視黃醇結(jié)合蛋白4的體外表達及其對豬卵母細胞成熟的影響
發(fā)布時間：2018-08-14 09:34
【摘要】：視黃醇結(jié)合蛋白（Retinol Binding Proteins，RBPs）是一種低分子量的載脂蛋白，近年來也將其稱作脂肪因子。RBP主要由肝臟細胞合成并分泌，主要作用是結(jié)合并轉(zhuǎn)運維生素A，存在六種結(jié)構(gòu)形式，分別為RBP1、RBP2、RBP3和RBP4、RBP5、RBP7。近年來研究發(fā)現(xiàn)，RBP4在動物的生長、發(fā)育及繁殖等過程中發(fā)揮重要作用。但天然的豬RBP4蛋白來源有限，并且難于提純，原核表達能夠有效彌補這一不足。本文主要研究內(nèi)容和結(jié)果如下： 1.豬RBP4原核表達載體的構(gòu)建、表達、純化及復(fù)性 利用RT-PCR方法，獲得豬RBP4基因編碼序列，構(gòu)建原核表達載體pEASY-E1-RBP4，轉(zhuǎn)化入大腸桿菌BL21(DE3)pLysS中，IPTG誘導(dǎo)表達出分子量為21kDa的包涵體蛋白。使用8M尿素變性包涵體，經(jīng)Ni-NTA親和層析柱純化后，采用尿素溶液透析復(fù)性，復(fù)性的RBP4處理豬卵泡顆粒細胞實驗結(jié)果表明，原核表達的豬RBP4蛋白復(fù)性后具有良好的生物學(xué)活性。Western blot結(jié)果顯示，復(fù)性后的蛋白能與商品化鼠抗RBP4抗體特異性結(jié)合，具有較好的抗原性和特異性。 2.豬RBP4多克隆抗體的制備 復(fù)性的豬RBP4蛋白免疫家兔，收集兔抗血清。間接ELISA方法鑒定其效價為1:10000，Western blot結(jié)果顯示，豬肝臟組織蛋白和純化復(fù)性RBP4蛋白均能與制備的多抗形成特異性條帶，表明原核表達蛋白RBP4具有很好的免疫原性，抗體效價及特異性較高。 3. RBP4對豬卵母細胞體外成熟的影響 為了研究RBP4對豬卵母細胞體外成熟的影響，在卵母細胞成熟培養(yǎng)液中添加0，10，100ng/mL純化的豬RBP4蛋白處理豬COCs42h。統(tǒng)計卵母細胞第一極體的排出情況。結(jié)果表明，10，100ng/mL的RBP4對豬卵母細胞第一極體的排出具有顯著抑制作用。再利用Real-time PCR檢測外源性RBP4處理卵母細胞體外成熟42h中GDF-9及BMP-15的表達規(guī)律。結(jié)果表明，10，100ng/mL組GDF-9及BMP-15的表達量均顯著低于對照組。 本研究成功構(gòu)建了pEASY-E1-RBP4原核表達載體，攜帶重組質(zhì)粒的大腸桿菌經(jīng)IPTG誘導(dǎo)后能高效表達豬RBP4蛋白。建立了豬RBP4包涵體蛋白純化、復(fù)性工藝，復(fù)性的蛋白具有良好的生物學(xué)活性。同時制備得到了效價和特異性較高的兔抗豬RBP4抗體。原核表達的RBP4處理體外成熟的豬COCs，，顯著抑制第一極體排出，顯著下調(diào)了GDF-9和BMP-15基因的表達水平。
[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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q78;S852.2
本文編號：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.
【學(xué)位授予單位】：吉林大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：Q78;S852.2
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