The Preliminary Study Of Cyprinus Carpio Peptidoglycan Recognition Protein (PGRP) In Innate Immunity

Compared with adaptive immunity,innate immunity,as an immunoprotection mechanism that animals born with,is much more earlier in activation and faster in protecting reactions.Innate immunity in teleost mainly works through the combination of their pattern recognize receptor and specific pathogen-associated molecular patterns that existed in outside pathogen to active relevant cell signal pathways corresponding with eliminating pathogen that invading organism.In this case,it might be faster in dealing with microbe invasion in an early stage.As a pattern recognize receptor,peptidoglycan recognition protein(PGRP)is able to specifically recognize and bond to peptidoglycan existing on microbe cell wall and then activating signal pathways related to innate immunity to trigger downstream effective protein or other molecular to fight against those invasion even kill bacterial by themselves.In human,the short-type PGRP can act this way while the long-type PGRP with long transcript,performs not only as pattern recognize receptor,but also acts like N-acetylmuramoyl-L-alanine amidase due to its convert phage T7 like amidase domain.By far,there are many kinds of peptidoglycan recognition protein now being cloned and identified in teleost.And researches on what role they perform in innate immunity is on the process.In my experiments here,I focused on one of the most popular kind of freshwater fish in our country,which is Cyprinus carpio,and first dealing with Cyprinus carpio in two immunostimulation measures such as intraperitoneal injection and soak test.Real-time PCR analyses were then performed with immune tissues at different time point post stimulation to figure out different expression pattern of PGRP genes from time to locations.PGRP5 mRNA expression was apparently up regulated in Cyprinus carpios? spleen,headkidney,foregut and hindgut after intraperitoneal injection treatment.At 6 hours post injection to the top expression of 4.88,6,3.44,5.58 times in experimental tissues mentioned before.While PGRP6 mRNA shared the samevariation tendency.At 1 day post injection,PGRP6 mRNA expression in spleen upregulated to top of 19 times compared to control group.In foregut,PGRP6 upregulated to top of 17.54 times compared to control group.In hindgut PGRP6 mRNA expression in spleen upregulated to top of 36.2 times compared to control group at 6 hours post injection.When it came to analyses of sock test tissues,PGRP5 mRNA expression was apperently upregulated in liver,top to 5.87 times compared to control group at 7 days post stimulation and top to 4.27 times at 3 days post stimulation in hindgut.There were no significant variation in the rest of tissues challenged.PGRP6 mRNA expression upregulated in liver,spleen and hindgut post stimulation.In liver,PGRP6 mRNA expression upregulated to top of 14.67 times at 6hours post stimulation compared to control group,in spleen,11.8 times to the top at1 day post stimulation,23 times to the top at 12 hours post stimulation in hindgut.Results above showed either PGRP5 or PGRP6 played an important part in immune response to bacterial invasion.Then I performed PGRP6 gene expression successfully in vitro and carried out several analyses to its amidase biological activity.Results showed that PGRP6 in Cyprinus carpio has N-acetylmuramoyl-L-alanine amidase activity similar to phage T7 hydrolytic enzyme and it is able to bind to bacteria cell wall then somehow hydrolyse them.