来航鸡FOXL2基因的真核表达及蛋白纯化
发布时间：2019-05-12 13:20
【摘要】：FOXL2是第一个被认定在维持卵巢功能方面发挥重要作用的人类常染色体基因，也是在脊椎动物中最早被发现的卵巢分化的性别二态性标记。FOXL2基因不但在抗击卵泡凋亡、维持卵巢储备功能方面发挥着重要作用，而且其正常表达与否可能与鸡产蛋性能的高低有关。目前，随着人们对FOXL2功能研究的逐渐深入，FOXL2蛋白的需求量也不断增大，但是市场上现有的FOXL2产品存在少而贵的特点，不能很好的满足于应用。基于此不足，本实验室曾采用大肠杆菌表达体系对来航鸡FOXL2基因进行了表达，但蛋白量较少且活性低。故本研究利用酵母菌表达系统对来航鸡FOXL2基因进行真核表达及纯化，，以期获得大量的高活性表达蛋白。 本研究根据GenBank上收录的来航鸡FOXL2的全基因序列（登录号：JF＿708868.1），通过生物信息学比对分析其保守序列及真核表达载体的多克隆酶切位点，利用Primmer5.0来设计合成一对引物P1、P2。以纯系SPF来航鸡全血DNA为模板，经PCR扩增带有酶切位点的FOXL2基因，并将其与pMD18-T载体连接，获得重组克隆质粒pMD18-T-FOXL2。再以pMD18-T-FOXL2为模板，经PCR扩增带有酶切位点的FOXL2基因，定向克隆到真核表达载体pPICZaA中，构建pPICZaA-FOXL2重组真核表达载体。用SacI将重组pPICZaA-FOXL2线性化后，电转GS115毕赤酵母菌。28℃培养5d，PCR鉴定和测序正确后，进行高拷贝子筛选，将筛选到的高拷贝转化子加甲醇28℃诱导表达96h。应用SDS-PAGE和Western blot方法分析重组蛋白的表达情况，鉴定重组蛋白的抗原特异性，然后采用His镍柱亲和层析获得重组蛋白的纯化产物。 研究结果表明，成功扩增出来航鸡FOXL2基因，经PCR、双酶切和测序结果比对分析未发生氨基酸突变现象，成功构建了重组克隆载体和表达载体。重组表达质粒转入毕赤酵母菌后经甲醇诱导，由于重组蛋白以胞外分泌的形式进行表达，取上清SDS-PAGE分析显示，表达出与预期大小相一致的目的蛋白条带，其分子量为37kD。Western-blot鉴定结果显示，重组蛋白能被His标签单抗所识别，具有良好的反应原性。重组蛋白经纯化后，分别得到浓度为1.2mg/mL重组目的蛋白。FOXL2基因的克隆、表达及纯化为进一步探索该基因的生物学功能奠定了基础。
[Abstract]:FOXL2 is the first human autosomal gene to play an important role in maintaining ovarian function. FOXL2 gene is not only the first sex dimorphism marker of ovarian differentiation found in vertebrates. FOXL2 gene is not only in the fight against follicular apoptosis. Maintaining ovarian reserve function plays an important role, and its normal expression may be related to the egg laying performance of chickens. At present, with the deepening of the study of FOXL2 function, the demand for FOXL2 protein is also increasing, but the existing FOXL2 products in the market have few and expensive characteristics, which can not be well satisfied with the application. Because of this deficiency, we used Escherichia coli expression system to express FOXL2 gene of Leghorn chicken, but the amount of protein was less and the activity was low. In this study, the yeast expression system was used to express and purify the FOXL2 gene of Leghorn chicken in order to obtain a large number of highly active protein. According to the whole gene sequence (accession number: JF_708868.1) of Laihang chicken FOXL2 collected on GenBank, the conserved sequence and polyclonal restriction site of eukaryotic expression vector were analyzed by bioinformatics comparison. A pair of primers P _ 1, P _ 2 were designed and synthesized by Primmer5.0. The whole blood DNA of pure line SPF was used as template. The FOXL2 gene with enzyme site was amplified by PCR and ligated with pMD18-T vector to obtain the recombinant clone plasmid pMD18-T-FOXL2.. Then using pMD18-T-FOXL2 as template, FOXL2 gene with restriction site was amplified by PCR and cloned into eukaryotic expression vector pPICZaA to construct recombinant eukaryotic expression vector pPICZaA-FOXL2. The recombinant pPICZaA-FOXL2 was linearized by SacI and electrotransferred to Pichia pastoris at 28 鈩
本文编号：2475421
[Abstract]:FOXL2 is the first human autosomal gene to play an important role in maintaining ovarian function. FOXL2 gene is not only the first sex dimorphism marker of ovarian differentiation found in vertebrates. FOXL2 gene is not only in the fight against follicular apoptosis. Maintaining ovarian reserve function plays an important role, and its normal expression may be related to the egg laying performance of chickens. At present, with the deepening of the study of FOXL2 function, the demand for FOXL2 protein is also increasing, but the existing FOXL2 products in the market have few and expensive characteristics, which can not be well satisfied with the application. Because of this deficiency, we used Escherichia coli expression system to express FOXL2 gene of Leghorn chicken, but the amount of protein was less and the activity was low. In this study, the yeast expression system was used to express and purify the FOXL2 gene of Leghorn chicken in order to obtain a large number of highly active protein. According to the whole gene sequence (accession number: JF_708868.1) of Laihang chicken FOXL2 collected on GenBank, the conserved sequence and polyclonal restriction site of eukaryotic expression vector were analyzed by bioinformatics comparison. A pair of primers P _ 1, P _ 2 were designed and synthesized by Primmer5.0. The whole blood DNA of pure line SPF was used as template. The FOXL2 gene with enzyme site was amplified by PCR and ligated with pMD18-T vector to obtain the recombinant clone plasmid pMD18-T-FOXL2.. Then using pMD18-T-FOXL2 as template, FOXL2 gene with restriction site was amplified by PCR and cloned into eukaryotic expression vector pPICZaA to construct recombinant eukaryotic expression vector pPICZaA-FOXL2. The recombinant pPICZaA-FOXL2 was linearized by SacI and electrotransferred to Pichia pastoris at 28 鈩
本文编号：2475421
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