论文部分内容阅读
结合免疫球蛋白(binding immunoglobulin protein,BiP),是Hsp70(70 kilodalton heatshock proteins)蛋白家族的成员之一,是内质网应激(endoplasmic reticulum stress,ERS)主要的调节器。为了研究BiP的分子结构与生物学功能的关系,首先,分析BiP的DNA序列和蛋白质空间构象,接着利用重叠PCR方法分别克隆ATPase结构域缺失体和peptide-binding结构域缺失体(BiPa和BiPp),成功构建带myc标签的真核表达载体,转染LO2细胞和SMMC-7721细胞,应用免疫印迹方法检测其在细胞内的表达;运用MTT法、BrdU免疫组化法及流式细胞仪分别检测细胞的增殖和凋亡;检测BiP及两个组成结构域对SMMC-7721细胞增殖和凋亡的影响。酶切、电泳和DNA测序结果显示,BiP全长和两个缺失体的真核表达载体构建成功;免疫印迹检测到BiP三种真核载体在LO2细胞和SMMC-7721细胞中均能正确表达;MTT和BrdU免疫组化结果表明,BiP全长和缺失体BiPa、BiPp三种真核载体均能有效促进SMMC-7721的增殖,各组间细胞增殖率结果分析提示ATPase功能域可能抑制细胞增殖,而peptide-binding结构域可能促进细胞的增殖;流式细胞仪结果显示,转染BiP、BiPa、BiPp三种真核载体均可以促进SMMC-7721的凋亡,缺失体BiPa与全长BiP组间凋亡率差异不显著;而缺失体BiPp组细胞凋亡率明显低于全长BiP组,两组差异具有显著性(P<0.05);提示ATPase功能域与细胞凋亡可能不相关,而peptide-binding结构域可能促进细胞的凋亡。上述结果表明,BiP的两个组成结构域:ATPase功能域与peptide-binding结构域,独立存在或者共同存在时,调控细胞增殖与凋亡具有差异性;当两个结构域共同存在时,它们会协同调控细胞的增殖与凋亡,具体的分子机制还需要进一步的实验研究和探讨。
Binding of immunoglobulin protein (BiP), a member of the Hsp70 (70 kilodalton heatshock proteins) family of proteins, is a major regulator of endoplasmic reticulum stress (ERS). In order to study the relationship between molecular structure and biological function of BiP, we first analyzed the DNA sequence of BiP and the spatial conformation of the protein, then cloned ATPase domain deleted and peptide-binding domain deleted (BiPa and BiPp) , Successfully constructed myc tagged eukaryotic expression vector, transfected LO2 cells and SMMC-7721 cells, Western blotting was used to detect the expression in cells; using MTT assay, BrdU immunohistochemistry and flow cytometry were detected Proliferation and apoptosis of SMMC-7721 cells were detected by BiP and two constitutive domains. The results of restriction enzyme digestion, electrophoresis and DNA sequencing showed that the eukaryotic expression vector with full length and two deletions of BiP was constructed successfully. The three eukaryotic vector of BiP were detected by Western blot in LO2 cells and SMMC-7721 cells. The results of MTT and BrdU immunohistochemistry showed that all three eukaryotic vectors of BiP, BiP and BiPp can effectively promote the proliferation of SMMC-7721. The analysis of cell proliferation rate among groups showed that the ATPase domain may inhibit cell proliferation, The results of flow cytometry showed that the transfection of three eukaryotic vectors, BiP, BiPa and BiPp, could promote the apoptosis of SMMC-7721 cells. The results of flow cytometry showed that the transcripts of BiP, BiPa and BiPp The apoptotic rate was not significantly different between the BiPp group and the full-length BiPp group (P <0.05), suggesting that the ATPase domain might not be related to apoptosis, whereas the peptide The -binding domain may promote cell apoptosis. The above results show that the two components of BiP domain: ATPase domain and peptide-binding domain, exist independently or co-exist, the regulation of cell proliferation and apoptosis are different; when the two domains co-exist, they will Collaborative regulation of cell proliferation and apoptosis, the specific molecular mechanism still needs further experimental study and discussion.