【摘要】 为了对细粒棘球绦虫F-盒富含亮氨酸的重复蛋白2(Echinococcus granulosus F-box and leucine rich repeat protein 2,EgFBXL2)进行生物信息学分析，制备EgFBXL2的多克隆抗体并定位其在幼虫阶段的表达组织，试验对EgFBXL2进行生物信息学分析；基于NCBI已公布的细粒棘球绦虫源FBXL2基因序列，通过PCR扩增获得EgFBXL2基因的完整编码区，经测序验证后，对其进行原核诱导表达，并利用Western-blot验证其反应原性。用纯化后的蛋白免疫Balb/c小鼠，制备多克隆抗体，并利用ELISA方法检测抗体效价。用该抗体通过免疫荧光定位FBXL2在细粒棘球绦虫幼虫原头蚴及囊泡中的表达情况。结果表明：EgFBXL2基因编码区全长为1 656 bp, FBXL2由551个氨基酸组成，理论分子量为59 890.25 u,理论等电点(pI)为7.63;不稳定指数为43.41;脂肪指数较高(为98.80),整体亲水性平均值(GRAVY)为0.049;含有47个潜在磷酸化位点，不含跨膜结构域及信号肽；为F-box＿SF超家族的成员，存在AMN1结构域；含有8个B细胞抗原表位；二级结构以α螺旋为主(52.09%),并含有大量无规卷曲(43.92%)及少量延伸链(3.99%),无β-转角；三级结构存在大量α螺旋。EgFBXL2在大肠杆菌BL21(DE3)中成功表达出大小为63 ku的重组蛋白，且以包涵体形式存在，最佳诱导表达条件为15℃、0.1 mmol/L IPTG诱导16 h;经组氨酸标签镍离子蛋白纯化柱纯化与复性后，该蛋白表现出良好的免疫反应性。利用纯化蛋白免疫小鼠，制备得到效价为1∶102 400的多克隆抗体；EgFBXL2在细粒棘球绦虫原头蚴的顶突、实质、皮层和生发层中广泛表达，在囊泡生发层中亦有表达。说明试验成功克隆并表达了EgFBXL2基因，制备了高效价多克隆抗体，初步明确了其表达特征。更多还原

【Abstract】 In order to perform bioinformatics analysis of the Echinococcus granulosus F-box and leucine-rich repeat protein 2(EgFBXL2), prepare polyclonal antibodies, and locate its expression in the larval stage, the bioinformatics analysis was conducted on EgFBXL2, the complete coding sequence of the EgFBXL2 gene was amplified by PCR based on the published Echinococcus granulosus-derived sequence in NCBI. After sequencing verification, prokaryotic expression were performed, and its immunoreactivity was confirmed by Western-blot. Balb/c mice were immunized with the purified protein to prepare polyclonal antibodies, and the antibody titer was determined by ELISA. The expression of FBXL2 in protoscoleces and cysts of Echinococcus granulosus larvae was located using immunofluorescence with the prepared antibody. The results showed that the EgFBXL2 gene had a coding sequence length of 1 656 bp; FBXL2 consisted of 551 amino acids, with a theoretical molecular weight of 59 890.25 u and a theoretical isoelectric point(pI) of 7.63. Its instability index was 43.41; the aliphatic index was relatively high at 98.80, and the grand average of hydropathicity(GRAVY) was 0.049. The protein contained 47 potential phosphorylation sites, without transmembrane domains or signal peptides. As a member of the F-box＿SF superfamily, it harbored the AMN1 domain. In addition, it possessed 8 potential B-cell epitopes. For the secondary structure, α-helices accounted for the major proportion(52.09%), followed by a large number of random coils(43.92%) and a small amount of extended strands(3.99%), while no β-turns were detected. A large number of α-helices were also observed in its tertiary structure.EgFBXL2 was successfully expressed as a recombinant protein of 63 ku in Escherichia coli BL21(DE3), and it existed in the form of inclusion bodies, with optimal induction conditions of 15 ℃ and 0.1 mmol/L IPTG for 16 hours. After purification and renaturation using Ni-NTA, the protein exhibited good immunoreactivity. Polyclonal antibodies with a titer of 1∶102 400 were produced by immunizing mice with the purified protein. EgFBXL2 was widely expressed in the rostellum, parenchyma, tegument, and germinal layer of Echinococcus granulosus protoscoleces, as well as in the germinal layer of cysts. The results indicated that the EgFBXL2 gene was successfully cloned and expressed, high-titer polyclonal antibodies were prepared, and its expression characteristics were preliminarily clarified.更多还原