公开(公告)号：US20070150976A1

公开(公告)日：2007-06-28

申请号：US10585976

申请日：2004-12-09

申请人： Daichang Yang ,  Kevin Hennegan ,  Ning Huang

发明人： Daichang Yang ,  Kevin Hennegan ,  Ning Huang

IPC分类号： A01H5/00 ,  C12N15/82 ,  C12N5/04

CPC分类号： C12N15/8221 ,  C12N15/8234 ,  C12N15/8257

摘要： The expression of heterologous peptides or polypeptides in the seeds of monocot plants is optimized by generating fusion protein constructs in which monocot plant seed storage proteins are used as fusion protein carriers for the heterologous peptides or polypeptides. The heterologous peptides or polypeptides are preferably small, about 10 kDa or less and/or between 5 and 100 amino acids in length. These heterologous peptides or polypeptides may be used in human and animal nutritional and therapeutic compositions.

摘要翻译： 通过产生融合蛋白构建体来优化单子叶植物种子中的异源肽或多肽的表达，其中单子叶植物种子储存蛋白用作异源肽或多肽的融合蛋白载体。 异源肽或多肽优选长度小，约10kDa或更少和/或5至100个氨基酸。 这些异源肽或多肽可用于人和动物营养和治疗组合物中。

公开(公告)号：US20080010697A1

公开(公告)日：2008-01-10

申请号：US10584225

申请日：2003-12-23

申请人： Daichang Yang ,  Kevin Hennegan ,  Ning Huang

发明人： Daichang Yang ,  Kevin Hennegan ,  Ning Huang

IPC分类号： A01H1/02 ,  C12N15/87

CPC分类号： C12N9/2462 ,  C07H21/04 ,  C12N15/8216 ,  C12N15/8221 ,  C12N15/8257

摘要： The invention is directed to expression of non-plant proteins in rice plants. Expression is optimized by use of a non-rice promoter of a monocot protein gene and its corresponding signal peptide for expression of the non-plant protein in rice plant at high yields. The invention is useful for making human proteins, polypeptides and peptides in rice seeds. The expressed protein product can be isolated from the rice seed for administration to humans or other animals.

摘要翻译： 本发明涉及水稻植物中非植物蛋白的表达。 通过使用单子叶植物蛋白基因的非水稻启动子及其相应的信号肽来优化表达，以高产量在水稻植物中表达非植物蛋白。 本发明可用于在水稻种子中制备人类蛋白质，多肽和肽。 表达的蛋白质产物可以从水稻种子中分离以供给人或其他动物。

公开(公告)号：US20050229273A1

公开(公告)日：2005-10-13

申请号：US11083617

申请日：2005-03-16

申请人： Ning Huang ,  Daichang Yang ,  Yong-Sic Hwang ,  Robert Schmidt

发明人： Ning Huang ,  Daichang Yang ,  Yong-Sic Hwang ,  Robert Schmidt

IPC分类号： A23K1/14 ,  A23K1/16 ,  A23L1/185 ,  A23L1/30 ,  A23L1/305 ,  C07K14/415 ,  C07K14/485 ,  C07K14/65 ,  C07K14/79 ,  C07K14/81 ,  C12N9/08 ,  C12N9/36 ,  C12N15/29 ,  C12N15/82 ,  A01H1/00 ,  A01H5/00

CPC分类号： C12N9/2462 ,  A23K10/30 ,  A23K20/147 ,  A23L7/20 ,  A23L33/105 ,  A23L33/19 ,  C07K14/415 ,  C07K14/485 ,  C07K14/65 ,  C07K14/79 ,  C07K14/8125 ,  C12N9/0065 ,  C12N15/8216 ,  C12N15/8222 ,  C12N15/8234 ,  C12N15/8257

摘要： The invention relates to transgenic plants that demonstrate enhanced expression of a plant transcription factor under the control of a seed specific promoter such that expression of the transcription factor activates transcription of a native or non-native coding sequence in the plant. The invention further relates to a method of generating such transgenic plants and methods for enhancing the level of expression of a selected heterologous protein in seeds of such transgenic plants.

