食物过敏是一个全世界关注的公共卫生问题。如何降低大豆过敏原含量,保证大豆食品安全,已成为日益关注的问题。大豆种子过敏原包括种子储藏蛋白、结构蛋白和防御相关蛋白,其中7S球蛋白组分中的Gly m Bd 28K,Gly m Bd 30K及β-伴球蛋白的...食物过敏是一个全世界关注的公共卫生问题。如何降低大豆过敏原含量,保证大豆食品安全,已成为日益关注的问题。大豆种子过敏原包括种子储藏蛋白、结构蛋白和防御相关蛋白,其中7S球蛋白组分中的Gly m Bd 28K,Gly m Bd 30K及β-伴球蛋白的Gly m Bd 60K是3种主要的过敏原。目前通过对过敏原的理化性质、过敏原性和基因结构的认识,运用传统育种及基因工程技术等方法,在减少大豆的过敏原性方面已取得一定的进展。文章对大豆过敏原的类型及特性、3种主要过敏原的理化性质、基因结构以及低过敏原种质创新等方面的研究报道进行了综述。展开更多
根据文献并结合生物信息学方法选取大豆主要过敏原Gly m Bd 30k的抗原表位区,采用PCR方法扩增出该抗原表位区基因片段,并通过酶切连接分别构建单体的pET表达载体(pET-sGly)和二聚体的pET表达载体(pET-dGly),重组质粒转化到大肠杆菌BL21(...根据文献并结合生物信息学方法选取大豆主要过敏原Gly m Bd 30k的抗原表位区,采用PCR方法扩增出该抗原表位区基因片段,并通过酶切连接分别构建单体的pET表达载体(pET-sGly)和二聚体的pET表达载体(pET-dGly),重组质粒转化到大肠杆菌BL21(DE3)plysS,经IPTG诱导后进行SDS-PAGE分析;同时用Ni2+离子亲和层析柱纯化表达的sGly和dGly抗原表位蛋白,用western-blotting和ELISA检测重组蛋白的免疫原性。结果表明:表达的单体sGly蛋白以可溶性表达为主,而其二聚体dGly蛋白以包涵体形式存在,纯化的sGly和dGly蛋白都具有较好的免疫原性,重组蛋白sGly的抗原性更佳。成功构建的大豆主要过敏原Gly m Bd 30k蛋白的抗原表位区单体sGly蛋白及其二聚体dGly蛋白的工程菌,为研制大豆主要过敏原的单克隆抗体以制备用于大豆主要过敏原检测的试剂奠定了基础。展开更多
A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome a...A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags （ESTs）, we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription.展开更多
本文应用生物信息学技术,通过使用三种软件SOPMA、Swiss-model和DNAStar来预测大豆主要过敏原Gly m Bd 30K的抗原表位,结果发现80-85、103-106、217-220、355-360这四段氨基酸残基序列是可能的抗原表位,为后续的蛋白表位实验提供了依据...本文应用生物信息学技术,通过使用三种软件SOPMA、Swiss-model和DNAStar来预测大豆主要过敏原Gly m Bd 30K的抗原表位,结果发现80-85、103-106、217-220、355-360这四段氨基酸残基序列是可能的抗原表位,为后续的蛋白表位实验提供了依据,大大提高了工作效率。展开更多
利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆Gly m Bd 30K的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了...利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆Gly m Bd 30K的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了其原核表达载体。该基因含有长度为1 140 bp的开放阅读框,编码379个氨基酸。该蛋白质的相对分子质量为42 758,等电点为5.08。序列同源性分析发现其与数据库中已知的Gly m Bd30K基因同源性很高,因此认为其是大豆的过敏原基因,在GenBank数据库中的登录号为EU883600。克隆的大豆主要过敏原Gly m Bd 30K基因及构建的原核表达载体,为大豆主要过敏原Gly m Bd 30K的重组表达和免疫活性鉴定等奠定基础。展开更多
