| Pig liver carboxylesterase is abundant in pig liver and belongs to serine hydrolase.It is the most complex enzyme family in mammalian enzymes which is comprised by a variety of isoenzymes whose gene sequence are highly homologous.Our research team has cloned 108 kinds of PLE isoenzymes from Tong Cheng swine and Large White swine,including 5 reported subtypes and 103 new found subtypes.According to the amino acid types at 25 sites in 5 mutation regions,The PLE subtypes are re-divided into 7 categories(A ? G),and each category is distinguished by Arabic numerals.Previous studies have demonstrated that human and mouse carboxylesterases can hydrolyze endocannabinoids,and it was found that the two main subtypes of PLE,PLEA1 and PLE-B9,can hydrolyze the anti-inflammatory mediator cannabinoid(2-AG)with high activity to produce arachidonic acid(AA)and thus up-regulate the level of inflammation On the basis of previous studies,the main subtype of PLE,which plays a role in the inflammatory response of Tong Cheng swine and Large White swine,was further targeted in this paper.The specific methods and main results are as follows:Twelve isotypes with high abundance of PLE isozyme in Tong Cheng swine and large white swine were selected for the study,namely,PLE-A1,PLE-B9,PLE-C1,PLEC4,PLE-D1,PLE-E4,PLE-F1,PLE-F4,PLE-G3,PLE-G5,PLE-G14 and PLE-G16(covering various types of A-G).Firstly,eukaryotic overexpressed plasmids of 12 isoforms of PLE were constructed(previously constructed by the research group of PLE-A1 and PLE-B9),Universal si RNA synthesis was designed.and of all subtypes of a pair of highly efficient silencing PLE was screened by WB test.In cell inflammation model(293T+PAM)on the expression and PLE and silence each subtype,detection of proinflammatory factor(IL-1?,IL-6,TNF-?)level,found that 12 subtypes ascending levels of proinflammatory factor effect each are not identical,prompt PLE different subtypes hydrolysis cannabinoid ability,for further research on Tong Cheng swine and Large White swine regulate inflammation reaction mainly PLE subtypes laid a solid foundation.Secondly,prokaryotic expression plasmids of 12 isoforms of PLE were constructed and purified to obtain 12 isoform recombinant proteins(previously constructed by the research group of PLE-A1 and PLE-B9).By measuring the hydrolytic activity of the common substrate p-NPA,it was proved that the obtained recombinant proteins were all active,and the hydrolytic activity of different isoforms to the common substrate was different.The specific activity of 12 isoforms of in vitro hydrolyzed cannabinoids(2-AG and AEA)was detected by LC-MS / MS,and it was found that the ability of hydrolyzing cannabinoids in vitro varied greatly among different isoforms and was proportional to the activity of hydrolyzing p-NPA to some extent.The results showed that the proinflammatory effect of different isoforms of PLE in inflammation was related to their ability to hydrolyze cannabinoids.Finally,LPS was used to directly stimulate pig primary alveolar macrophages(PAM)to induce an inflammation model,various subtypes enzyme were added respectively,to detect proinflammatory factor(IL-1?,IL-6,TNF-?,IL-12,etc.)m RNA and protein level,protein chip results consistent with the results of m RNA,no matter in the level of m RNA or protein level,different subtypes of inflammation contribution to the hydrolysis cannabinoid ability.The PLE subtypes and abundance of Ton Cheng swine and Large White swine at different ages are different,so the results of protein chip were combined with the PLE expression profile of Tong Cheng swine and Large White swine,thus determining the main subtypes that play a key role in inflammation at different ages of Tong Cheng swine and Large White swine.Finally,the key subtypes in the inflammatory response of Large White swine are identified as PLE-A1,PLE-B9 and PLE-G5,and the key subtypes in the inflammatory response of Tong Cheng swine are determined as PLE-B9,PLE-C4,PLE-G3 and PLE-G5. |
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