重组旋毛虫plancitoxin-1-like活性位点突变体蛋白的核酸酶活性

Nuclease activity of the recombinant plancitoxin-1-like proteins with mutations in the active site from Trichinella spiralis

摘要:

旋毛虫plancitoxin-1-like (Ts-Pt) 是旋毛虫125种DNaseⅡ家族蛋白中唯一具有典型DNaseⅡ活性区域HKD基序的核酸酶，且普遍认为，组氨酸位点是DNaseⅡ的活性氨基酸位点。为研究Ts-Pt活性位点突变体蛋白的核酸酶活性，利用重叠PCR方法获得Ts-Pt活性位点突变体片段，以pET-28a(+)为载体构建重组表达质粒并在大肠杆菌中诱导表达。重组Ts-Pt突变体蛋白经亲和层析纯化后进行SDS-PAGE分析。利用琼脂糖凝胶电泳法和核酸酶酶谱分析重组Ts-Pt突变体蛋白的核酸酶活性。成功构建含Ts-Pt突变体重组质粒的基因工程菌，SDS-PAGE和亲和层析纯化结果显示，重组Ts-Pt突变体蛋白呈包涵体表达。重组蛋白经复性后并没有表现出核酸酶活性，但核酸酶酶谱分析结果显示，包涵体表达的重组Ts-Pt突变体蛋白表现出降解DNA的能力。同时，N端和C端活性位点H及HCK和DHSK突变并不影响Ts-Pt的核酸酶活性，研究结果为进一步研究庞大的DNaseⅡ家族蛋白在旋毛虫发育和感染方面的作用提供一定的参考。

Abstract:

Although there are 125 predicted DNase Ⅱ-like family genes in the Trichinella spiralis genome, plancitoxin-1-like (Ts-Pt) contains the HKD motif, a typical conserved region of DNase Ⅱ, in N- and C-terminal. It is generally believed that histidine is the active site in DNase Ⅱ. To study the nuclease activity of recombinant Ts-Pt with mutations in the active site from T. spiralis, different fragments of the mutated Ts-Pt genes were cloned using overlap PCR technique and inserted into the expressing vector pET-28a(+), and transformed into Escherichia coli Rosseta (DE3). The fusion proteins were purified by Ni-NTA affinity chromatography and SDS-PAGE. Nuclease activity of the recombinant proteins was detected by agarose gel electrophoresis and nuclease-zymography. The recombinant plasmids harboring the mutated Ts-Pt genes were constructed and expressed as inclusive body in a prokaryotic expression system. After renaturation in vitro, the recombinant proteins had no nuclease activity according to agarose gel electrophoresis. However, the expressed proteins as inclusive body displayed the ability to degrade DNA after renaturation in gel. And the nuclease activity was not affected after subjected to mutation of active site in N- and C-termini of Ts-Pt. These results provide the basis to study the relationship between DNase Ⅱ-like protein family and infection of T. spiralis.