Gly m Bd 30K蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应。因此,发掘低Gly m Bd 30K蛋白含量优异种质对于培育优质大豆品种具有重要意义。为了获得致敏蛋白Gly m Bd 30K低含量的优异种质,根据Gly m ...Gly m Bd 30K蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应。因此,发掘低Gly m Bd 30K蛋白含量优异种质对于培育优质大豆品种具有重要意义。为了获得致敏蛋白Gly m Bd 30K低含量的优异种质,根据Gly m Bd 30K蛋白的190-379aa多肽序列制备多克隆抗体;对来源于山西省的29份种质,利用Western blot技术对其Gly m Bd 30K蛋白含量进行检测;并扩增和分析Gly m Bd 30K基因序列;利用荧光定量PCR技术对筛选出的低Gly m Bd 30K含量种质进行表达分析。结果表明:从参试种质中鉴定出2份Gly m Bd 30K蛋白含量低的种质,Gly m Bd 30K蛋白低含量种质的鉴定效率为6.9%,分别是来自太原市的大豆种质134(ZDD02046)和大青豆(ZDD02174),其Gly m Bd 30K基因序列与Willams82相比,在启动子上都有TA重复序列变异,134(ZDD02046)有8次TA重复,大青豆(ZDD02174)有42次TA重复,表达分析结果显示,134(ZDD02046)的转录水平显著低于大青豆(ZDD02174),推测启动子上TA多态性可能影响了其转录水平。本研究建立Gly m Bd 30K蛋白含量测定的方法,鉴定出2份低蛋白含量的优异种质,为今后选育优质蛋白组合的大豆新品种提供了技术和材料支撑。展开更多
The Mg-3.0Nd-0.2Zn-0.4Zr (NZ30K) alloys were prepared by direct-chill casting (DCC) and sand mould casting (SMC) processes,respectively and their microstructures and mechanical properties were investigated.The results...The Mg-3.0Nd-0.2Zn-0.4Zr (NZ30K) alloys were prepared by direct-chill casting (DCC) and sand mould casting (SMC) processes,respectively and their microstructures and mechanical properties were investigated.The results indicate that casting method plays a remarkable influence on the microstructure and mechanical properties of as-cast NZ30K alloy.The grain size increases from 35-40μm in the billets made by the DCC to about 100-120μm in the billets by the SMC.The aggregation of Mg12Nd usually found at the triple joints of grain boundaries in the billets prepared by SMC while is not observable from the billets by DCC.The tensile strengths and elongations of the billets are 195.2 MPa and 15.5% by DCC,and 162.5 MPa and 3.2% by SMC,respectively.The tensile strength of the alloy by DCC is remarkably enhanced by T6 heat treatment,which reached 308.5 MPa.Fracture surfaces of NZ30K alloy have been characterized as intergranular fracture by SMC and quasi-cleavage fracture by DCC,respectively.展开更多
以栽培大豆(G.max)核心种质经聚类随机选择样本为主,以栽培大豆保留种质和野生大豆(G.soja)为对照,利用小鼠单克隆抗体Gly m Bd 30K(F5)和Gly m Bd 28K(C5)分别检测大豆的30K和28K过敏蛋白抗原,目的是发掘过敏蛋白缺失的品种资源,明确...以栽培大豆(G.max)核心种质经聚类随机选择样本为主,以栽培大豆保留种质和野生大豆(G.soja)为对照,利用小鼠单克隆抗体Gly m Bd 30K(F5)和Gly m Bd 28K(C5)分别检测大豆的30K和28K过敏蛋白抗原,目的是发掘过敏蛋白缺失的品种资源,明确其分布规律和特点,检测大豆核心种质的代表性,为资源和育种研究提供参考。鉴定结果表明,在参试的60份野生大豆和421份栽培大豆中没有发现30K过敏蛋白缺失的种质,但28K过敏蛋白缺失率分别为13.3%和37.8%。栽培大豆核心种质28K过敏蛋白缺失比率的变化趋势与栽培大豆保留种质总体规律基本相同,在3个生态区的缺失比率差异均不显著,说明核心种质在28K过敏蛋白缺失这个性状上具有代表性。SSR标记分析发现,在缺失过敏蛋白的种质中,80%相似系数在0.44以下,表明这些种质遗传基础较为广泛,可充分利用类群内或类群间差异较大的材料作为亲本配制杂交组合,培育缺失28K过敏蛋白优质品种。展开更多
