嗜线虫致病杆菌HB310几丁质酶基因chi70的克隆和表达

Cloning and protokaryotic expression of chitinase gene chi70 from Xenorhabdus nematophila HB310

为了了解嗜线虫致病杆菌Xenorhabdus nematophila几丁质酶的功能,本研究通过PCR技术从X.nematophila HB310菌株中克隆得到了几丁质酶基因chi70,对该基因进行了生物信息学分析。结果表明该基因全长1947 bp,编码648个氨基酸,表达的Chi70蛋白不含信号肽。将chi70基因进行原核表达,获得大量重组Chi70蛋白。采用二硝基水杨酸法(DNS法)测定重组蛋白酶活,结果表明几丁质酶Chi70在pH7.0时活性最高,为8.815 U/mL。采用生测方法测定Chi70对苏云金芽孢杆菌HD-73(Bt HD-73)的增效活性,结果表明单独使用Bt HD-73时对2龄棉铃虫幼虫的LC50为14.80μg/m L,添加Chi70后的Bt HD-73对2龄棉铃虫幼虫的LC50为5.66μg/m L,实验结果说明Chi70对Bt具有明显的增效作用。

In order to study the function of chitinase from Xenorhabdus nematophila, the chi70 chitinase gene from X. nematophila HB 310 strain was cloned by PCR and analyzed by bioinformatics. The results showed that the chiTO gene has an 1947 bp open reading frame, encoding 648 amino acids. The Chi70 protein did not contain any signal peptide. The chi70 gene was successfully expressed in prokaryotic expression system. The recombinant Chi70 protein showed the highest chitinase enzyme activity of 8. 815 U/mL at pH 7.0 through 3, 5 -Dinitrosalicylic acid colorimetric method （DNS method ）. The synergistic activity of Chi70 to Bt HD -73was tested. The recombinant ChiT0 chitinase showed strong synergistic effect to Bt HD - 73 with a LCs0 of 5.66 μg/mL, however LC50 of only Bt HD - 73 was 14. 80μ g/mL against second larvae of Helicoverpa armigera.