植物甜味蛋白在哺乳动物乳腺中的分泌表达
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摘要
甜味是人类生来就喜欢的味道，蔗糖是目前应用最为广泛的甜味剂，是生活中必不可少的食物添加剂，可以补充能量，延迟疲劳。然而，摄入糖过量会引发许多疾病，如龋齿、心血管疾病、糖尿病等。因此寻找经济健康的甜味替代剂，满足人类对甜味的需求，一直是人们关注的焦点。甜味蛋白是一类高甜度、低热量的天然甜味添加剂，目前发现的甜味蛋白有以下几种：Brazzein, Curculin,Mabinlin, Miraculin, Monellin,Pentadin和Thaumatin，其中，Brazzein是从非洲植物Pentadi plandra brazzeana Baillion的果实中分离到的一种甜味蛋白。Brazzein由54个氨基酸残基组成，其分子量是所有甜味蛋白中最小的，其甜度是蔗糖的2000倍（按重量计）并具有很好的热稳定性，即使在80℃下加热4小时也不会丧失甜味。但是由于受到产地种植的限制，植物成分分离提取复杂，化学合成成本又比较昂贵，难以实现Brazzein的大量生产。近几年采用生物工程方法获得甜味蛋白成为研究的热点。乳腺生物反应器不仅可以生产出生物活性高，无污染的蛋白，且具有蛋白质翻译后修饰的功能确保了蛋白天然活性。因此，本课题中，我们将对利用动物乳腺生物反应器进行植物甜味蛋白生产的可行性进行探索。
已有研究报道利用转基因技术在转基因猪中成功表达了菠菜的不饱和酶脂肪酸Δ12，这说明植物蛋白可以在哺乳动物细胞中表达[1]]。为了探索植物甜味蛋白能否在动物细胞中和动物乳腺中表达。首先，我们根据密码子的偏好性对植物甜味蛋白brazzein和thaumatin基因进行了优化，并在基因前端加入信号肽使其可分泌到乳汁中，以优化修饰后的基因，构建全细胞表达载体pCAG-Brazzein-neo、pCAG-Thaumatin和乳腺特异表达载体pBC1-Brazzein和pBC1-thaumatin。并将pCAG-brazzein-neo和pCAG-thaumatin-neo转染HEK293细胞中，在细胞内和细胞培养基上清中都能够检测到Brazzein和thaumatin蛋白的表达。为了进一步验证其在动物活体内的表达活性，我们将DNA注射到兔子乳腺导管内，通过SDS-PAGE检测到蛋白可以在兔子乳汁中表达。确定了植物甜味蛋白在动物体内具有表达活性后，利用嵌合体技术生产了含Brazzein基因的转基因小鼠。通过转染和筛选获得了稳定表达Brazzein的干细胞细胞株，从中选取3株进行嵌合体小鼠制作，经传代建系后获得了3种稳定表达Brazzein的转基因小鼠品系。这3种Brazzein转基因小鼠品系都能够检测到Brazzein的在小鼠乳汁中稳定表达其中2种表现出甜味活性。
植物甜味蛋白在小鼠乳汁中的成功表达，第一次证实了植物基因可以在动物乳腺中表达，并生产出具有生物活性的蛋白。能够生产具有甜味活性的甜味蛋白Brazzein转基因小鼠成功地制备，为进一步利用大动物如牛、羊等大规模生产高效活性的甜味蛋白以及利用动物乳腺生物反应器表达其他植物蛋白奠定了基础。
Sweetness is the innate favorite taste of human beings. Sugar, the most popularsweetener, is essential in daily food. However, excessive sugar intake has beenassociated with several lifestyle-related diseases including caries (dental cavities),obesity, diabetes mellitus, and many more undesirable effects. Finding healthier andmore economical alternatives to sugars has received increasing attention to fulfill thegrowing demand. Sweet Protein is a kind of protein with high sweetness, low caloriesand high thermostability. So far, many sweet proteins including Brazzein, Curculin,Mabinlin, Miraculin, Monellin, Pentadin and Thaumatin have been identified. Amongthem, Brazzein, which comes from the pulp of the edible fruit of the African plantPentadiplandra brazzeana Baill, is a protein that is2,000times sweeter than sucroseby weight and has an in-diminishable sweetness profile after incubation at80℃forup to4hrs. Howerver, Brazzein can be grown only in tropical area, and the process ofpurifying Brazzein is very complicated thus limiting the production scale. Recently,the main efforts have be turned to seek producing sweet protein by geneengineering. Mammary gland bioreactor is a good factory to produce proteins with thehigh bioactivities and no toxicity. Hence, in this report, we attempt to explore thefeasibility by using mammary gland bioreactor to produce plant sweet protein.
Previously, the functional expression of Δ12fatty acid desaturase (FAD2) fromspinach plants has been achieved in transgenic pigs, indicating that a large quantity ofplant protein being expressed in mammalian animal cells.To test whether the plantgene can be expressed in mammalian cells, we designed the construction with thefollowing strategy. In order to express brazzein and thaumatin well in animalmammary glands, the gene sequence was optimized by codon usage. To allowsecretion of brazzein into the milk, we used the goat secretion signal β-casein.