文摘食物过敏是一个全世界关注的公共卫生问题。如何降低大豆过敏原含量,保证大豆食品安全,已成为日益关注的问题。大豆种子过敏原包括种子储藏蛋白、结构蛋白和防御相关蛋白,其中7S球蛋白组分中的Gly m Bd 28K,Gly m Bd 30K及β-伴球蛋白的Gly m Bd 60K是3种主要的过敏原。目前通过对过敏原的理化性质、过敏原性和基因结构的认识,运用传统育种及基因工程技术等方法,在减少大豆的过敏原性方面已取得一定的进展。文章对大豆过敏原的类型及特性、3种主要过敏原的理化性质、基因结构以及低过敏原种质创新等方面的研究报道进行了综述。
文摘根据文献并结合生物信息学方法选取大豆主要过敏原Gly m Bd 30k的抗原表位区,采用PCR方法扩增出该抗原表位区基因片段,并通过酶切连接分别构建单体的pET表达载体(pET-sGly)和二聚体的pET表达载体(pET-dGly),重组质粒转化到大肠杆菌BL21(DE3)plysS,经IPTG诱导后进行SDS-PAGE分析;同时用Ni2+离子亲和层析柱纯化表达的sGly和dGly抗原表位蛋白,用western-blotting和ELISA检测重组蛋白的免疫原性。结果表明:表达的单体sGly蛋白以可溶性表达为主,而其二聚体dGly蛋白以包涵体形式存在,纯化的sGly和dGly蛋白都具有较好的免疫原性,重组蛋白sGly的抗原性更佳。成功构建的大豆主要过敏原Gly m Bd 30k蛋白的抗原表位区单体sGly蛋白及其二聚体dGly蛋白的工程菌,为研制大豆主要过敏原的单克隆抗体以制备用于大豆主要过敏原检测的试剂奠定了基础。
基金This work was supported by the National Basic Research Program of China, the National Scientific and Technological Project and the National Natural Science Foundation under grant No. 2005 CB 121000, 2005 B A711A07 and 30471313, respectively.
文摘A group of lipoproteins with molecular sizes of approximately 30 kDa, referred to as 30K proteins, axe synthesized in fat body cells in the fifth instar larvae of silkworm, Bombyx mori. Analyzing the silkworm genome and its expressed sequence tags （ESTs）, we found 10 genes encoding 30K proteins, which are mainly distributed in three subfamilies. Of these, seven coding proteins were found to harbor the degrading sites of 30kP protease A, although the number of degrading sites may be different. As some potential core promoters and regulatory elements were supposed to be essential for gene transcription, the expression profiles of these genes were examined by semi-quantitative reverse transcription polymerase chain reaction. Eight 30K protein genes were detected to express luxuriantly in the fat body, while two were hardly expressed. Such results suggest that these 30K proteins may have different functions, and their adjacent regulatory elements play a crucial role in regulating their transcription.
