人早孕因子表达与促细胞生长模型的初步建立

发布时间：2018-06-01 20:44

本文选题：早孕因子 + 原核表达　；参考：《郑州大学》2011年硕士论文

【摘要】：早孕因子(early pregnancy factor, EPF)是哺乳动物妊娠早期母体血清中最早出现的一种免疫抑制因子。具有免疫抑制和生长调节两种功能,为目前最早确认妊娠的生化指标之一。小鼠受精后6h,猪、牛、山羊24 h,人受精48 h即可从母体内测出EPF,这些远早于人绒毛膜促性腺激素(hCG)测定,而且EPF的活性可以在妊娠期存在很长时间,因此对EPF的活性检测可用于牛、马的早期妊娠诊断。EPF在孕妇的超早孕诊断中准确率达88.6%,因此,EPF在早孕诊断方面的应用,将对人类及畜牧业有重要的意义。为获得人早孕因子重组蛋白,提取人肝癌SMMC-7721细胞总RNA,对早孕因子基因进行RT-PCR扩增、TA克隆和测序,通过PCR技术扩增人早孕因子基因,克隆入原核表达载体pGEX-6p-1和pET-28a,分别与GST基因和his基因相融合,构建重组表达质粒,转化大肠杆菌表达菌株BL21,经异丙基-β-D-硫代半乳糖苷(IPTG)诱导表达,以SDS-PAGE和Western blot分析重组蛋白的表达,通过GST树脂和纯化表达蛋白。结果成功构建重组表达质粒pGEX6p-EPF,在大肠杆菌中诱导表达了GST-EPF融合蛋白,表达蛋白分子量为37.3 kDa,并能被抗GST单克隆抗体特异识别,通过GST亲和层析纯化,制备得到EPF重组蛋白,pET28-EPF重组质粒成功构建,在大肠杆菌中诱导表达了his-EPF融合蛋白,表达蛋白分子量为17kDa,并能被抗his单抗特异识别,通过镍柱纯化得蛋白。玫瑰花环抑制实验证明了蛋白活性。为研究EPF的生长因子作用,建立EPF对细胞有明显促生长作用的细胞模型,将不同浓度梯度的EPF分别加入Marc145、NSO、PK15、BHK及人肝癌SMMC-7721细胞中,通过显微镜观察和细胞计数判断EPF对细胞数目的影响,发现在Marc145和NSO细胞系中细胞数目有明显增多,初步筛选出Marc145和NSO两种细胞系作为促生长细胞模型。
[Abstract]:Early pregnancy factor, EPF) is one of the earliest immunosuppressive factors in mammalian maternal serum. It has two functions of immunosuppression and growth regulation, which is one of the earliest biochemical indexes to confirm pregnancy. EPFs were detected from the mother at 6 h after fertilization in mice, 24 hours in pigs, cattle and goats, and 48 hours in human fertilization. These results were far earlier than those of human chorionic gonadotropin (hCGG), and the activity of EPF could exist for a long time during pregnancy. Therefore, the detection of EPF activity can be used in the diagnosis of bovine and horse early pregnancy. The accuracy of EPF in the diagnosis of super early pregnancy of pregnant women is 88.6. therefore, the application of EPF in the diagnosis of early pregnancy will be of great significance to human beings and animal husbandry. In order to obtain the recombinant protein of human early pregnancy factor and extract the total RNAs of human hepatoma SMMC-7721 cells, the gene of early pregnancy factor was cloned and sequenced by RT-PCR amplification, and the gene of human early pregnancy factor was amplified by PCR. The plasmid was cloned into prokaryotic expression vector pGEX-6p-1 and pET-28a and fused with GST gene and his gene respectively. The recombinant expression plasmid was constructed and transformed into Escherichia coli expression strain BL21. The expression was induced by isopropyl- 尾 -D- thiogalactoside (IPTG). The expression of the recombinant protein was analyzed by SDS-PAGE and Western blot, and the expressed protein was purified by GST resin. Results the recombinant expression plasmid pGEX6p-EPFwas successfully constructed, and the GST-EPF fusion protein was induced and expressed in Escherichia coli. The molecular weight of the expressed protein was 37.3 kDa. it could be specifically recognized by monoclonal antibody against GST and purified by GST affinity chromatography. The recombinant plasmid of EPF recombinant protein pET28-EPF was successfully constructed, and his-EPF fusion protein was induced and expressed in Escherichia coli. The molecular weight of the expressed protein was 17kDa. the protein could be specifically recognized by anti his monoclonal antibody. The protein was purified by nickel column. The activity of protein was proved by rosette inhibition experiment. In order to study the effect of EPF on the growth factor and to establish a cell model with obvious growth promoting effect of EPF, EPF with different concentration gradient was added to the cells of Marc145 NSOP PK15 BHK and SMMC-7721 cells of human hepatocellular carcinoma respectively. The effect of EPF on cell number was determined by microscopic observation and cell count. It was found that the number of cells in Marc145 and NSO cell lines increased obviously. Two cell lines, Marc145 and NSO, were preliminarily selected as growth promoting cell models.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

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