视黄醇结合蛋白4的体外表达及其对猪卵母细胞成熟的影响
发布时间：2018-08-14 09:34
【摘要】：视黄醇结合蛋白（Retinol Binding Proteins，RBPs）是一种低分子量的载脂蛋白，近年来也将其称作脂肪因子。RBP主要由肝脏细胞合成并分泌，主要作用是结合并转运维生素A，存在六种结构形式，分别为RBP1、RBP2、RBP3和RBP4、RBP5、RBP7。近年来研究发现，RBP4在动物的生长、发育及繁殖等过程中发挥重要作用。但天然的猪RBP4蛋白来源有限，并且难于提纯，原核表达能够有效弥补这一不足。本文主要研究内容和结果如下： 1.猪RBP4原核表达载体的构建、表达、纯化及复性 利用RT-PCR方法，获得猪RBP4基因编码序列，构建原核表达载体pEASY-E1-RBP4，转化入大肠杆菌BL21(DE3)pLysS中，IPTG诱导表达出分子量为21kDa的包涵体蛋白。使用8M尿素变性包涵体，经Ni-NTA亲和层析柱纯化后，采用尿素溶液透析复性，复性的RBP4处理猪卵泡颗粒细胞实验结果表明，原核表达的猪RBP4蛋白复性后具有良好的生物学活性。Western blot结果显示，复性后的蛋白能与商品化鼠抗RBP4抗体特异性结合，具有较好的抗原性和特异性。 2.猪RBP4多克隆抗体的制备 复性的猪RBP4蛋白免疫家兔，收集兔抗血清。间接ELISA方法鉴定其效价为1:10000，Western blot结果显示，猪肝脏组织蛋白和纯化复性RBP4蛋白均能与制备的多抗形成特异性条带，表明原核表达蛋白RBP4具有很好的免疫原性，抗体效价及特异性较高。 3. RBP4对猪卵母细胞体外成熟的影响 为了研究RBP4对猪卵母细胞体外成熟的影响，在卵母细胞成熟培养液中添加0，10，100ng/mL纯化的猪RBP4蛋白处理猪COCs42h。统计卵母细胞第一极体的排出情况。结果表明，10，100ng/mL的RBP4对猪卵母细胞第一极体的排出具有显著抑制作用。再利用Real-time PCR检测外源性RBP4处理卵母细胞体外成熟42h中GDF-9及BMP-15的表达规律。结果表明，10，100ng/mL组GDF-9及BMP-15的表达量均显著低于对照组。 本研究成功构建了pEASY-E1-RBP4原核表达载体，携带重组质粒的大肠杆菌经IPTG诱导后能高效表达猪RBP4蛋白。建立了猪RBP4包涵体蛋白纯化、复性工艺，复性的蛋白具有良好的生物学活性。同时制备得到了效价和特异性较高的兔抗猪RBP4抗体。原核表达的RBP4处理体外成熟的猪COCs，，显著抑制第一极体排出，显著下调了GDF-9和BMP-15基因的表达水平。
[Abstract]:Retinol binding protein (Retinol Binding) is a kind of low molecular weight apolipoprotein, which is also called adipoprotein. RBP is mainly synthesized and secreted by liver cells. Its main function is to bind and transport vitamin A, which has six structural forms. RBP1, RBP2, RBP3, RBP4, RBP5, RBP7. In recent years, it has been found that RBP4 plays an important role in the growth, development and reproduction of animals. But natural porcine RBP4 protein has limited source and is difficult to be purified. Prokaryotic expression can effectively make up for this deficiency. The main contents and results of this paper are as follows: 1. Construction, expression, purification and renaturation of porcine RBP4 prokaryotic expression vector the encoding sequence of porcine RBP4 gene was obtained by RT-PCR. The prokaryotic expression vector pEASY-E1-RBP4 was constructed and transformed into E. coli BL21 (DE3) pLysS to induce the expression of inclusion body proteins with molecular weight of 21kDa. Pig follicle granulosa cells were treated with 8M urea denatured inclusion body, purified by Ni-NTA affinity chromatography, and renatured by urea solution dialysis. The recombinant porcine RBP4 protein showed good biological activity after renaturation. Western blot results showed that the refolded protein could specifically bind to the commercial mouse anti RBP4 antibody, and had good antigenicity and specificity. 2. Preparation of porcine RBP4 polyclonal antibody renatured porcine RBP4 protein was immunized with rabbit antiserum. The titer of indirect ELISA assay was 1: 10 000 Western blot. The results showed that both porcine liver tissue protein and purified RBP4 protein could form specific bands with the prepared polyclonal antibodies, indicating that the prokaryotic expression protein RBP4 had a good immunogenicity. The titer and specificity of antibody were higher. Effects of RBP4 on Porcine oocytes maturation in Vitro in order to study the effect of RBP4 on Porcine oocytes maturation in vitro, porcine COCs were treated with purified porcine RBP4 protein for 42 h. Statistics on the discharge of the first polar body of oocytes. The results showed that 10100ng / mL RBP4 significantly inhibited the first polar body excretion of porcine oocytes. Real-time PCR was used to detect the expression of GDF-9 and BMP-15 in oocytes treated with exogenous RBP4 for 42 h. The results showed that the expression of GDF-9 and BMP-15 in 10 100 ng / mL group was significantly lower than that in control group. In this study, the prokaryotic expression vector of pEASY-E1-RBP4 was successfully constructed. E. coli carrying recombinant plasmid was induced by IPTG to express porcine RBP4 protein efficiently. The purification and renaturation of porcine RBP4 inclusion body protein were established. At the same time, rabbit anti-pig RBP4 antibody with high titer and specificity was prepared. The prokaryotic expression of RBP4 significantly inhibited the first polar body excretion and significantly down-regulated the expression of GDF-9 and BMP-15 genes in mature porcine COCs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S852.2
