Defence Institute of Physiology and Allied Sciences, Defence Research and Development Organisation, Delhi 110 054, India.
Res Vet Sci. 2010 Apr;88(2):258-62. doi: 10.1016/j.rvsc.2009.09.004.
The present work on Bubalus bubalis (buffalo) was designed to study heat shock protein 70 (HSP70) induction in lymphocytes, its purification and characterization. HSP70 induction and expression kinetics at different temperatures and time durations were also studied. HSP70 purification was carried out by immunoaffinity chromatography using adenosine di-phosphate (ADP-agarose column) and the characterization of the purified protein was done using western blotting by mouse monoclonal anti-HSP70. The molecular weight of HSP70 of buffalo lymphocytes was found to be approximately of 68 kDa and was less than that of bovine brain HSP70. The purified HSP70 was assessed using indirect inhibition enzyme-linked immunosorbent assay (ELISA). A good amount of HSP70 (1430 eta g HSP70/100 microl) was recovered after purification, out of the total 2040.40 eta g of HSP70/100 microl of cell supernatant. To assess the impact of temperature and time dependent variability in the induction and expression pattern of HSP70, buffalo lymphocytes were subjected to three different temperature treatments, viz.: (I) 38 degrees C for 48 h and further exposed the same cells at 45 degrees C for 3 h, (II) 42 degrees C for 3 h, and (III) 45 degrees C for 3 h, respectively. The respective cell viability was found to be 68%, 63%, and 51%. The HSP70 levels were 58.30+/-4.37, 42.59+/-9.04 and 21.95+/-6.79 ng/million cells, respectively, at three temperature exposures. The results indicates that higher intensity and duration of temperature exposure cause higher HSP70 induction in buffalo lymphocytes to maintain cellular homeostasis with a threshold of thermal dose for maximum HSP70 expression. However immediate induction of HSP70 in the lymphocytes was dependent on magnitude of thermal exposure (stress level) and time of thermal exposure (stress duration). The present study on HSP70 and its induction will help likely to solve the problems related to the present scenario of thermo-adaptability in buffaloes.
本研究旨在探讨水牛淋巴细胞热休克蛋白 70（HSP70）的诱导、纯化及特性。还研究了不同温度和时间持续时间下 HSP70 的诱导和表达动力学。通过免疫亲和层析用二磷酸腺苷（ADP-琼脂糖柱）纯化 HSP70，并用小鼠单克隆抗 HSP70 的 Western blot 对纯化蛋白进行特性分析。发现水牛淋巴细胞 HSP70 的分子量约为 68 kDa，小于牛脑 HSP70。使用间接抑制酶联免疫吸附试验（ELISA）评估纯化的 HSP70。从 2040.40 eta g HSP70/100 microl 的细胞上清液中，纯化后回收了相当数量的 HSP70（1430 eta g HSP70/100 microl）。为了评估 HSP70 诱导和表达模式随温度和时间的变化的影响，将水牛淋巴细胞分别进行三种不同温度处理，即：（I）38°C 48 小时，然后将相同的细胞暴露于 45°C 3 小时，（II）42°C 3 小时，（III）45°C 3 小时。分别发现细胞存活率为 68%、63%和 51%。在三种温度暴露下，HSP70 水平分别为 58.30+/-4.37、42.59+/-9.04 和 21.95+/-6.79 ng/百万个细胞。结果表明，较高的温度强度和持续时间导致水牛淋巴细胞中 HSP70 的诱导增加，以维持细胞内稳态，达到最大 HSP70 表达的热剂量阈值。然而，淋巴细胞中 HSP70 的即刻诱导取决于热暴露的幅度（应激水平）和热暴露的时间（应激持续时间）。本研究关于 HSP70 及其诱导将有助于解决水牛热适应性方面的问题。
