α-苦瓜素蛋白的原核表达及其抗体制备

Prokaryotic Expression of Alpha-momorcharin and Preparation of Its Polyclonal Antibody

α-苦瓜素是一种典型的Ⅰ型核糖体失活蛋白,具有多种生物活性,有着广泛的应用前景。本研究通过原核表达技术成功获得α-苦瓜素蛋白及制备了血清抗体。首先通过RT-PCR方法从苦瓜籽中克隆到α-苦瓜素全长基因,然后将该基因连接至原核表达载体p ET-28a(+)上,导入大肠杆菌Rosetta菌株中并少量诱导表达,筛选出重组蛋白的最适表达条件。在37℃、终浓度1 mmol/L IPTG的诱导条件下,可成功诱导出分子量约为29.7 k D左右的可溶性重组蛋白,与预期α-苦瓜素大小一致。按照最适条件进行大量诱导α-苦瓜素蛋白的表达,利用Ni-NTA柱对α-苦瓜素重组蛋白进行纯化,最后以纯化的α-苦瓜素蛋白免疫注射新西兰大白兔制备抗血清。Western blotting分析表明,制备的抗血清具有较好的特异性和灵敏度,可以特异性检测苦瓜籽中的α-苦瓜素蛋白。本研究结果为下一步探究α-苦瓜素调控植物防御生物胁迫的分子机理提供指导。

Alpha-momorcharin is a typical type I ribosome-inactivating protein with a variety of biological activities and has broad application prospects. In this study, alpha-momorcharin protein was successfully obtained by prokaryotic expression technology and serum antibodies were prepared. Firstly, The alpha-momorcharin gene from which the signal peptide was removed was cloned from bitter gourd seeds by RT-PCR. Then the gene was ligated into the prokaryotic expression vector pET-28 a(+), Introduced into E. coli Rosetta strain and induced to express in a small amount. The optimal expression conditions of recombinant protein were selected. Under the condition of induction at 37℃ and a final concentration of 1 mmol/L IPTG, a soluble recombinant protein with a molecular weight of about 29.7 kD was successfully induced, which was consistent with the expected size of alpha-momorcharin. The alpha-momorcharin protein was induced in a large amount according to the optimal conditions. The alpha-momorcharin recombinant protein was purified by Ni-NTA column. Finally, the purified alpha-momorcharin protein was injected into New Zealand white rabbit to prepare antiserum. Western blotting analysis showed that the prepared antiserum had good specificity and sensitivity, and can be used to detect the alpha-momorcharin protein in bitter melon. The results of this study provides a reference for the next step to explore the molecular mechanism of alpha-balsamin regulating plant defense against biological stress.