摘要翻译： 本发明涉及在种子特异性启动子控制下表现出植物转录因子表达增强的转基因植物，使得转录因子的表达激活植物中天然或非天然编码序列的转录。 本发明还涉及产生这种转基因植物的方法和用于增强这种转基因植物种子中所选择的异源蛋白质表达水平的方法。

公开(公告)号：US07589252B2

公开(公告)日：2009-09-15

申请号：US11083617

申请日：2005-03-16

申请人： Ning Huang ,  Daichang Yang ,  Yong-Sic Hwang ,  Robert J. Schmidt

发明人： Ning Huang ,  Daichang Yang ,  Yong-Sic Hwang ,  Robert J. Schmidt

IPC分类号： C12N15/82 ,  C12N5/04

CPC分类号： C12N9/2462 ,  A23K10/30 ,  A23K20/147 ,  A23L7/20 ,  A23L33/105 ,  A23L33/19 ,  C07K14/415 ,  C07K14/485 ,  C07K14/65 ,  C07K14/79 ,  C07K14/8125 ,  C12N9/0065 ,  C12N15/8216 ,  C12N15/8222 ,  C12N15/8234 ,  C12N15/8257

摘要： The invention relates to transgenic plants that demonstrate enhanced expression of a plant transcription factor under the control of a seed specific promoter such that expression of the transcription factor activates transcription of a native or non-native coding sequence in the plant. The invention further relates to a method of generating such transgenic plants and methods for enhancing the level of expression of a selected heterologous protein in seeds of such transgenic plants.

摘要翻译： 本发明涉及在种子特异性启动子控制下表现出植物转录因子表达增强的转基因植物，使得转录因子的表达激活植物中天然或非天然编码序列的转录。 本发明还涉及产生这种转基因植物的方法和用于增强这种转基因植物种子中所选择的异源蛋白质表达水平的方法。

公开(公告)号：US07723571B2

公开(公告)日：2010-05-25

申请号：US11810863

申请日：2007-06-06

申请人： Daichang Yang ,  Tingting Xie

发明人： Daichang Yang ,  Tingting Xie

IPC分类号： C12N15/82

CPC分类号： C12N15/8257 ,  C07K14/65

摘要： The present invention provides a method of using cereal non-storage protein as fusion carrier to highly express small peptides in host endosperm cells. The method includes the steps of providing an endosperm-specific promoter and a DNA leading sequence encoding an endosperm-specific signal peptide; providing the gene of a non-storage protein as fusion carrier and an target gene; constructing a expression vector containing the promoter and DNA leading sequence, the gene of the fusion carrier, and a target gene; and expressing the expression vector in a host endosperm cell. Also provided in the invention are a vector constructed there from and the use thereof.

摘要翻译： 本发明提供了使用谷物非储存蛋白作为融合载体以在宿主胚乳细胞中高度表达小肽的方法。 该方法包括提供胚乳特异性启动子和编码胚乳特异性信号肽的DNA前导序列的步骤; 提供非储存蛋白的基因作为融合载体和靶基因; 构建含有启动子和DNA前导序列的表达载体，融合载体的基因和靶基因; 并在宿主胚乳细胞中表达载体。 本发明还提供了构建在其上的载体及其用途。

公开(公告)号：US09255138B2

公开(公告)日：2016-02-09

申请号：US13206844

申请日：2011-08-10

申请人： Daichang Yang ,  Yang He ,  Guangfei Li

发明人： Daichang Yang ,  Yang He ,  Guangfei Li

IPC分类号： C07K1/14 ,  C07K14/765

CPC分类号： C07K14/765

摘要： A method for extracting recombinant human serum albumin (rHSA) from transgenic rice grain is provided, comprising the steps of: 1) grinding dehusked rice containing rHSA into milled rice grain with a fineness of 80˜120 mesh, which is mixed with a extraction buffer in a w/v ratio of 1:5, then extracting at 55˜60° C. for 1˜3 hours to obtain mixture I; said extraction buffer comprises 10˜30 mM phosphate buffer, 10˜20 mM sodium acetate, 15˜30 mM ammonium sulfate and 5˜20 mM sodium caprylate and has a pH of 6.5˜8; 2) adjusting the pH of mixture I to 4.0˜4.5, followed by precipitating at room temperature for 3˜12 hours to obtain mixture II; 3) filtering the mixture II and collecting the filtrate, to obtain a solution containing high concentration of rHSA. The concentration of rHSA in the resultant solution is 650˜660 μg/mL, which increases by 1.15 times comparing to the extraction amount before improvement, and the amount of non-target protein is reduced by 2.46 times. The method provides a basis for subsequent purification of rHSA.

