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

作　　者：付立霞[1] 冀德君[1] 卢徐斌[1] 韩先干[2] 魏文志[1] 

机构地区：[1]扬州大学动物科学与技术学院江苏省人畜共患病学重点实验室,江苏扬州225009 [2]中国农业科学院上海兽医研究所,上海200241

出　　处：《生物工程学报》2016年第12期1654-1663,共10页Chinese Journal of Biotechnology

基　　金：江苏省人兽共患病学重点实验室项目(No.R1510);上海市科技兴农重点攻关项目(No.2015HNG1-9);江苏省乳品生物技术与安全控制重点实验室课题(No.K14009)资助~~

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c SNA或u SNA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、c SNA和u SNA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c SNA即可在宿主菌胞内被检出,而u SNA需1 h;相较之下,SNA和c SNA在培养液上清中的被检出时间要晚1 h,u SNA则晚2 h。对三者的核酸酶活性检测均为:SNA﹥c SNA﹥u SNA,且胞外活性都显著低于胞内。此外,SNA和c SNA在诱导2 h后即将宿主基因组成功降解,而u SNA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、c SNA和u SNA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。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（c SNA） or Mycobacterium tuberculosis urease gene（u SNA） were constructed and evaluated in Escherichia coli.Results show that the percentages of inactivated E.coli by SNA,c SNA and u SNA after 4 h of induction were 99.9%,99.8% and 74.2%,respectively.Moreover,SNA and c SNA in the cytoplasm of host bacteria were initially detectable after 30 min of induction,whereas u SNA was after 1 h.In comparison,SNA and c SNA in culture supernatant were initially detectable 1 h later,whereas u SNA was 2 h later.The nuclease activity in the cytoplasm or supernatant was ranked as follows：SNA c SNA u SNA,and the activity in the supernatant was significantly lower than that in the cytoplasm.Furthermore,host genomic DNA was degraded by SNA or c SNA after 2 h of induction but not by u SNA even throughout the whole experiment.In conclusion,this study indicates that SNA,c SNA and u SNA 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.

关 键 词：菌蜕 cro基因 裂解基因E 金黄色葡萄球菌核酸酶A 脲酶基因 

分 类 号：Q78[生物学—分子生物学]