文摘本文应用生物信息学技术,通过使用三种软件SOPMA、Swiss-model和DNAStar来预测大豆主要过敏原Gly m Bd 30K的抗原表位,结果发现80-85、103-106、217-220、355-360这四段氨基酸残基序列是可能的抗原表位,为后续的蛋白表位实验提供了依据,大大提高了工作效率。
文摘利用RT-PCR克隆大豆主要过敏原Gly m Bd 30K的全长基因,根据序列设计带有酶切位点的特异性引物,扩增大豆Gly m Bd 30K的完整开放阅读框,与pET-28a载体连接,构建原核表达载体。结果表明:克隆了大豆主要过敏原Gly m Bd 30K基因,且构建了其原核表达载体。该基因含有长度为1 140 bp的开放阅读框,编码379个氨基酸。该蛋白质的相对分子质量为42 758,等电点为5.08。序列同源性分析发现其与数据库中已知的Gly m Bd30K基因同源性很高,因此认为其是大豆的过敏原基因,在GenBank数据库中的登录号为EU883600。克隆的大豆主要过敏原Gly m Bd 30K基因及构建的原核表达载体,为大豆主要过敏原Gly m Bd 30K的重组表达和免疫活性鉴定等奠定基础。
文摘Gly m Bd 30K蛋白是大豆中主要的免疫显性过敏原之一,会引起人和牲畜腹泻和肠道炎症等过敏反应。因此,发掘低Gly m Bd 30K蛋白含量优异种质对于培育优质大豆品种具有重要意义。为了获得致敏蛋白Gly m Bd 30K低含量的优异种质,根据Gly m Bd 30K蛋白的190-379aa多肽序列制备多克隆抗体;对来源于山西省的29份种质,利用Western blot技术对其Gly m Bd 30K蛋白含量进行检测;并扩增和分析Gly m Bd 30K基因序列;利用荧光定量PCR技术对筛选出的低Gly m Bd 30K含量种质进行表达分析。结果表明:从参试种质中鉴定出2份Gly m Bd 30K蛋白含量低的种质,Gly m Bd 30K蛋白低含量种质的鉴定效率为6.9%,分别是来自太原市的大豆种质134(ZDD02046)和大青豆(ZDD02174),其Gly m Bd 30K基因序列与Willams82相比,在启动子上都有TA重复序列变异,134(ZDD02046)有8次TA重复,大青豆(ZDD02174)有42次TA重复,表达分析结果显示,134(ZDD02046)的转录水平显著低于大青豆(ZDD02174),推测启动子上TA多态性可能影响了其转录水平。本研究建立Gly m Bd 30K蛋白含量测定的方法,鉴定出2份低蛋白含量的优异种质,为今后选育优质蛋白组合的大豆新品种提供了技术和材料支撑。
基金supported by the National High-tech R&D Program of China (863 Program),grant No.2009AA03Z521the foundation of Shanghai Rising-Star Program (A type),grant No. 09QA1403100
文摘The Mg-3.0Nd-0.2Zn-0.4Zr (NZ30K) alloys were prepared by direct-chill casting (DCC) and sand mould casting (SMC) processes,respectively and their microstructures and mechanical properties were investigated.The results indicate that casting method plays a remarkable influence on the microstructure and mechanical properties of as-cast NZ30K alloy.The grain size increases from 35-40μm in the billets made by the DCC to about 100-120μm in the billets by the SMC.The aggregation of Mg12Nd usually found at the triple joints of grain boundaries in the billets prepared by SMC while is not observable from the billets by DCC.The tensile strengths and elongations of the billets are 195.2 MPa and 15.5% by DCC,and 162.5 MPa and 3.2% by SMC,respectively.The tensile strength of the alloy by DCC is remarkably enhanced by T6 heat treatment,which reached 308.5 MPa.Fracture surfaces of NZ30K alloy have been characterized as intergranular fracture by SMC and quasi-cleavage fracture by DCC,respectively.
文摘以栽培大豆(G.max)核心种质经聚类随机选择样本为主,以栽培大豆保留种质和野生大豆(G.soja)为对照,利用小鼠单克隆抗体Gly m Bd 30K(F5)和Gly m Bd 28K(C5)分别检测大豆的30K和28K过敏蛋白抗原,目的是发掘过敏蛋白缺失的品种资源,明确其分布规律和特点,检测大豆核心种质的代表性,为资源和育种研究提供参考。鉴定结果表明,在参试的60份野生大豆和421份栽培大豆中没有发现30K过敏蛋白缺失的种质,但28K过敏蛋白缺失率分别为13.3%和37.8%。栽培大豆核心种质28K过敏蛋白缺失比率的变化趋势与栽培大豆保留种质总体规律基本相同,在3个生态区的缺失比率差异均不显著,说明核心种质在28K过敏蛋白缺失这个性状上具有代表性。SSR标记分析发现,在缺失过敏蛋白的种质中,80%相似系数在0.44以下,表明这些种质遗传基础较为广泛,可充分利用类群内或类群间差异较大的材料作为亲本配制杂交组合,培育缺失28K过敏蛋白优质品种。
