中文摘要：

【目的】克隆家蚕卵黄原蛋白受体（vitellogenin receptor,VgR）,分析其在昆虫细胞中的表达特征,了解正常VgR和突变VgR的表达模式,为阐明其生物学功能提供参考依据。【方法】基于家蚕VgR的基因编码序列设计特异引物,扩增的目的片段连接到载体pET28a上,构建原核表达载体,转化E.coil BL21（DE3）表达菌株,IPTG诱导获得目的蛋白;用镍柱亲和层析方法纯化诱导表达的家蚕VgR肽蛋白;将纯化后的肽蛋白制备BmVgR的兔源多克隆抗体;PCR扩增获得大造和突变型白妙卵（scanty vitellin,vit）的LBD1＋EGF1结构域（C11D和C11V）片段,连接到细胞表达载体上,通过细胞转染表达目的蛋白,采用细胞免疫组化检测大造和突变型vit的VgR在细胞里的表达情况;通过Western blot检测细胞培养液中大造和突变型vit VgR的表达量。【结果】原核表达获得了一个大小约为22 kD的融合蛋白,以包涵体形式表达;镍柱亲和层析初纯化得到BmVgR的肽蛋白,进一步切胶纯化后,以此为抗原制备BmVgR的兔源多克隆抗体,其抗体效价〉1﹕512 000,纯度为95%以上。转录水平检测表明Sf9和Spli221昆虫细胞中没有BmVgR和BmVg内源基因的表达;在Sf9昆虫细胞中成功表达了家蚕野生型和突变型vit BmVgR的结构域SP＋LBD1＋EGF1肽段,家蚕突变型vit在BmVgR第1个表皮生长因子同源区域的第3个ClassB区域有编码50个氨基酸残基的核酸序列缺失;细胞免疫组化试验表明,突变型和正常型的C11V和C11D都能在Sf9细胞质中正常表达;制备的BmVgR兔源多克隆抗体和Myc标签抗体同时检测到细胞表达的目的蛋白,表明试验所制备的VgR抗体特异性较好;由于SP＋LBD1＋EGF1肽段存在信号肽,细胞表达的蛋白会分泌进入细胞培养液中,Western blot对培养液进行蛋白检测,表明突变型vit存在氨基酸缺失,但并不影响肽蛋白的正常表达,且表达量没有明显差异。【结论】EGF1结构域的缺失并不?

英文摘要：

【Objective】 The objective of this study is to clone the silkworm BmVgR, to analyze its expression characteristics in insect cells and to investigate the expression patterns of the normal and mutational VgRs, thus providing a basis for its function identification. 【Method】 Specific primer sequences were designed according to BmVgR coding sequence, the amplified fragment was connected to the pET28 a, and then transformed into E. coil BL21（DE3）, the fusion protein was obtained by IPTG induction. Ni-NTA affinity chromatography was used to purify the BmVgR protein peptide. Then the anti-BmVgR rabbit polyclonal antibody was obtained by BmVgR protein peptide as an antigen. The LBD1＋EGF1（C11D and C11V） domain of the normal and mutational VgRs was connected to the cell expression vector, the target protein was expressed with cell transfection. Immunohistochemistry was used to analyze the expression characteristics of the normal and mutational VgRs in insect cells. Western blot was used to detect the expression levels of the normal and mutational VgRs in the cell culture medium.【Result】 Prokaryotic expression gained a size of about 22 kD fusion protein, which was expressed as inclusion bodies. BmVgR protein peptide was expressed and purified, the anti-BmVgR rabbit polyclonal antibody was obtained, its titer was higher than 1﹕512 000. Purity was more than 95%. The endogenous genes of BmVgR and BmVg did not express in Sf9 and Spli221. The normal and mutational BmVgR-SP＋LBD1＋EGF1 was successfully expressed in the Sf9 cell（the BmVgR of the vit mutant, which was mutated in the 3rd Class B region of the EGF1 domain, coding 50 amino acid residuces）. Immunohistochemistry showed that vit mutant and normal BmVgR could express normally in Sf9 cytoplasm. BmVgR rabbit polyclonal antibody and Myc-tagged antibody simultaneously detected the target protein, which indicated that the VgR antibody was better. As the SP ＋ LBD1 ＋ EGF1 peptide existed signal peptide, the expressed protein was secreted into th