摘要翻译： 提供了一种从转基因水稻提取重组人血清白蛋白（rHSA）的方法，包括以下步骤：1）将含有rHSA的脱壳米粉碎成细粉度为80〜120目的米粒，与提取缓冲液 以aw / v比为1:5，然后在55〜60℃下提取1〜3小时，得到混合物I; 所述提取缓冲液包含10〜30mM磷酸盐缓冲液，10〜20mM乙酸钠，15〜30mM硫酸铵和5〜20mM辛酸钠，pH为6.5〜8; 2）将混合物I的pH调节至4.0〜4.5，然后在室温下沉淀3〜12小时，得到混合物II; 3）过滤混合物II并收集滤液，得到含有高浓度rHSA的溶液。 所得溶液中rHSA的浓度为650〜360μg/ mL，与改善前的提取量相比增加1.15倍，非靶蛋白的量减少2.46倍。 该方法为随后的rHSA纯化提供依据。

公开(公告)号：US20120202973A1

公开(公告)日：2012-08-09

申请号：US13206844

申请日：2011-08-10

申请人： Daichang Yang ,  Yang He ,  Guangfei Li

发明人： Daichang Yang ,  Yang He ,  Guangfei Li

IPC分类号： C07K14/765 ,  C07K1/34

CPC分类号： C07K14/765

摘要： A method for extracting recombinant human serum albumin (rHSA) from transgenic rice grain is provided, comprising the steps of: 1) grinding dehusked rice containing rHSA into milled rice grain with a fineness of 80˜120 mesh, which is mixed with a extraction buffer in a w/v ratio of 1:5, then extracting at 55˜60° C. for 1˜3 hours to obtain mixture I; said extraction buffer comprises 10˜30 mM phosphate buffer, 10˜20 mM sodium acetate, 15˜30 mM ammonium sulfate and 5˜20 mM sodium caprylate and has a pH of 6.5˜8; 2) adjusting the pH of mixture I to 4.0˜4.5, followed by precipitating at room temperature for 3˜12 hours to obtain mixture II; 3) filtering the mixture II and collecting the filtrate, to obtain a solution containing high concentration of rHSA. The concentration of rHSA in the resultant solution is 650˜660 μg/mL, which increases by 1.15 times comparing to the extraction amount before improvement, and the amount of non-target protein is reduced by 2.46 times. The method provides a basis for subsequent purification of rHSA.

摘要翻译： 提供了一种从转基因水稻提取重组人血清白蛋白（rHSA）的方法，包括以下步骤：1）将含有rHSA的脱壳米粉碎成细粉度为80〜120目的米粒，与提取缓冲液 以aw / v比为1:5，然后在55〜60℃下提取1〜3小时，得到混合物I; 所述提取缓冲液包含10〜30mM磷酸盐缓冲液，10〜20mM乙酸钠，15〜30mM硫酸铵和5〜20mM辛酸钠，pH为6.5〜8; 2）将混合物I的pH调节至4.0〜4.5，然后在室温下沉淀3〜12小时，得到混合物II; 3）过滤混合物II并收集滤液，得到含有高浓度rHSA的溶液。 所得溶液中rHSA的浓度为650〜360μg/ mL，与改善前的提取量相比增加1.15倍，非靶蛋白的量减少2.46倍。 该方法为随后的rHSA纯化提供依据。

公开(公告)号：US20070289033A1

公开(公告)日：2007-12-13

申请号：US11810863

申请日：2007-06-06

申请人： Daichang Yang ,  Tingting Xie

发明人： Daichang Yang ,  Tingting Xie

IPC分类号： A01H5/00 ,  C12N15/82

CPC分类号： C12N15/8257 ,  C07K14/65

摘要： The present invention provides a method of using cereal non-storage protein as fusion carrier to highly express small peptides in host endosperm cells. The method includes the steps of providing an endosperm-specific promoter and a DNA leading sequence encoding an endosperm-specific signal peptide; providing the gene of a non-storage protein as fusion carrier and an target gene; constructing a expression vector containing the promoter and DNA leading sequence, the gene of the fusion carrier, and a target gene; and expressing the expression vector in a host endosperm cell. Also provided in the invention are a vector constructed there from and the use thereof.

