Serratia marcescens非特异性核酸内切酶的原核表达及其应用

Prokaryotic Expression and Application of Serratia marcescens Non-specific Endonuclease

目的在大肠杆菌中表达Serratia marcescens非特异性核酸内切酶(SMNE),并进行纯化、活性检测及应用。方法合成smne基因,应用PCR技术在基因的5′端引入6个组氨酸标签序列,将其插入分泌表达载体pET-20b(+)中,转化大肠杆菌BL21(DE3)pLysS,IPTG诱导表达。表达产物经镍离子螯合琼脂糖凝胶一步纯化后,检测其活性并计算比活。将纯化的SMNE用于重组腺病毒的制备,对外源性核酸进行降解,并采用Southern blot对外源性DNA残留量进行测定。结果重组表达质粒pET-20b-smne经PCR、双酶切和测序证明构建正确。重组蛋白的表达量为8.0 mg/L,纯化后纯度达95%,比活达1.1×106 U/mg。在重组腺病毒制备过程中使用后,成品中的外源性DNA残留量≤10 ng/5.0×1011 VP。结论已成功地在大肠杆菌中表达了SMNE,纯化的SMNE活性高,有望应用于重组生物制品制备过程中外源性核酸的去除。

Objective To express Serratia marcescens non-specific endonuclease （SMNE） in E. coli, purify the expressed product, determine its activity and investigate its usage. Methods The smne gene fragment was synthesized, to which 6xHis-tagged sequence was introduced at 5＇-terminus by PCR, and inserted into secretory expression vector pET-20b（＋）. The constructed recombinant plasmid pET-20b-smne was transformed to E. coli BI221 （DE3） pLysS for expression under induction of IPTG. The expressed product was purified by nickel ion chelating agarose gel chromatography and determined for activity, based on which the specific activity was calculated. The purified SMNE was used for the degradation of exogenous nucleic acid during preparation of recombinant adenovirus. The residual exogenous DNA content was determined by Southern blot. Results PCR, restriction analysis and sequencing proved that recombinant plasmid pET-20b-smne was constructed correctly. The recombinant SMNE at an expression level of 8. 0 mg/L reached a purity of 95% and a specific activity of 1. 1 ×10^6 U/rag after purification. The residual exogenous DNA content in the final product of prepared recombinant adenovirus was not more than 10 ng/5. 0 ×10^11 VP. Conclusion SMNE was successfully expressed in E. coli. The purified SMNE showed a high activity and might be used for the removal of exogenous nucleic acid during preparation of recombinant biologics.