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻭﺒﺎﻟﺘﺎﻟﻲ ﺘﺠﻤﻌﻬﺎ ﻋﻠﻰ ﺴﻁﺢ ﺍﻟﺤﺒﻴﺒﺎﺕ ﻜﻨﺘﻴﺠﺔ ﻻﻟﺘﺼﺎﻗﻬﺎ ﺃﻭ ﺍﺭﺘﺒﺎﻁﻬﺎ ﺒﺎﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ. ٩-٢-١ﺘﺤﻀﻴﺭ ﺸﺭﻴﺤﺔ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﻴﺩ ﻭﺘﺼﺒﻴﻐﻬﺎ ﺒﺼﺒﻐﺔ ﻜﺭﺍﻡ ﺘﹸﺅﺨﺫ ﻋﻴﻨﺔ ﻤﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﺒﻭﺍﺴﻁﺔ ﺍﻟﻨﺎﻗل Loopﺒﻌﺩ ﺭﺝ ﺍﻟﻌﺎﻟﻕ ﺠﻴﺩﺍ ﻭﺘﹸﻭﻀﻊ ﻋﻠﻰ ﺸﺭﻴﺤﺔ ﻨﻅﻴﻔﺔ ﻭﻤﻌﻘﻤﺔ ﻭﺘﺘﺭﻙ ﻋﺸﺭ ﺩﻗﺎﺌﻕ ﻟﺘﺠﻑ ﺜﻡ ﺘﹸﺼﺒﻎ ﺒﺼﺒﻐﺔ ﻜﺭﺍﻡ ﻭﺘﹸﻔﺤﺹ ﺘﺤﺕ ﺍﻟﻤﺠﻬﺭ ﺍﻟﻀﻭﺌﻲ ﻜﺸـﺭﻴﺤﺔ ﺴـﻴﻁﺭﺓ Controlﺒﻘﻭﺓ ﺘﻜﺒﻴﺭ .(Pop et al., 2006 ) X ٤٠ ١٠-٢-١ﺘﺤﻀﻴﺭ ﺸﺭﻴﺤﺔ ﻤﻌﻘﺩ ﺒﻜﺘﺭﻴﺎ)ﻋﺼﻴﺎﺕ( -ﻟﻜﺘﻴﻥ )ﻤﻘﻴﻴﺩ( ﺘﹸﺅﺨﺫ ﻨﻘﻠﺔ ﻤﻥ ﻗﻌﺭ ﺃﻨﺎﺒﻴﺏ ﺍﺨﺘﺒﺎﺭ )ﺘﺤﻀﻴﺭ ﻭﺴﻁ ﺘﻔﺎﻋل ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ-ﺍﻟﻌـﺎﻟﻕ ﺍﻟﺒﻜﺘﻴـﺭﻱ( ﻟﻠﻨـﻭﻉ ﺍﻟﺒﻜﺘﻴﺭﻱ ﻗﻴﺩ ﺍﻟﺩﺭﺍﺴﺔ ﺒﻌﺩ ﻤﺭﻭﺭ ﺍﻟﺩﻗﺎﺌﻕ ﺍﻟﻌﺸﺭ ﻭﺘﹸﺼﺒﻎ ﺒﺼﺒﻐﺔ ﻜﺭﺍﻡ ﻭﺘﹸﻔﺤﺹ ﺘﺤﺕ ﺍﻟﻤﺠﻬﺭ ﺍﻟﻀـﻭﺌﻲ ﺒﻘـﻭﺓ .(Pop et al., 2006 ) X٤٠ ٢ﺍﻟﻨﺘﺎﺌﺞ ﻭﺍﻟﻤﻨﺎﻗﺸﺔ ١- ٢ﺍﻟﺘﺤﺭﻱ ﻋﻥ ﻓﻌﺎﻟﻴﺔ ﺍﻟﻠﻜﺘﻴﻥ ﻓﻲ ﺒﺫﻭﺭ ﺒﻌﺽ ﺍﻟﻨﺒﺎﺘﺎﺕ ﺍﻟﺒﻘﻭﻟﻴﺔ ﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻟﻤﺒﻴﻨﺔ ﻓﻲ ﺍﻟﺸﻜل ) (١ﻭﺠﻭﺩ ﻓﻌﺎﻟﻴﺔ ﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻓﻲ ﺘﻠـﻙ ﺍﻟﻤﺼـﺎﺩﺭ ﻭﺒﻨﺴـﺏ ﻤﺘﻔﺎﻭﺘﺔ .ﺍﺫ ﻭﺠﺩ ﺃﻥ ﻤﺴﺘﺨﻠﺹ ﺒﺫﻭﺭ ﻨﺒﺎﺕ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ ﻗﺩ ﺴﺠل ﺃﻋﻠﻰ ﻓﻌﺎﻟﻴﺔ ﻟﻠﺒﺭﻭﺘﻴﻥ ﺍﺫ ﺒﻠﻐﺕ ﺍﻟﻔﻌﺎﻟﻴﺔ ١٩٢ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ ﺃﻭ ﻤﻌﻴﺎﺭﺍﹰ ﻭﺃﻋﻁﻰ ﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ٧ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻤﻘﺎﺭﻨـﺔﹰ ﺒﺎﻟﻤﺴﺘﺨﻠﺼـﺎﺕ ﺍﻟﻨﺒﺎﺘﻴـﺔ ﺍﻷﺨﺭﻯ ﺍﻟﻌﺎﺌﺩﺓ ﻟﻠﻌﺎﺌﻠﺔ ﻨﻔﺴﻬﺎ. اﻟﻔﻌﺎﻟﯿﺔ اﻟﻜﻠﯿﺔ اﻟﻔﻌﺎﻟﯿﺔ اﻟ ﻨﻮﻋﯿﺔ ﺸﻜل ) :(١ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﻜﻠﻴﺔ ﻭﺍﻟﻨﻭﻋﻴﺔ ﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﺴﺘﺨﻠﺹ ﻤﻥ ﺍﻟﺒﺫﻭﺭ ﺍﻟﻤﺠﻔﻔﺔ ﻟﺒﻌﺽ ﺍﻨﻭﺍﻉ ﻨﺒﺎﺘﺎﺕ ﺍﻟﻌﺎﺌﻠﺔ ﺍﻟﺒﻘﻭﻟﻴﺔ. ٢-٢ﺘﺤﺩﻴﺩ ﺘﺭﻜﻴﺯ ﺍﻓﻀل ﻤﺤﻠﻭل ﻤﻠﺤﻲ ﻟﻼﺴﺘﺨﻼﺹ 2107 ﻜﻤﺎ ﻤﻭﻀﺢ ﻓﻲ ﺍﻟﺠﺩﻭل ) (١ﻓﺈﻥ ﺍﻟﺘﺭﻜﻴﺯ ﺍﻟﻤﻠﺤﻲ %١٠ﻴﻌﻁﻲ ﺃﻋﻠﻰ ﻓﻌﺎﻟﻴﺔ ﺘﻼﺯﻨﻴﺔ ﻭﻫـﻲ ١٠٢٤ ﻤﻌﻴﺎﺭﺍﹰ ﻭﺃﻋﻠﻰ ﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ﻭﻗﺩﺭﻫﺎ ١١،٢ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻜﻤﺎ ﻜﺎﻨﺕ ﻜﻤﻴﺔ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻤﺴﺘﺨﻠﺼـﺔ ١،٨٢ ﻤﻠﻐﻡ/ﻤل ،ﺤﻴﺙ ﺃﻥ ﺍﻟﻘﻭﺓ ﺍﻷﻴﻭﻨﻴﺔ ﻟﻬﺎ ﺩﻭﺭ ﻤﻬﻡ ﻓﻲ ﻋﻤﻠﻴﺔ ﺍﻷﺴﺘﺨﻼﺹ ﻓﻀﻼ ﻋﻥ ﺘﺄﺜﻴﺭﻫﺎ ﻓﻲ ﻓﻌﺎﻟﻴﺔ ﺍﻟﺒـﺭﻭﺘﻴﻥ ﺍﻟﺘﻼﺯﻨﻴﺔ ،ﻭﻗﺩ ﺃﺸﺎﺭ ﺍﻟﺒﺎﺤﺙ Yufangﻭﺠﻤﺎﻋﺘﻪ ) (٢٠١٠ﺃﻥ ﺍﺴﺘﺨﻼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺼﻴﻨﻴﺔ ﺍﻟﻤﺼﺩﺭ ﺒﺘﺭﻜﻴﺯ ﻤﻠﺤﻲ ﻗﺩﺭﻩ ٠،٠٥ﻤﻭﻻﺭ ﻤﻥ ﻜﻠﻭﺭﻴﺩ ﺍﻟﺼﻭﺩﻴﻭﻡ NaClﻻ ﻴﻌﻁﻲ ﻓﻌﺎﻟﻴﺔ ﺠﻴﺩﺓ ﻤﻘﺎﺭﻨﺔ ﺒﺎﻟﺘﺭﻜﻴﺯ ٠،١٥ﻤﻭﻻﺭ ﻭﻋﺯﻱ ﺴﺒﺏ ﺫﻟﻙ ﺍﻟﻰ ﺃﻥ ﺍﺴﺘﺨﺩﺍﻡ ﻜﻠﻭﺭﻴﺩ ﺍﻟﺼﻭﺩﻴﻭﻡ ﺒﺘﺭﻜﻴﺯ ٠،٠٥ﻤﻭﻻﺭ ﻻﻴﺤـﺩﺙ ﺃﻨﻔﺼـﺎﻻﹰ ﻜﺎﻤﻼﹰ ﺒﻴﻥ ﺍﻟﻁﻭﺭ ﺍﻟﻤﺎﺌﻲ ﻭﺍﻻﻁﻭﺍﺭ ﺍﻟﻌﻀﻭﻴﺔ ﻤﻥ ﺠﻬﺔ ﻭﺒﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻭﺍﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﻓﻲ ﺍﻟﻤﺴﺘﺨﻠﺹ ﺍﻟﺨﺎﻡ ﻤﻥ ﺠﻬﺔ ﺃﺨﺭﻯ ﺃﻤﺎ ﻋﻨﺩ ﺍﻟﺘﺭﻜﻴﺯ ٠،١٠ﻤﻭﻻﺭ ﻓﻴﺤﺩﺙ ﺍﻹﻨﻔﺼﺎل ﻟﻜﻥ ﺒﺸﻜل ﻏﻴﺭ ﺘـﺎﻡ ﺒﻴﻨﻤـﺎ ﺍﺴـﺘﺨﺩﺍﻡ ﻜﻠﻭﺭﻴـﺩ ﺍﻟﺼﻭﺩﻴﻭﻡ ﺒﺎﻟﺘﺭﻜﻴﺯ ٠،١٥ﻤﻭﻻﺭ ﻴﺅﺩﻱ ﻷﻨﻔﺼﺎل ﺘﺎﻡ. ٣-٢ﺘﺨﺼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﻟﺯﻤﺭ ﺍﻟﺩﻡ ﺘﺸﻴﺭ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻟﻤﻭﻀﺤﺔ ﺒﺎﻟﺸﻜل ) (٢ﺍﻟﻰ ﺃﻥ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻗﻴﺩ ﺍﻟﺩﺭﺍﺴﺔ ﺫﻭ ﺍﻟﻔﺔ ﻋﺎﻟﻴﺔ ﻓـﻲ ﺘﻠـﺯﻴﻥ ﺯﻤﺭﺘﻲ ﺍﻟﺩﻡ Aﻭ ABﻜﻤﺎ ﻴﻠﺯﻥ ﺍﻟﺯﻤﺭﺘﻴﻥ Bﻭ Oﻟﻜﻥ ﺒﺎﻟﻔﺔ ﺃﻗل ،ﻫﺫﺍ ﻴﻭﻀﺢ ﺍﻻﻟﻔﺔ ﺍﻟﻌﺎﻟﻴﺔ ﻟﻠﻜﺘﻴﻥ ﻗﻴﺩ ﺍﻟﺘﺠﺭﺒﺔ ﺘﺠﺎﻩ ﺍﻟﺴﻜﺭﻴﺎﺕ Galactose, N-acetlyglucosamine, N-acetylgalactosamineﺤﻴﺙ ﺘﺘﻭﺍﺠـﺩ ﻫـﺫﻩ ﺍﻟﺴﻜﺭﻴﺎﺕ ﺒﺸﻜل ﻤﺴﺘﻘﺒﻼﺕ ﻜﺭﺒﻭﻫﻴﺩﺭﺍﺘﻴﺔ ﻋﻠﻰ ﺴﻁﻭﺡ ﺨﻼﻴﺎ ﺍﻟﺯﻤﺭﺘﻴﻥ Aﻭ ABﻓﻘﻁ ﻓﻲ ﺤﻴﻥ ﻟﻡ ﺘﺘﻭﺍﺠﺩ ﻫﺫﻩ ﺍﻟﻤﺴﺘﻘﺒﻼﺕ ﻋﻠﻰ ﺍﻟﺯﻤﺭ Oﻭ .(Kuku et al., 2004) B ﺠﺩﻭل ) :(١ﺃﻓﻀل ﺘﺭﻜﻴﺯ ﻤﻠﺤﻲ ﻻﺴﺘﺨﻼﺹ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﺒﻴﻀﺎﺀ ﺍﻟﻤﺠﻔﻔﺔ Phaseolus vulgaris L. c.v. white ﺘﺭﻜﻴﺯ ﺍﻟﻤﻠﺢ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻜﻠﻲ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﻜﻠﻴﺔ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﻨﻭﻋﻴﺔ % ﻤﻠﻐﻡ/ﻤل ﻤﻠﻐﻡ ﻤﻌﻴﺎﺭ ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ٥ ١،٦٤ ٨٢،١٤ ٨٠ ٠،٩٧ ١٠ ١،٨٢ ٩١،٤ ١٠٢٤ ١١،٢ ١٥ ٠،٩٤ ٤٧،١٤ ١٩٢ ٤،٠٧ ٢٠ ٠،٦٨ ٣٤،٢ ١٩٢ ٥،٦ 2108 ﺸﻜل) :(٢ﺘﺨﺼﺹ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻌﺯﻭل ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ Phaseolus vulgaris L. cv. whiteﺍﻟﻤﺠﻔﻔﺔ ﻟﺯﻤﺭ ﺩﻡ ﺍﻻﻨﺴﺎﻥ. ٤-٢ﺍﻟﺘﻨﻘﻴﺔ ١-٤-٢ﺍﻟﺘﺭﺴﻴﺏ ﺒﻜﺒﺭﺘﺎﺕ ﺍﻻﻤﻭﻨﻴﻭﻡ ﺘﹸﺸﻴﺭ ﺍﻟﻨﺘﺎﺌﺞ ﺍﻟﻤﻭﻀﺤﺔ ﺒﺎﻟﺠﺩﻭل ) (٢ﺍﻟﻰ ﺃﻥ ﺍﻟﺒﺭﻭﺘﻴﻥ ﺴﺠل ﺃﻋﻠﻰ ﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ﻗﺩﺭﻫﺎ ٣٧،٦١ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻋﻨﺩ ﻨﺴﺒﺔ ﺇﺸﺒﺎﻉ %٧٠ﻓﻲ ﺤﻴﻥ ﺒﺩﺃﺕ ﺒﺎﻹﻨﺨﻔﺎﺽ ﻋﻨﺩ ﺍﻟﺘﺭﻜﻴﺯ ﺒﻨﺴﺒﺔ ﺇﺸﺒﺎﻉ %٨٠ﺒﻔﻌﺎﻟﻴـﺔ ﻟـﻡ ﺘﺘﺠﺎﻭﺯ ٢٠،٨٩ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻭﺃﺨﺘﻔﺕ ﺍﻟﻔﻌﺎﻟﻴﺔ ﻨﻬﺎﺌﻴﺎﹰﻋﻨﺩ ﺍﻟﺘﺭﻜﻴﺯ ﺒﻨﺴﺒﺔ ﺇﺸﺒﺎﻉ %٩٠ﻜﻤﺎ ﻫﻭ ﻭﺍﻀﺢ ﻓـﻲ ﺍﻟﺸﻜل ) ،(٣ﻟﺫﺍ ﻋﺩ ﺍﻟﺘﺭﻜﻴﺯ ﺒﻜﺒﺭﻴﺘﺎﺕ ﺍﻷﻤﻭﻨﻴﻭﻡ ﺒﻨﺴﺒﺔ ﺇﺸﺒﺎﻉ %٧٠ﻫﻭ ﺍﻷﻓﻀل ﻭﺍﺴﺘﺨﺩﻡ ﺒﺎﻟﺘﺠﺎﺭﺏ ﺍﻟﻼﺤﻘﺔ. % ٢٠ % ٣٠ % ٥٠ % ٦٠ % ٧٠ % ٨٠ ﻧﺴﺒﺔ اﻹﺷﺒﺎع ﺑﻜﺒﺮﯾﺘﺎت اﻷﻣﻮﻧﯿﻮم % ٤٠ % ٩٠ ﺸﻜل ) :(٣ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻌﺯﻭل ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ Phaseolus vulgaris L. cv. Whiteﺍﻟﻤﺠﻔﻔﺔ ﺍﻟﻤﺭﺴﺏ ﺒﻨﺴﺏ ﺇﺸﺒﺎﻉ ﻤﺨﺘﻠﻔﺔ ﺒﻜﺒﺭﻴﺘﺎﺕ ﺍﻷﻤﻭﻨﻴﻭﻡ. ٢-٤-٢ﺍﻟﺘﻨﻘﻴﺔ ﺒﺎﻟﺘﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻤﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ DEAE-cellulose ﻋﻘﺏ ﻋﻤﻠﻴﺘﺎ ﺍﻟﺘﺭﺴﻴﺏ ﺒﻜﺒﺭﻴﺘﺎﺕ ﺍﻷﻤﻭﻨﻴﻭﻡ ﻭﺍﻟﺩﻴﻠﺯﺓ ﺨﻁﻭﺓ ﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺘﺒﺎﺩل ﺍﻷﻴﻭﻨﻲ ﺒﺄﺴـﺘﻌﻤﺎل ﺍﻟﻤﺒﺎﺩل ﺍﻷﻴﻭﻨﻲ ﺍﻟﻤﻭﺠﺏ ﺜﻨﺎﺌﻲ ﺃﺜﻴل ﺃﻤﻴﻨﻭ ﺃﺜﻴل ﺴﻴﻠﻠﻭﺯ DEAE-celluloseﻭﻜﺎﻥ ﻨﺎﺘﺞ ﻋﻤﻠﻴﺔ ﺍﻟﺘﺭﺤﻴل ﻋﻠـﻰ ﻫﻼﻡ DEAE-celluloseﻅﻬﻭﺭ ﻗﻤﺘﻴﻥ ﺒﺭﻭﺘﻴﻨﻴﺘﻴﻥ ﻋﻨﺩ ﺍﻟﻘﺭﺍﺀﺓ ﻋﻠﻰ ﻁﻭل ﻤﻭﺠﻲ ٢٨٠ﻨﺎﻨﻭﻤﻴﺘﺭ ،ﻭﺍﺤﺩﺓ ﻓـﻲ ﻤﺭﺤﻠﺔ ﺍﻟﻐﺴل Washingﺃﻨﺤﺼﺭﺕ ﺒﺎﻷﺠﺯﺍﺀ ﺍﻟﻤﻨﻔﺼﻠﺔ ٣٧-١٣ﻜﻤﺎ ﻓﻲ ﺍﻟﺸﻜل ) ،(٤ﻜﻤﺎ ﻅﻬﺭﺕ ﻗﻤﺔ ﻭﺍﺤﺩﺓ ﺃﻴﻀﺎ ﻗﻲ ﻤﺭﺤﻠﺔ ﺍﻷﺴﺘﺭﺩﺍﺩ Elutionﻭﺍﻨﺤﺼﺭﺕ ﺒﺎﻷﺠﺯﺍﺀ ٨٠-٦٣ﻭﺍﻟﺫﻱ ﺘﻡ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺘﺩﺭﺝ ﻤﻠﺤﻲ ﺨﻁـﻲ ﻟﻜﻠﻭﺭﻴﺩ ﺍﻟﺼﻭﺩﻴﻭﻡ ٠،٧-٠ﻤﻭﻻﺭﻱ ،ﻭﻋﻨﺩ ﺘﻘﺩﻴﺭ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﻓﻲ ﺍﻟﻘﻤﺘﻴﻥ ﺍﻟﻨﺎﺘﺠﺘﻴﻥ ﻭﺠﺩ ﺃﻥ ﺍﻟﻘﻤﺔ ﺍﻷﻭﻟﻰ ﻓﻘﻁ ﺘﺤﻭﻱ ﻓﻌﺎﻟﻴﺔ ﺘﻼﺯﻨﻴﺔ ﻭﻫﻲ ﺍﻟﻘﻤﺔ ﺍﻟﻨﺎﺘﺠﺔ ﻤﻥ ﻋﻤ ﻠﻴﺔ ﺍﻟﻐﺴل ﻭﺃﻨﺤﺼﺭ ﻭﺠﻭﺩ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺒﺎﻷﺠﺯﺍﺀ ،٢٦-١٥ﻭﺘﻡ ﺘﻘﺩﻴﺭ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﺃﺘﺠﺎﻩ ﺨﻼﻴﺎ ﺍﻟﺩﻡ ﺍﻟﺤﻤﺭﺍﺀ ﻟﻸﻨﺴﺎﻥ ﻟﻜل ﺠﺯﺀ ﺜﹸﻡ ﺠﻤﻌﺕ ﺍﻷﻨﺎﺒﻴﺏ ﺍﻟﺘﻲ ﺃﻋﻁـﺕ ﻓﻌﺎﻟﻴـﺔ ﺘﻼﺯﻨﻴﺔ ﻟﻐﺭﺽ ﺤﺴﺎﺏ ﺍﻟﺤﺠﻡ ﻭﺘﻘﺩﻴﺭ ﺍﻟﻔﻌﺎﻟﻴﺔ ﺍﻟﺘﻼﺯﻨﻴﺔ ﻭﺘﻘﺩﻴﺭ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ،ﺘﻡ ﺒﻌﺩ ﺫﻟﻙ ﺘﺭﻜﻴﺯ ﺍﻟﺒﺭﻭﺘﻴﻥ ﻤﻥ ﺨﻼل ﺩﻴﻠﺯﺘﻪ ﺃﺴﺘﻌﺩﺍﺩﺍﹰ ﻟﺨﻁﻭﺓ ﺍﻟﺘﻨﻘﻴﺔ ﺒﺎﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ،ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻰ ﻋﺩﺩ ﻤﺭﺍﺕ ﺘﻨﻘﻴـﺔ ﻗـﺩﺭﻫﺎ ٦،٤١ ﻭﺤﺼﻴﻠﺔ ﻗﺩﺭﻫﺎ %٥،٣ﻭﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ٤٥،٥ﻭﺤﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ . 2109 ﺠﺩﻭل ) :(٢ﺘﺄﺜﻴﺭ ﻨﺴﺏ ﺇﺸﺒﺎﻉ ﻤﺨﺘﻠﻔﺔ ﻤﻥ ﻜﺒﺭﻴﺘﺎﺕ ﺍﻻﻤﻭﻨﻴﻭﻡ ﻓﻲ ﺘﺭﻜﻴﺯ ﻭﻓﻌﺎﻟﻴﺔ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﺴﺘﺨﻠﺹ ﻤﻥ ﺒﺫﻭﺭ ﻨﺒﺎﺕ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﻤﺠﻔﻔﺔ ﻧﺴﺒﺔ اﻹﺷﺒﺎع % ٢٠ ٣٠ ٤٠ ٥٠ ٦٠ ٧٠ ٨٠ ٩٠ ﺗﺮﻛﯿﺰ اﻟﺒﺮوﺗﯿﻦ ﻣﻠﻐﻢ/ﻣﻞ ٢٠،٣٥ ٢١،٣٨ ١٨،٠٤ ١٢،٥١ ٣١،٦٧ ٣٢،٩٧ ٥٨،٨ - Phaseolus vulgaris L. cv. White ﺗﺮﻛﯿﺰ اﻟﺒﺮوﺗﯿﻦ اﻟﻜﻠﻲ ﻣﻠﻐﻢ ٤٠٧ ٤٢٧،٦ ٣٦٠،٨ ٢٥٠،٢ ٦٣٣،٤ ٦٥٣،٤ ١١٧٦ - اﻟﻔﻌﺎﻟﯿﺔ اﻟﻜﻠﯿﺔ ﻣﻌﯿﺎر ١٩٢ ١٩٢ ٤٤٨ ٤٤٨ ٥١٢٠ ٢٤٥٧٦ ٢٤٥٧٦ - اﻟﻔﻌﺎﻟﯿﺔ اﻟﻨﻮﻋﯿﺔ وﺣﺪة ﺗﻼزﻧﯿﺔ/ﻣﻠﻐﻢ ٠،٤٧ ٠،٤٤ ١،٢٤ ١،٧٩ ٨،٠٨ ٣٧،٦١ ٢٠،٨٩ - ٣-٤-٢ﺍﻟﺘﻨﻘﻴﺔ ﺒﺎﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻫﻼﻡ Sephadex G-200 ﺇﻥ ﺃﺴﺘﻌﻤﺎل ﺨﻁﻭﺍﺕ ﺘﻨﻘﻴﺔ ﺃﺨﺭﻯ ﻴﺤﻘﻕ ﺩﺭﺠﺔ ﺃﻋﻠﻰ ﻤﻥ ﺍﻟﻨﻘﺎﻭﺓ ﻟﺫﺍ ﺍﺴﺘﻜﻤﻠﺕ ﻋﻤﻠﻴﺔ ﺘﻨﻘﻴﺔ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘـﻴﻥ ﺍﻟﻤﻌﺯﻭل ﻤﻥ ﺒﺫﻭﺭ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﻤﺠﻔﻔﺔ ﺒﺨﻁﻭﺘﻴﻥ ﺃﻀﺎﻓﻴﺘﻴﻥ ﺒﻁﺭﻴﻘﺔ ﻜﺭﻭﻤﺎﺘﻭﻏﺭﺍﻓﻴﺎ ﺍﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ﺒﺎﺴـﺘﺨﺩﺍﻡ ﻫﻼﻡ Sephadex G-200ﺤﻴﺙ ﺘﻡ ﻤﻭﺍﺯﻨﺔ ﺍﻟﻌﻤﻭﺩ ﺒﺩﺍﺭﺉ ﺍﻟﺘﺭﺱ ﺍﻟﺤﺎﻤﻀـﻲ Tris-HClﺒﺘﺭﻜﻴـﺯ ٠،٠٢ ﻤﻭﻻﺭﻱ ﺫﻱ ﺭﻗﻡ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ ٨ﻭﺒﺘﺭﻜﻴﺯ ﻤﻠﺤﻲ ٠،١٥ﻤﻭﻻﺭﻱ ﻤﻥ ﻜﻠﻭﺭﻴﺩ ﺍﻟﺼﻭﺩﻴﻭﻡ ،ﻓﺄﻋﻁﺕ ﺨﻁﻭﺓ ﺍﻟﺘﻨﻘﻴﺔ ﺍﻷﻭﻟﻰ ﺒﺎﺴﺘﺨﺩﺍﻡ ﻫﻼﻡ Sephadex G-200ﻓﻌﺎﻟﻴﺔ ﻨﻭﻋﻴﺔ ﻭﺤﺼﻴﻠﺔ ﻭﻋﺩﺩ ﻤﺭﺍﺕ ﺘﻨﻘﻴﺔ ﻗﺩﺭﻫﺎ ٥٧،٠٧ﻭﺤـﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻭ %٢،٣ﻭ ، ٨،٠٣ﻋﻠﻰ ﺍﻟﺘﻭﺍﻟﻲ ،ﻜﻤﺎ ﻤﻭﻀﺢ ﺒﺎﻟﺸﻜل ) (٥ﺤﻴﺙ ﺘﻅﻬﺭ ﻗﻤﺔ ﺒﺭﻭﺘﻴﻨﻴﺔ ﻭﺍﺤﺩﺓ ﻭﻗﻤﺔ ﻓﻌﺎﻟﻴﺔ ﺘﻼﺯﻨﻴﺔ ﻭﺍﺤﺩﺓ. ٤-٤-٢ﺍﻟﺘﻘﻴﻴﺩ ﺃﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻜﻔﺎﺀﺓ ﻋﻤﻠﻴﺔ ﺍﻟﺘﻘﻴﻴﺩ ﺤﻴﺙ ﺃﻋﻁﻰ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﻓﻌﺎﻟﻴـﺔ ﻨﻭﻋﻴـﺔ ﻗـﺩﺭﻫﺎ ٩٩،٥ﻭﺤـﺩﺓ ﺘﻼﺯﻨﻴﺔ/ﻤﻠﻐﻡ ﻭﻓﻌﺎﻟﻴﺔ ﻤﺘﺒﻘﻴﺔ %٢،٣ﻭﺒﻌﺩﺩ ﻤﺭﺍﺕ ﺘﻨﻘﻴﺔ ﻗﺩﺭﻫﺎ .١٤،٠١ﺍﻅﻬﺭﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻗﺩﺭﺓ ﻋﺎﻟﻴﺔ ﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﻓﻲ ﺘﺜﺒﻴﻁ ﻨﻤﻭ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﺼﻴﺎﺕ Bacillus subtilisﺤﻴﺙ ﻜﺎﻨﺕ ﻗﺭﺍﺀﺓ ﺠﻬﺎﺯ ﺍﻟﻤﻁﻴﺎﻑ ﻋﻠﻰ ﻁﻭل ﻤﻭﺠﻲ ٦٦٠ ﻨﺎﻨﻭﻤﻴﺘﺭ ﻫﻲ ٠,٦١٧ﻭﺒﻌﺩ ﻤﺭﻭﺭ ﻋﺸﺭ ﺩﻗﺎﺌﻕ ﻜﺎﻨﺕ ﺍﻟﻘﺭﺍﺀﺓ ﻗﺩ ﺍﻨﺨﻔﻀﺕ ﺇﻟﻰ ٠,٤٥٢ﻫﺫﺍ ﺩﻟﻴل ﻋﻠﻰ ﺍﺭﺘﺒـﺎﻁ ﺍﻟﻠﻜﺘﻴﻥ ﻤﻊ ﻜﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﺴﻁﺢ ﺨﻠﻴﺔ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﺼﻴﺎﺕ ﻭﺘﺭﺴﻴﺒﻬﺎ ﺍﻟﻰ ﻗﻌﺭ ﺍﻻﻨﺒﻭﺒﺔ ﻭﻟﺘﺄﻜﻴﺩ ﻤﺎ ﺘﻡ ﺍﻟﺤﺼﻭل ﻋﻠﻴﻪ ﻤﻥ ﻨﺘﺎﺌﺞ ﺘﻡ ﺘﺤﻀﻴﺭ ﺸﺭﺍﺌﺢ ﻟﻔﺤﺼﻬﺎ ﻋﻠﻰ ﺍﻟﻤﺠﻬﺭ ﺍﻟﻀﻭﺌﻲ ﺒﺎﻻﺴﺘﻌﺎﻨﺔ ﺒﺼﺒﻐﺔ ﻜﺭﺍﻡ ﻭﻜﺎﻨﺕ ﺍﻟﻨﺘﺎﺌﺞ ﻫﻭ ﻅﻬﻭﺭ ﺘﺠﻤﻌﺎﺕ ﻟﻠﺨﻼﻴﺎ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻜﻤﺎ ﻤﻭﻀﺢ ﺒﺎﻟﺸﻜل ).(٦ 2110 8 1.4 7 1.2 أﻷﻣﺘﺼﺎﺻﯿﺔ أﻟﻔﻌﺎﻟﯿﺔ اﻟﻨﻮﻋﯿﺔ أﻟﻔﻌﺎﻟﯿﺔ اﻟﻨﻮﻋﯿﺔ ) وﺣﺪة ﺗﻼزﻧﯿﺔ /ﻣﻠﻐﻢ ( 5 0.8 4 0.6 3 0.4 2 0.2 1 0 0 13 17 21 25 29 33 37 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 5 9 1 ﺣﺠﻢ ﺸﻜل ) :(٤ﻜﺭﻭﻤﺎﺘﻭﻜﺭﺍﻓﻴﺎ ﺍﻟﺘﺒﺎﺩل ﺍﻻﻴﻭﻨﻲ ﺒﺎﺴﺘﺨﺩﺍﻡ ﺍﻟﻤﺒﺎﺩل ﺍﻷﻴﻭﻨﻲ ) (DEAE-celluloseﺒﺎﺒﻌﺎﺩ )(2.5×50 ﺴﻡ ﻟﺘﻨﻘﻴﺔ ﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻤﻥ ﺍﻟﺒﺫﻭﺭ ﺍﻟﻤﺠﻔﻔﺔ ﻟﻨﺒﺎﺕ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ،Phaseolus vulgaris L. cv. Whiteﺤﻴﺙ ﺘﻡ ﻏﺴل ﺍﻟﻌﻤﻭﺩ ﺒﺩﺍﺭﺉ ﺍﻟﺘﺭﺱ ﺍﻟﺤﺎﻤﻀﻲ ) (PH 8،0.02Mﻭﻜﺎﻥ ﺍﻻﺴﺘﺭﺩﺍﺩ ﺒﺘﺩﺭﺝ ﻤﻠﺤﻲ ﺨﻁﻲ ) 0.7M (NaClﻭﺒﺴﺭﻋﺔ ﺠﺭﻴﺎﻥ ١٥ﻤﻠﻴﻠﺘﺭ\ﺴﺎﻋﺔ ﻭﺒﻭﺍﻗﻊ 5ﻤل ﻟﻠﺠﺯﺀ ﺍﻟﻭﺍﺤﺩ. 1.6 12 أﻷﻣﺘﺼﺎﺻﯿﺔ 1.4 10 اﻟﻔﻌﺎﻟﯿﺔ اﻟﻨﻮﻋﯿﺔ 8 1 0.8 6 0.6 4 أﻷﻣﺘﺼﺎﺻﯿﺔ ٢٨٠ﻧﺎﻧﻮﻣﯿﺘﺮ أﻟﻔﻌﺎﻟﯿﺔ اﻟﻨﻮﻋﯿﺔ ) وﺣﺪة ﺗﻼزﻧﯿﺔ /ﻣﻠﻐﻢ ( 1.2 0.4 2 0.2 0 0 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 8 7 6 5 4 3 2 1 ﺣﺠﻢ أﻷﺳﺘﺮداد ) ﻣﻞ ( ﺸﻜل ) :(٥ﻜﺭﻭﻤﺎﺘﻭﻜﺭﺍﻓﻴﺎ ﺍﻟﺘﺭﺸﻴﺢ ﺍﻟﻬﻼﻤﻲ ﻟﺒﺭﻭﺘﻴﻥ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻨﻘﻰ ﺠﺯﺌﻴﺎﹰ ﻤﻥ ﺒﺫﻭﺭ ﻨﺒﺎﺕ ﺍﻟﻔﺎﺼﻭﻟﻴﺎ ﺍﻟﻤﺠﻔﻔﺔ L. Phaseolus vulgaris cv. Whiteﺒﺎﺴﺘﺨﺩﺍﻡ ﻫﻼﻡ ) (Sephadex G-200ﻭﺒﺄﺒﻌﺎﺩ )(50×1.25ﺴﻡ، ﺤﻴﺙ ﺘﻡ ﺍﻟﻐﺴل ﺒﻤﺤﻠﻭل ﺩﺍﺭﺉ ﺍﻟﺘﺭﺱ ﺍﻟﺤﺎﻤﻀﻲ 0.02ﻤﻭﻻﺭﻱ ﻭﺒﺭﻗﻡ ﻫﻴﺩﺭﻭﺠﻴﻨﻲ ٨ﻭﺒﺴﺭﻋﺔ ﺠﺭﻴﺎﻥ15 ﻤﻠﻴﻠﺘﺭ\ﺴﺎﻋﺔ ﻭﺒﻭﺍﻗﻊ 5ﻤﻠﻴﻠﺘﺭ ﻟﻠﺠﺯﺀ ﺍﻟﻭﺍﺤﺩ. 