Screen And Identify Differentially Expressed Proteins Of Plasma In Cold Stressed Rats

Cold stimulation as a common cold stressor in livestock production, easy cause animals incold stress condition, resulting in reduced quality of animal products and increased animalmortality. It has become one of the key factors to restrict the rapid development of breedingindustry in our country. The researchers also pay attention to study the mechanism of cold stressboth at home and abroad. At present, the study of mechanism only emphasize the role of asingle or few molecules in cold stress, and lack of complete and systematic research, which isdifficult to accurately assess the cold stress state of animals. Therefore, screening differentiallyexpressed plasma proteins in cold stress rats based on iTRAQ combined with massspectrometry technology, in order to find suitable diagnostic markers of cold stress at themolecular level, and provide theoretical basis to prevent and control cold stress.92health SPF Wistar rats were used as the research object, which only12weeks of age andthe weight is about340g. The rats were randomly divided into control group (Z group) andcold stress group (L group). The temperature of room raising is（24.0±0.1）℃, and the coldstress temperature is（4.0±0.1）℃, duration of cold stress was12h. After the cold stress, thelevels of IL-2and IL-4in serum of rats were measured by ELISA, and the levels of HSP70andmRNA of HSP70in peripheral-blood lymphocyte of rats were measured by Western Blot andqRT-PCR, and to determine whether the rats be in cold stress status, then to successful establishthe cold stress model of rats. According to the conditions of cold stress model in rats to begincold stress test. After the cold stress, separated plasma and screened differentially expressedplasma proteins in cold stress rats by iTRAQ technology. Through the COG functionclassification, the enrichment of GO and Pathway analysis to find the differentially expressedproteins in cold stress. Then, to verify the differentially expressed proteins by Western Blot. Theresults were showed as following:1. Establishment of cold stress model of ratsThe results showed that the level of IL-2in serum of cold stress group increased significantlycompared with control group (P<0.05), and the level of IL-4in serum of cold stress groupincreased quite significantly compared with control group (P<0.01). The levels of HSP70andmRNA of HSP70in peripheral-blood lymphocyte of cold stress group were significantly increased compared with control group (P<0.05). At the same time, combine with the results ofthe team members: the levels of IL-6、TNF-α and CORT in serum of cold stress group increasedquite significantly compared with control group(P<0.01), the level of ACTH in serum of coldstress group increased significantly compared with control group (P<0.01), while the IL-10hadno significant difference. In conclusions, cold stress model of rats was established successfully.2. Screen differentially expressed plasma proteins in cold stress rats by iTRAQ technologyTotally,39differentially expressed proteins were screened, including29up-regulatedproteins and10down-regulated proteins. The important differentially expressed proteins Rap1band ITGB in cold stress were screened by bioinformatics analysis.3. Verified the differentially expressed proteins Rap1and ITGB by Western BlotThe level of Rap1b in plasma of cold stress group were significantly increased comparedwith control group (P<0.05), and the level of ITGB in plasma of cold stress group increasedquite significantly compared with control group (P<0.01).The conclusion is as follows: cold stress model of rats was established successfully. Thedifferentially expressed plasma proteins were successfully screened in cold stress rats, and theimportant differentially expressed proteins Rap1b and ITGB in cold stress were screened bybioinformatics analysis. The results of Rap1b and ITGB were positive by Western Blot, andthey were consistent with the results of the iTRAQ technology.