本文编号：2182443
[Abstract]:Retinol binding protein (Retinol Binding) is a kind of low molecular weight apolipoprotein, which is also called adipoprotein. RBP is mainly synthesized and secreted by liver cells. Its main function is to bind and transport vitamin A, which has six structural forms. RBP1, RBP2, RBP3, RBP4, RBP5, RBP7. In recent years, it has been found that RBP4 plays an important role in the growth, development and reproduction of animals. But natural porcine RBP4 protein has limited source and is difficult to be purified. Prokaryotic expression can effectively make up for this deficiency. The main contents and results of this paper are as follows: 1. Construction, expression, purification and renaturation of porcine RBP4 prokaryotic expression vector the encoding sequence of porcine RBP4 gene was obtained by RT-PCR. The prokaryotic expression vector pEASY-E1-RBP4 was constructed and transformed into E. coli BL21 (DE3) pLysS to induce the expression of inclusion body proteins with molecular weight of 21kDa. Pig follicle granulosa cells were treated with 8M urea denatured inclusion body, purified by Ni-NTA affinity chromatography, and renatured by urea solution dialysis. The recombinant porcine RBP4 protein showed good biological activity after renaturation. Western blot results showed that the refolded protein could specifically bind to the commercial mouse anti RBP4 antibody, and had good antigenicity and specificity. 2. Preparation of porcine RBP4 polyclonal antibody renatured porcine RBP4 protein was immunized with rabbit antiserum. The titer of indirect ELISA assay was 1: 10 000 Western blot. The results showed that both porcine liver tissue protein and purified RBP4 protein could form specific bands with the prepared polyclonal antibodies, indicating that the prokaryotic expression protein RBP4 had a good immunogenicity. The titer and specificity of antibody were higher. Effects of RBP4 on Porcine oocytes maturation in Vitro in order to study the effect of RBP4 on Porcine oocytes maturation in vitro, porcine COCs were treated with purified porcine RBP4 protein for 42 h. Statistics on the discharge of the first polar body of oocytes. The results showed that 10100ng / mL RBP4 significantly inhibited the first polar body excretion of porcine oocytes. Real-time PCR was used to detect the expression of GDF-9 and BMP-15 in oocytes treated with exogenous RBP4 for 42 h. The results showed that the expression of GDF-9 and BMP-15 in 10 100 ng / mL group was significantly lower than that in control group. In this study, the prokaryotic expression vector of pEASY-E1-RBP4 was successfully constructed. E. coli carrying recombinant plasmid was induced by IPTG to express porcine RBP4 protein efficiently. The purification and renaturation of porcine RBP4 inclusion body protein were established. At the same time, rabbit anti-pig RBP4 antibody with high titer and specificity was prepared. The prokaryotic expression of RBP4 significantly inhibited the first polar body excretion and significantly down-regulated the expression of GDF-9 and BMP-15 genes in mature porcine COCs.
【学位授予单位】：吉林大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：Q78;S852.2
【参考文献】
相关期刊论文 前4条
1 朱吉;孙建帮;谢菊兰;彭英林;;湖南黑猪RBP4基因与产仔数的相关性分析[J];家畜生态学报;2011年03期
2 方敏,黄华j;包涵体蛋白体外复性的研究进展[J];生物工程学报;2001年06期
3 金宏;视黄醇转运的分子机制[J];生理科学进展;1997年02期
4 王世春,刘云,李永辉,徐琪寿;视黄醇结合蛋白的分离纯化[J];生物技术通讯;2004年01期
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