棉铃虫中肠Cry1A受体蛋白氨肽酶N1在Tn细胞系的表达

Expression of Aminopeptidase N (APN1),the Main Receptor Protein for Bacillus thuringiensis Cry1A Toxin from Helicoverpa armigera Larval Midgut in Tn Cells

【目的】将棉铃虫中肠Bt受体蛋白在真核表达系统中成功表达。【方法】用PCR方法分别扩增出对Cry1Ac毒素敏感和抗性棉铃虫的中肠氨肽酶N1(APN1)基因的去信号肽片段,将其克隆至pUC19载体,测定核酸序列后,克隆入Bac-to-Bac表达系统中杆状病毒转移载体pFastBacHTB中多角体基因启动子的下游,筛选出重组质粒pFastBacHTB/APN1并转化大肠杆菌DH10Bac,在体内进行重组,经抗性和蓝白斑筛选,获得杆状病毒重组载体Bacmid/APN1。转染粉纹夜蛾(Trichoplusia ni)细胞(Tn-5B1-4),获得含APN1基因的重组杆状病毒,重组病毒感染Tn细胞后,得到表达的蛋白。【结果】SDS-PAGE分析和点杂交显示,目的基因得到了成功表达。【结论】APN1基因在Tn细胞中成功表达,为今后继续研究其功能和与抗性的关系奠定了基础。

【Objective】The present study was conducted to successfully express the Bt （Bacillus thuringiensis） toxin receptor protein located on internal membrane of larval midgut of cotton bollworm （Helicoverpa armigera Hbner）,within eukaryotic expression system. 【Method】 PCR method was used to separately amplify the fragments without signal peptide of Aminopeptidase N1 gene from midguts of susceptible and resistant H. armigera,and separately cloned into pUC 19 vector. After sequencing the gene,the fragments encoding for APN1 without signal peptide were cloned into the Bac-to-Bac baculovirus expression system transfer vector pFastBacHTB under the polyhedron gene promoter. The recombinant transposing plasmid pFastBacHTB/APN1 was screened and then transformed into E scherichia coli DH10Bac. It was cultured in LB plate which contained Te,Kan,Ge,X-gal and IPTG. The resulting recombinant bacmid was transfected into cells of the insect Trichoplusia ni （Tn） and recombinant baculovirus was obtained. The lysate of cells infected with recombinant baculovirus was analyzed by SDS-PAGE and blot analysis. 【Result】The results showed that the recombinant baculovirus was fully capable of expressing APN1. 【Conclusion】The APN1 gene was successfully expressed in Tn cell has established a foundation for its function research and its relationship of resistance to Bt.