金黄色葡萄球菌核酸酶A与不同外源DNA片段融合表达的作用比较

Comparison of effects of staphylococcal nuclease A fused with different exogenous DNA fragments

摘要:

金黄色葡萄球菌核酸酶A (Staphylococcal nuclease A, SNA) 可用于菌蜕制备中宿主菌的进一步灭活及残存遗传物质的去除。关于SNA在去除了信号肽后是否仍能分泌至胞外以及是否需要融合其他氨基酸片段才能在宿主菌胞质中发挥作用尚存争议。为厘清这一争议，分别构建一系列含SNA、SNA与λ噬菌体cro基因或结核杆菌脲酶基因部分序列的融合片段cSNA或uSNA的温控质粒并对其在大肠杆菌内的作用进行评价。结果显示，SNA、cSNA和uSNA的表达产物对大肠杆菌的4 h灭活率分别为99.9%、99.8%和74.2%。升温诱导30 min后，SNA和cSNA即可在宿主菌胞内被检出，而uSNA需1 h；相较之下，SNA和cSNA在培养液上清中的被检出时间要晚1 h，uSNA则晚2 h。对三者的核酸酶活性检测均为：SNA﹥cSNA﹥uSNA，且胞外活性都显著低于胞内。此外，SNA和cSNA在诱导2 h后即将宿主基因组成功降解，而uSNA则在整个实验期间均未能使宿主基因组完全降解。这表明SNA、cSNA和uSNA的表达产物均具有核酸酶活性，可被动释放至胞外，外源DNA片段的融入反而会降低SNA的核酸酶活性。

Abstract:

Staphylococcal nuclease A (SNA) may be used to produce bacterial ghosts for further inactivation of host bacteria and elimination of residual genetic materials. It is still controversial if SNA without signal peptide can be secreted to extracellular matrix and if fusion with other peptide is required for its function in the cytoplasm of host bacteria. To clarify this dispute, a series of temperature-inducible plasmids carrying SNA alone or SNA fused with partial sequences of λ phage cro gene (cSNA) or Mycobacterium tuberculosis urease gene (uSNA) were constructed and evaluated in Escherichia coli. Results show that the percentages of inactivated E. coli by SNA, cSNA and uSNA after 4 h of induction were 99.9%, 99.8% and 74.2%, respectively. Moreover, SNA and cSNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction, whereas uSNA was after 1 h. In comparison, SNA and cSNA in culture supernatant were initially detectable 1 h later, whereas uSNA was 2 h later. The nuclease activity in the cytoplasm or supernatant was ranked as follows: SNA > cSNA > uSNA, and the activity in the supernatant was significantly lower than that in the cytoplasm. Furthermore, host genomic DNA was degraded by SNA or cSNA after 2 h of induction but not by uSNA even throughout the whole experiment. In conclusion, this study indicates that SNA, cSNA and uSNA expressed in host bacteria all have nuclease activity, the enzymes can be released to culture media, and fusion with exogenous peptide negatively reduces the nuclease activity of SNA.