摘要翻译： 本发明提供了使用谷物非储存蛋白作为融合载体以在宿主胚乳细胞中高度表达小肽的方法。 该方法包括提供胚乳特异性启动子和编码胚乳特异性信号肽的DNA前导序列的步骤; 提供非储存蛋白的基因作为融合载体和靶基因; 构建含有启动子和DNA前导序列的表达载体，融合载体的基因和靶基因; 并在宿主胚乳细胞中表达载体。 本发明还提供了构建在其上的载体及其用途。

公开(公告)号：US20120165509A1

公开(公告)日：2012-06-28

申请号：US13206884

申请日：2011-08-10

申请人： Daichang Yang ,  Yang He ,  Guangfei Li ,  Jingru Liu

发明人： Daichang Yang ,  Yang He ,  Guangfei Li ,  Jingru Liu

IPC分类号： C07K1/18 ,  C07K14/765

CPC分类号： C07K1/18 ,  B01J20/3285 ,  C07K14/765

摘要： A method for separating and purifying recombinant human serum albumin (rHSA) from transgenic rice grain, sequentially comprising the steps of: 1) subjecting crude extract of rHSA to cation exchange chromatography to obtain primary product I; 2) subjecting the primary product I to anion exchange chromatography to obtain secondary product II; 3) subjecting the secondary product II to hydrophobic chromatography to obtain purified rHSA. The method may further comprise a step of ceramic hydroxyapatite chromatography prior to the hydrophobic chromatography. The method has the advantages of low cost and easy operation. The resultant rHSA has a purity of about 99% by HPLC.

摘要翻译： 一种从转基因水稻籽粒中分离纯化重组人血清白蛋白（rHSA）的方法，其顺序包括以下步骤：1）使rHSA的粗提物进行阳离子交换色谱以获得初级产物I; 2）使初级产物I进行阴离子交换色谱以获得二次产物II; 3）使二次产物II进行疏水色谱以获得纯化的rHSA。 该方法还可以包括在疏水层析之前的陶瓷羟基磷灰石层析步骤。 该方法成本低，操作方便。 所得的rHSA通过HPLC纯度为约99％。

公开(公告)号：US09023990B2

公开(公告)日：2015-05-05

申请号：US13206884

申请日：2011-08-10

申请人： Daichang Yang ,  Yang He ,  Guangfei Li ,  Jingru Liu

发明人： Daichang Yang ,  Yang He ,  Guangfei Li ,  Jingru Liu

IPC分类号： C07K1/14 ,  C07K14/765 ,  A61K38/00

CPC分类号： C07K1/18 ,  B01J20/3285 ,  C07K14/765

摘要： A method for separating and purifying recombinant human serum albumin (rHSA) from transgenic rice grain, sequentially comprising the steps of: 1) subjecting crude extract of rHSA to cation exchange chromatography to obtain primary product I; 2) subjecting the primary product I to anion exchange chromatography to obtain secondary product II; 3) subjecting the secondary product II to hydrophobic chromatography to obtain purified rHSA. The method may further comprise a step of ceramic hydroxyapatite chromatography prior to the hydrophobic chromatography. The method has the advantages of low cost and easy operation. The resultant rHSA has a purity of about 99% by HPLC.

摘要翻译： 一种从转基因水稻籽粒中分离纯化重组人血清白蛋白（rHSA）的方法，其顺序包括以下步骤：1）使rHSA的粗提物进行阳离子交换色谱以获得初级产物I; 2）使初级产物I进行阴离子交换色谱以获得二次产物II; 3）使二次产物II进行疏水色谱以获得纯化的rHSA。 该方法还可以包括在疏水层析之前的陶瓷羟基磷灰石层析步骤。 该方法成本低，操作方便。 所得的rHSA通过HPLC纯度为约99％。