We constructed the brazzein and thaumatin expressing vectors,pCAG-Brazzein-neo and pCAG-Thaumatin with CAG promoter for universal expression in all the tissues. Two vectors (pBC1-Brazzein and pBC1-thaumatin)which can be specifically expressed in mammary glad also were constructed. Theuniversal expression vectors were tansfected into in HEK293cells and and the sweetprotein expression have beed confirmed by both SDS-PAGE and Westernblot. To testif the mammary glad-specific expression vectors can be expressed in vivo, we directlyinjected the DNA into the rabbit mammary gland duct and the expression wasdecerned by SDS-PAGE and WB.To examine the expression of a plant protein inmammals, we generated three chimeric mouse line specifically expressing the plantprotein in the mammary gland. We transfected the vector into mouse embryo stem(mES) cell line and produced germline chimera mice. Expression of brazzein in themilk was confirmed by Western blot and immunohistochemisty. The milk oftransgenic mice is tasted sweet. These results demonstrate that a plant gene for asweet protein is able to be expressed in mammals, opening up the possibility ofproducing plant-protein-sweetened milk from those large animals as cattle and goats.
已有研究报道利用转基因技术在转基因猪中成功表达了菠菜的不饱和酶脂肪酸Δ12，这说明植物蛋白可以在哺乳动物细胞中表达[1]]。为了探索植物甜味蛋白能否在动物细胞中和动物乳腺中表达。首先，我们根据密码子的偏好性对植物甜味蛋白brazzein和thaumatin基因进行了优化，并在基因前端加入信号肽使其可分泌到乳汁中，以优化修饰后的基因，构建全细胞表达载体pCAG-Brazzein-neo、pCAG-Thaumatin和乳腺特异表达载体pBC1-Brazzein和pBC1-thaumatin。并将pCAG-brazzein-neo和pCAG-thaumatin-neo转染HEK293细胞中，在细胞内和细胞培养基上清中都能够检测到Brazzein和thaumatin蛋白的表达。为了进一步验证其在动物活体内的表达活性，我们将DNA注射到兔子乳腺导管内，通过SDS-PAGE检测到蛋白可以在兔子乳汁中表达。确定了植物甜味蛋白在动物体内具有表达活性后，利用嵌合体技术生产了含Brazzein基因的转基因小鼠。通过转染和筛选获得了稳定表达Brazzein的干细胞细胞株，从中选取3株进行嵌合体小鼠制作，经传代建系后获得了3种稳定表达Brazzein的转基因小鼠品系。这3种Brazzein转基因小鼠品系都能够检测到Brazzein的在小鼠乳汁中稳定表达其中2种表现出甜味活性。
植物甜味蛋白在小鼠乳汁中的成功表达，第一次证实了植物基因可以在动物乳腺中表达，并生产出具有生物活性的蛋白。能够生产具有甜味活性的甜味蛋白Brazzein转基因小鼠成功地制备，为进一步利用大动物如牛、羊等大规模生产高效活性的甜味蛋白以及利用动物乳腺生物反应器表达其他植物蛋白奠定了基础。
Sweetness is the innate favorite taste of human beings. Sugar, the most popularsweetener, is essential in daily food. However, excessive sugar intake has beenassociated with several lifestyle-related diseases including caries (dental cavities),obesity, diabetes mellitus, and many more undesirable effects. Finding healthier andmore economical alternatives to sugars has received increasing attention to fulfill thegrowing demand. Sweet Protein is a kind of protein with high sweetness, low caloriesand high thermostability. So far, many sweet proteins including Brazzein, Curculin,Mabinlin, Miraculin, Monellin, Pentadin and Thaumatin have been identified. Amongthem, Brazzein, which comes from the pulp of the edible fruit of the African plantPentadiplandra brazzeana Baill, is a protein that is2,000times sweeter than sucroseby weight and has an in-diminishable sweetness profile after incubation at80℃forup to4hrs. Howerver, Brazzein can be grown only in tropical area, and the process ofpurifying Brazzein is very complicated thus limiting the production scale. Recently,the main efforts have be turned to seek producing sweet protein by geneengineering. Mammary gland bioreactor is a good factory to produce proteins with thehigh bioactivities and no toxicity. Hence, in this report, we attempt to explore thefeasibility by using mammary gland bioreactor to produce plant sweet protein.
Previously, the functional expression of Δ12fatty acid desaturase (FAD2) fromspinach plants has been achieved in transgenic pigs, indicating that a large quantity ofplant protein being expressed in mammalian animal cells.To test whether the plantgene can be expressed in mammalian cells, we designed the construction with thefollowing strategy. In order to express brazzein and thaumatin well in animalmammary glands, the gene sequence was optimized by codon usage. To allowsecretion of brazzein into the milk, we used the goat secretion signal β-casein.
We constructed the brazzein and thaumatin expressing vectors,pCAG-Brazzein-neo and pCAG-Thaumatin with CAG promoter for universal expression in all the tissues. Two vectors (pBC1-Brazzein and pBC1-thaumatin)which can be specifically expressed in mammary glad also were constructed. Theuniversal expression vectors were tansfected into in HEK293cells and and the sweetprotein expression have beed confirmed by both SDS-PAGE and Westernblot. To testif the mammary glad-specific expression vectors can be expressed in vivo, we directlyinjected the DNA into the rabbit mammary gland duct and the expression wasdecerned by SDS-PAGE and WB.To examine the expression of a plant protein inmammals, we generated three chimeric mouse line specifically expressing the plantprotein in the mammary gland. We transfected the vector into mouse embryo stem(mES) cell line and produced germline chimera mice. Expression of brazzein in themilk was confirmed by Western blot and immunohistochemisty. The milk oftransgenic mice is tasted sweet. These results demonstrate that a plant gene for asweet protein is able to be expressed in mammals, opening up the possibility ofproducing plant-protein-sweetened milk from those large animals as cattle and goats.
引文
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