【摘要】 从家蚕cDNA文库中扩增到家蚕BmAGO2基因完整的ORF序列,通过Lasergene软件分析获得BmAGO2的抗原表位区(命名为Kago2),构建含Kago2的表达载体,转化大肠杆菌诱导表达,融合蛋白经镍柱亲和层析纯化后免疫新西兰大白兔制备多克隆抗体,ELISA和Western blotting检测抗血清的效价可达到1∶25 600以上,抗体特异性较好。表达谱分析结果显示,BmAGO2蛋白在家蚕卵期、蛹期、蛾期和五龄幼虫期均表达,但在蛹期表达量要偏低。而在五龄幼虫各组织中BmAGO2蛋白的表达差异较明显,BmAGO2蛋白在头部和表皮中大量表达,丝腺、马氏管和脂肪中也有少量表达,而在血淋巴、气管和中肠中未检测到该蛋白的表达。结合BmAGO2表达谱分析结果,进一步利用其抗体通过免疫共沉淀方法从BmAGO2蛋白表达量高的家蚕头部和表皮中分离出BmAGO2蛋白及结合的RNA,并逆转录成cDNA。以家蚕miRNA潜在的靶基因Bmem4,Bm(spl)-like,Bmemc,Bmmγ,Bmmβ2作为检测基因,荧光定量PCR分析获得的BmAGO2结合RNA,和对照组RNA相比,Bmem4,Bm(spl)-like,Bmemc,Bmmγ,Bmβ2基因在BmAGO2结合RNA中的含量分别富集了3.93、1.31、2.4、1.31、0.97倍。miRNA靶位点分析表明,Bmem4和Bmemc存在多个miRNA结合位点,极有可能是miRNA的靶基因。目前还没有有效的家蚕miRNA靶基因高通量鉴定方法,本实验为家蚕miRNA靶基因的高通量筛选提提供了可靠地实验途径。更多还原

【Abstract】 This study extends to complete ORF sequence of silkworm BmAGO2 gene from silkworm cDNA library,obtains antigen epitope area(named as Kago2)of BmAGO2 through Lasergene software analysis,establishes an expression vector containing Kago2 and transforms escherichia coli induction expression.New Zealand white rabbits are immunized with fusion protein subject to nickel column affinity chromatography and purification and polyclonal antibody is prepared.ELISA and Western blotting test that the valence of antiserum can be over 1∶25,600 and it has a good antibody specificity.The result of expression profiling shows that BmAGO2 protein is expressed in egg stage,pupal period,moth period and five-instar larval phase of silkworm,but its expression quantity is lower in pupal period.The expression of BmAGO2 has significant difference in each tissue of five-instar larvae.BmAGO2 protein is greatly expressed in head and epidermis and slightly expressed in silk gland,malpighian tubule and fat.The expression of this protein is not detected in hemolymph,weasand and midgut.In combination with the result of BmAGO2 expression profiling,this study further uses its antibody to separate BmAGO2 protein and binding RNA from silkworm head and epidermis with high expression quantity of BmAGO2 protein with the method of co-immunoprecipitation and make it into cDNA through reverse transcription.According to the comparison of BmAGO2 binding RNA obtained through fluorescent quantitative PCR analysis with silkworm miRNA potential target gene Bmem4,Bm(spl)-like,Bmemc,Bmmγ,Bmmβ2as test genes and RNA in the control group,contents of Bmem4,Bm(spl)-like,Bmemc,Bmmγ,Bmβ2genes in BmAGO2 binding RNA are enriched respectively by 3.93,1.31,2.4,1.31 and 0.97 times.miRNA target site analysis shows that Bmem4 and Bmemc have multiple miRNA binding sites and they are very likely to be target genes of miRNA.There is no effective high throughput identification method of silkworm miRNA target gene currently.This experiment provides a reliable experimental approach for high throughput screening of silkworm miRNA target gene.更多还原