耐热荧光素酶的表达及热稳定焦磷酸测序体系的建立

Expression of thermostable recombiant Luciola lateralis luciferase and development of heat-stable pyrosequencing system

摘要:

焦磷酸测序是一种基于生物发光反应的实时DNA序列测定技术，其反应体系中的荧光素酶决定测序反应的灵敏度。传统焦测序反应使用的是野生型北美荧光素酶Photinus pyralis luciferase (PpL)，该酶热稳定性较差，因此由该酶配制的焦磷酸测序反应体系对热不稳定，影响焦磷酸测序的灵敏度。为了建立热稳定的焦磷酸测序反应体系，全合成了耐热突变日本萤火虫荧光素酶的编码序列，并将其插入到表达载体pET28a(+)中构建重组质粒，将重组质粒转化到大肠杆菌中，筛选得到的阳性重组菌成功表达了N端带有6×His (组氨酸) 标签的耐热突变日本萤火虫荧光素酶。利用镍亲和层析法，纯化得到了分子量约为60 kDa且比活为4.29×1010 RLU/mg的重组蛋白。对重组耐热日本萤火虫荧光素酶的热稳定性研究结果表明，该酶在50 ℃时仍具有活性，在40 ℃下孵育25 min后活性保留了90％以上。与基于野生型北美荧光素酶的焦磷酸测序体系相比，由耐热日本萤火虫荧光素酶配制的测序反应体系对热稳定性有了极大的提高，该测序反应液在37 ℃放置1 h后仍能够得到正确的测序结果。研究结果为建立稳定可靠的现场及时快速焦磷酸测序反应体系奠定了基础。

Abstract:

Pyrosequencing is a tool based on bioluminescence reaction for real-time analyzing DNA sequences. The sensitivity of pyrosequencing mainly depends on luciferase in reaction mixture. However, the instability of pyrosequencing reagents caused by fragile wild Photinus pyralis luciferase (PpL) in conventional pyrosequencing usually leads to unsatisfied results, which limits the application of pyrosequencing. In order to improve the stability of pyrosequencing reagents, the coding sequences of mutant thermostable Luciola lateralis luciferase (rt-LlL) was synthesized, and inserted into the plasmid of pET28a(+) to express the thermostable rt-LlL with a 6×His-tag in the N terminal. The purified rt-LlL with the molecular mass of 60 kDa was obtained by Ni-affinity chromatography. The specific activity of rt-LlL was determined as 4.29×1010 RLU/mg. Moreover, the thermostability of rt-LlL was investigated, and the results showed that rt-LlL had activity at 50 °C, and remained 90% of activity after incubated at 40 °C for 25 min. Finally, rt-LlL was used to substitute commercial Photinus pyralis luciferase in conventional pyrosequencing reagent to get thermostable pyrosequencing reagent. Comparing with conventional pyrosequencing reagent, the thermostable pyrosequencing reagent is more stable, and it’s activity would not lose when incubated at 37 °C for 1 h. This study laid foundation of establishing reliable and stable pyrosequencing system which would be applied in Point-of-Care Testing.