一株产抗菌蛋白基因工程菌株与应用

Engineering strain producing anti-fungal protein genes and application

Abstract

本发明公开了一株产抗菌蛋白基因工程菌株及其构建方法与应用,所述菌株为pET32a(+)-Ap1-BL21,保藏编号为CGMCC?No.11542。其构建方法如下:将抗菌蛋白AP1基因和质粒pET32a(+)分别用BamH?I和XholI双酶切,用T4DNA连接酶连接后转化E.coli?BL21感受态细胞中,获得带有抗菌蛋白基因的工程菌株pET32a(+)-Ap1-BL21。本发明的基因工程菌株含有抗菌蛋白基因,在30℃诱导时间短,表达率高(43.2%),纯化蛋白产量高(124.82mg/L),回收率高(92%),经ITPG诱导、HIS柱纯化和肠激酶酶切后的蛋白对植物病原菌(大豆根腐病菌、水稻恶苗病菌)有很好的抑制作用,该菌株为重组蛋白农药的研发提供了新型的菌株资源。 CGMCC No. 11542 20151021
The invention discloses an engineering strain producing anti-fungal protein genes and a construction method and application thereof. The strain is pET32a(+)-Ap1-BL21, and the preservation serial number is CGMCC No.11542. According to the construction method, double enzyme digestion is carried out on anti-fungal protein AP1 genes and plasmid pET32a(+) through BamH I and XholI, and the anti-fungal protein AP1 genes and the plasmid pET32a(+) are connected through T4DNA ligase and then converted into E.coli BL21 competent cells to obtain the engineering strain pET32a(+)-Ap1-BL21 with the anti-fungal protein genes. The gene engineering strain contains the anti-fungal protein genes, induction time at the temperature of 30 DEG C is short, the expression rate (43.2%) is high, the purification protein yield (124.82 mg/L) is high, the recovery rate (92%) is high, protein has a good inhibiting effect on phytopathogen such as soybean root rot bacteria and fusarium fujikuroi after ITPG inducting, HIS column purifying and enterokinase restriction enzyme digesting are carried out, and the strain provides a novel strain resource for research and development of recombinant protein pesticide.

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