盐芥ThMSD基因在大肠杆菌中的表达及特性研究

Expression and Characterization of ThMSD from Thellungiella halophila in Escherichia coli

超氧化物歧化酶(SODs)是真核生物中广泛存在的含金属抗氧化酶,包括Cu/Zn-SOD、Mn-SOD和Fe-SOD 3种。植物线粒体中的Mn-SOD在植物抗逆中发挥重要作用。ThMSD是前期工作中从极度抗逆植物盐芥中克隆到的Mn-SOD编码基因。将ThMSD连接到原核表达载体pET30a(+),转化到BL21,获得重组菌BL21(pET30a-ThMSD)。SDS-PAGE分析表明,重组菌在32kDa处有明显的诱导条带,与理论大小一致。Western blotting分析表明,诱导的条带能和抗His标签的单克隆抗体发生特异性反应。对3个重组子的可溶性全蛋白进行SOD活性分析,发现均高于空白对照菌。选择能大量表达ThMSD的重组子进行大量诱导表达,通过镍亲和层析纯化目的蛋白。对纯化的ThMSD重组蛋白进行热稳定性分析,结果表明,55℃下ThMSD仍有50%以上活性,42℃处理40 min后仍保持60%以上活性。在重组菌BL21(pET30a-ThMSD)对NaCl的抗性分析实验中,当培养基中盐浓度高于正常值时,在所测定阶段重组菌的生长速度均高于对照菌。可见,经原核表达的重组蛋白ThMSD具有较强的SOD活性及热稳定性,表达ThMSD基因的BL21菌株提高了对NaCl的耐受力。推测盐芥ThMSD基因与盐芥极强的抗逆性有密切联系。

Superoxide dismutases （SODs） are ubiquitous antioxidative enzymes found in eukaryotic and differ by the named metal cofactor, Cu/Zn, Mn or Fe. Plant mitochondria Mn-SOD play a vital role in fight aginst stresses. ThMSD, a gene of Mn-SOD was cloned from Thellungiella halophila with dramatic stresses tolerance in earlier research. ThMSD was ligated to a prokaryotic expression vector pET30a（ ＋ ） and transferred to E. coli BI21 to get recombinant BL21 （pET3Oa-ThMSD）. SDS-PAGE analysis showed an induced expression product band, with a molecular weight of about 32 kDa, which was consistent with the expected value, in all recombinant strains. Western blotting confirmed this band with anti-His monoclonal antibody. The results of SOD activity assay of soluble fractions of total protein from 3 recombinant strains showed that much higher SOD activity than control. The strain with highest expression level of ThMSD was selected and was cultured in large scale and target protein was purified using a Ni2~ Chelating column. The effect of temperature on the enzyme activity of ThMSD was tested, the result showed that ThMSD enzyme still had more than 50% residual activities at 55℃. The thermal stability of purified ThMSD was examined at 42℃, the result showed that SOD activity of ThMSD reduced with the time elapse, but it retained more than 60% residual activities after 40 min. In assay of NaCl tolerance of BL21 （ pET3Oa-ThMSD）, the recombinant strain can grow faster than control in higher concentration of NaC1. From above, the recombinant ThMSD protein had high SOD activity and high thermal stability, and BL21 transformed with ThMSD have increased NaC1 tolerance. It was concluded that ThMSD may connect with extreme stresses tolerance of Thellungiella halophila.