2111 أﻷﻣﺘﺼﺎﺻﯿﺔ ٢٨٠ﻧﺎﻧﻮﻣﯿﺘﺮ 6 1 ( ﺘﻭﻀﺢ ﺍﻟﺼﻭﺭﺓ ﺍﻟﻤﻭﺠﻭﺩﺓ ﺇﻟﻰ ﺍﻟﻴﺴﺎﺭ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﺒﺎﻟﻜﻠﻭﺘﺭﺍﻟﺩﻴﻬﺎﻴﺩ ﻭﻤﺼﺒﻎ ﺒﺼﺒﻐﺔ ﻜﺭﺍﻡ )ﺴﻴﻁﺭﺓ:(٦) ﺸﻜل .ﻓﻲ ﺤﻴﻥ ﺘﻭﻀﺢ ﺍﻟﺼﻭﺭﺓ ﺇﻟﻰ ﺍﻟﻴﻤﻴﻥ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﺼﻴﺎﺕ ﻭﻫﻲ ﻤﺘﺠﻤﻌﺔ ﻨﺘﻴﺠﺔ ﻷﺭﺘﺒﺎﻁﻬﺎ ﺒﺎﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﺩ ﺍﻷﺴﺘﻨﺘﺎﺠﺎﺕ٣ ﻭﺫﻟﻙ ﻤﻥ ﺨﻼلBacillus subtilis ﻨﺴﺘﻨﺘﺞ ﻤﻤﺎ ﺘﻘﺩﻡ ﺃﻥ ﻟﻠﻜﺘﻴﻥ ﺍﻟﻘﺩﺭﺓ ﻋﻠﻰ ﺘﺜﺒﻴﻁ ﻨﻤﻭ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﺼﻴﺎﺕ ﺍﻻﺭﺘﺒﺎﻁ ﺒﺎﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺕ ﺍﻟﻤﺘﻭﺍﺠﺩﺓ ﻋﻠﻰ ﺴﻁﺢ ﺍﻟﺨﻼﻴﺎ ﻭﺒﻤﺎ ﺃﻥ ﺍﻟﻠﻜﺘﻴﻥ ﻤﻘﻴﻴﺩ ﻓﺄﻥ ﺍﺭﺘﺒﺎﻁ ﺍﻟﻠﻜﺘﻴﻥ ﺒﺎﻟﻤﺴـﺘﻘﺒﻼﺕ ﻓﺄﻨﻪ ﺴﻴﺭﺩﻉ ﺃﻭ ﻴﻤﻨﻊ ﺤﺭﻜﺔBacillus subtilis ﺍﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺘﻴﺔ ﺍﻟﻤﻭﺠﻭﺩﺓ ﻋﻠﻰ ﺴﻁﺢ ﺨﻼﻴﺎ ﺒﻜﺘﺭﻴﺎ ﺍﻟﻌﺼﻴﺎﺕ ﺍﻟﺨﻼﻴﺎ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻭﺒﺎﻟﺘﺎﻟﻲ ﻓﺄﻨﻪ ﺴﻴﻤﻨﻊ ﺍﻟﺨﻼﻴﺎ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻤﻥ ﺍﻷﻏﺘﺫﺍﺀ ﻭﺍﻟﺒﺤﺙ ﻋﻥ ﻏﺫﺍﺌﻬﺎ ﻭﺘﻜﻭﻥ ﺍﻟﻨﺘﻴﺠـﺔ ﻫـﻭ ﻓﺄﻨﻪ ﻋﻨﺩ٦٦٠ ﺃﻤﺎ ﺴﺒﺏ ﺍﻨﺨﻔﺎﺽ ﺍﻟﻘﺭﺍﺀﺓ ﻋﻨﺩ ﺍﻟﻘﻴﺎﺱ ﺒﺠﻬﺎﺯ ﺍﻟﻤﻁﻴﺎﻑ ﺍﻟﻀﻭﺌﻲ ﻋﻠﻰ ﻁﻭل ﻤﻭﺠﻲ،ﺘﺜﺒﻴﻁ ﻟﻠﻨﻤﻭ ﺍﺭﺘﺒﺎﻁ ﺍﻟﻠﻜﺘﻴﻥ ﺍﻟﻤﻘﻴﻴﺩ ﺒﺎﻟﻤﺴﺘﻘﺒﻼﺕ ﺍﻟﻜﺭﺒﻭﻫﻴﺩﺭﺍﺘﻴﺔ ﻟﻠﺨﻼﻴﺎ ﺍﻟﺒﻜﺘﻴﺭﻴﺔ ﻭﺒﻔﻌل ﺍﻟﺠﺎﺫﺒﻴﺔ ﻓﺄﻨﻬﺎ ﺴﺘﺘﺭﺴﺏ ﺇﻟﻰ ﻗـﺎﻉ .ﺃﻨﺒﻭﺒﺔ ﺍﻷﺨﺘﺒﺎﺭ ﻗﺘﻘل ﻋﻜﻭﺭﺓ ﺍﻟﻭﺴﻁ ﻭﺘﻘل ﻗﺭﺍﺀﺓ ﺠﻬﺎﺯ ﺍﻟﻤﻁﻴﺎﻑ ﺍﻟﻀﻭﺌﻲ ﺍﻟﻤﺼﺎﺩﺭ Bradford, M. 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