بررسي و بهينه سازي ميزان توليد توكسين وبا توسط باكتري ويبريو كلرا در دوره هاي زماني مختلف

Evaluation and optimization of cholera toxin production by Vibrio cholerae in different time periods

ﺳﻢ ﺑﺎﮐﺘﺮي وﯾﺒﺮﯾﻮ ﮐﻠﺮا ﯾﮑﯽ از ﻣﻮرد ﻣﻄﺎﻟﻌﻪ ﺗﺮﯾﻦ ﺳﻤﻮم ﺑﺎﮐﺘﺮﯾﺎﯾﯽ ﺧﺎﻧﻮاده AB5 اﺳﺖ ﮐﻪ ﺑﻪ دﻟﯿﻞ ﺗﻮاﻧﺎﯾﯽ آن در اﻓﺰاﯾﺶ ﭘﺎﺳﺦ اﯾﻤﻨﯽ ﻧﺴﺒﺖ ﺑﻪ اﻧﺘﯽ ژن ﺗﺰرﯾﻖ ﺷﺪه ﺑﺎ آن ﺑﻪ ﻃﻮر ﮔﺴﺘﺮده ﺑﻪ ﻋﻨﻮان ﯾﮏ ادﺟﻮاﻧﺖ ﻣﻮﮐﻮزال ﻗﻮي ﻣﻮرد اﺳﺘﻔﺎده ﻗﺮار ﻣﯽ ﮔﯿﺮد. اﯾﻦ ﺗﻮﮐﺴﯿﻦ از دو زﯾﺮ واﺣﺪ، ﯾﮏ زﯾﺮواﺣﺪ ﭘﻨﺘﺎﻣﺮﯾﮏ ﺑﻪ ﻧﺎم زﯾﺮ واﺣﺪ B و ﯾﮏ زﯾﺮواﺣﺪ A ﺗﺸﮑﯿﻞ ﺷﺪه اﺳﺖ. اﯾﻦ ﺗﻮﮐﺴﯿﻦ از ﻃﺮﯾﻖ اﺗﺼﺎل ﺑﻪ ﮔﯿﺮﻧﺪه ﮔﺎﻧﮕﻠﻮزﯾﺪي GM1 ﺑﻪ ﺳﻠﻮل ﻫﺪف وارد ﻣﯽ ﮔﺮدد. در اﯾﻦ ﭘﮋوﻫﺶ ﺑﺎﮐﺘﺮي وﯾﺒﺮﯾﻮ ﮐﻠﺮا درون 100 ﺳﯽ ﺳﯽ ﻣﺤﯿﻂ AKI و Craig’s ﺑﻪ ﺻﻮرت دو ﮔﺮوه دوﺗﺎﯾﯽ ﮐﺸﺖ داده ﺷﺪ. ﺳﭙﺲ ﯾﮏ ﮔﺮوه درون اﻧﮑﻮﺑﺎﺗﻮر 37 درﺟﻪ ﺳﺎﻧﺘﯽ ﮔﺮاد و ﮔﺮوه دﯾﮕﺮ درون اﻧﮑﻮﺑﺎﺗﻮر 30 درﺟﻪ ﺳﺎﻧﺘﯽ ﮔﺮاد و ﺑﻪ ﻣﺪت 72 ﺳﺎﻋﺖ ﻗﺮار داده ﺷﺪ. از ﺗﻤﺎﻣﯽ ﻣﺤﯿﻂ ﺑﻪ ﺻﻮرت ﻣﻨﻈﻢ از ﻟﺤﻈﻪ آﻏﺎز دوره اﻧﮑﻮﺑﺎﺳﯿﻮن ﺑﻪ ﻣﻨﻈﻮر ﺑﺮرﺳﯽ ﻣﯿﺰان ﺗﻮﻟﯿﺪ ﺗﻮﮐﺴﯿﻦ ﻧﻤﻮﻧﻪ ﺑﺮداري ﺷﺪ. در ﻧﻬﺎﯾﺖ ﻣﯿﺰان ﺗﻮﻟﯿﺪ ﺗﻮﮐﺴﯿﻦ ﺑﻪ وﺳﯿﻠﻪ آزﻣﻮن اﻻﯾﺰا GM1 ﻣﻮرد ﺑﺮرﺳﯽ ﻗﺮار ﮔﺮﻓﺖ. ﻧﺘﺎﯾﺞ ﻧﺸﺎن داد ﮐﻪ ﺑﺎﮐﺘﺮي در ﻫﺮ دو ﻣﺤﯿﻂ ﺗﻮاﻧﺎﯾﯽ ﺗﻮﻟﯿﺪ ﺗﻮﮐﺴﯿﻦ ﺑﻪ ﻣﯿﺰان ﻧﺴﺒﺘﺎ ﺑﺎﻻ را دارد. ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﺗﻮﻟﯿﺪ ﺗﻮﮐﺴﯿﻦ در ﻣﺤﯿﻂ ﮐﺸﺖ Craig’s در دﻣﺎي 30 درﺟﻪ ﺳﺎﻧﺘﯽ ﮔﺮاد ﺑﻌﺪ از 50 ﺳﺎﻋﺖ ﻣﺸﺎﻫﺪه ﺷﺪ. ﻫﻤﭽﻨﯿﻦ ﻧﺘﺎﯾﺞ ﺣﺎﺻﻞ از ﺗﺴﺖ اﻻﯾﺰا GM1 ﻧﺸﺎن داد ﮐﻪ ﺑﺎﮐﺘﺮي ﭘﺲ از 50 ﺳﺎﻋﺖ ﺑﯿﺸﺘﺮﯾﻦ ﺗﻮﻟﯿﺪ را در ﻣﺤﯿﻂ Craig’s و در دﻣﺎي 30 درﺟﻪ ﺳﺎﻧﺘﯽ ﮔﺮاد دارد اﯾﻦ در ﺣﺎﻟﯽ اﺳﺖ ﮐﻪ ﺑﯿﺸﺘﺮﯾﻦ ﻣﯿﺰان ﺗﻮﻟﯿﺪ ﺗﻮﮐﺴﯿﻦ در ﻣﺤﯿﻂ AKI ﭘﺲ از 24 ﺳﺎﻋﺖ و در دﻣﺎ 37 درﺟﻪ ﺳﺎﻧﺘﯽ ﮔﺮاد ﺑﺪﺳﺖ ﻣﯽ آﯾﺪ.

Vibrio cholerae is one of the most studied bacterial toxins in the AB5 family and is widely used as a potent mucosal adjuvant because of its ability to enhance the immune response to the antigen injected with it. This toxin consists of two subunits, a pentameric subunit called subunit B, and a subunit A. This toxin enters the target cell by binding to the GM1 ganglionic receptor .In this study Vibrio cholerae were cultured in 100 ml of AKI and Craig's medium in two groups. One group was then incubated at 37 ° C and the other group was incubated at 30 ° C for 72 h. All media were sampled on a regular basis from the beginning of the incubation period to evaluate the toxin production. Finally, the amount of toxin production was evaluated by ELISA GM1 test. The results showed that the bacteria in both culture medium were capable of producing relatively high levels of toxin. The highest toxin production was observed in Craig's medium at 30 ° C after 50 h. The results of ELISA GM1 test showed that the bacterium had the highest production in Craig's medium after 50 hours at 30 ° C, while the highest toxin production in AKI medium after 24 hours at 37° C.