Expression Of HSPs In The Peripheral Blood Lymphocytes Of Beef Cattle By Transportation Durations And Cloning Of HSP70Gene

Abstract:Stress is a nonspecific response which is generated by stressful stimuli, the development of livestock and poultry breeding industry, and the prosperity of Livestock Trade at home and abroad, livestock transport has become the essential link in the animal husbandry. The experiment randomly choose6head Xia Nanniu （（?）） which were raised in the same environment, with bovine animal transport vehicles to transport stress, transportation time for before transportation1h、Oh;transportation2h、3h、9h、10h、15h、20h;after transportation10h、20h、30h. The road is mixed road including City Road、State Road、high speed, speed control in60-70km/h, the transportation without water and food. Experimental beef were taken blood on time, then separated plasma、serum and lymphocyte, the plasma and serum were stored at-20℃for ACTH and COR analysis. Lymphocytes were subsequently stored at-80℃, through the RT-PCR cloned genes by transport stress inducted in peripheral blood lymphocytes, for HSPs and HSPs mRNA transcript analysis, the current study is examines the expression kinetics of HSP70, HSP27, HSP90, and the corresponding mRNA transcripts of these compounds in the peripheral blood lymphocytes of beef cattle during different transportation processes of varying duration. Additionally, the relationship between HSPs, COR, and ACTH are examined in order to provide the link between stress during transportation processes and the regulation of various HSPs.We cloned one beef cattle gene of transport stress induced by using RT-PCR method: HSP70, The full length cDNA of HSP70is985bp. BLASTX results displayed, the genes of encoding nucleotide sequence has a high homolog with multiple animal HSP70, with cattle AY662497.1, sheep EF197836.1and wild boar NM₂13766.1sequences homologous respectively reached99%,99%and97%.Hormone levels was analyzed by radioimmunoassay, study influence of long-distance transport stress on beef hormone levels. The results show that, experimental bovine serum Cor is relatively low before transportation, it was increased significantly （P<0.01） at transportation10h,15h and20h, then slowly return to normal level at arrived. Experimental bovine serum adrenocorticotropic hormone low before the transportation, it was significantly increased （P<0.01）at transportation3h, the rising trend continued until after transportation20h, then return to normal level after transportation30h. Real-time fluorescence quantitative RT-PCR technology, study of HSPs mRNA transcript levels in lymphocytes of transportation stress bovine, it provided the experimental data for HSP mRNA as stress biological labels. Results show that, HSP70mRNA transcription level increased significantly （P<0.05） at transportation3h、9h in cattle peripheral blood lymphocytes, then the expression level returned to before transportation level; HSP27mRNA transcript levels significantly increased （P<0.05） at transportation3h; HSP90mRNA transcription level began increase only after transportation10h, and significantly increased （P<0.01） at after transportation the20h, reaches maximum value. Collected peripheral blood lymphocytes were subjected to protein quantification.The levels of HSP70. HSP27and HSP90in the peripheral blood lymphocytes of transported cattle were measured using commercially available ELISA kits. Quantification of samples was performed using a standard curve. The β-actin was used to mediate bias caused by the protein extraction procedure. ELISA results showed that, the expression of HSP70increased significantly （P<0.01） at transportation2h、3h and9h in experience cattle peripheral blood lymphocytes, then the expression levels returned to before transportation level. The expression trend of HSP27is similar with HSP70, HSP27was significantly increased （P<0.01） at transportation9h, and subsequently reduced and recovered to the levels of before transportation. HSP90was significantly increased （P<0.01） at the transportation9h.