CN106636189A - 一种表达鲑鱼降钙素的方法及其专用表达盒 - Google Patents
一种表达鲑鱼降钙素的方法及其专用表达盒 Download PDFInfo
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- CN106636189A CN106636189A CN201710015217.3A CN201710015217A CN106636189A CN 106636189 A CN106636189 A CN 106636189A CN 201710015217 A CN201710015217 A CN 201710015217A CN 106636189 A CN106636189 A CN 106636189A
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Abstract
本发明公开了一种表达鲑鱼降钙素的方法及其专用表达盒。本发明提供的方法依次包括如下步骤：(1)将重组载体导入受体植物，得到转基因植物；(2)通过培养所述转基因植物得到鲑鱼降钙素；所述重组载体含有表达盒；所述表达盒自上游至下游依次包括如下元件：植物种子特异表达蛋白基因的启动子、融合基因和终止序列；所述融合基因中含有区段乙；所述区段乙编码鲑鱼降钙素的前体；所述鲑鱼降钙素的前体为氨基酸序列是序列表中序列5自N末端起第158至190位所示的蛋白质。实验证明，采用本发明提供的方法可以得到生物学活性较高的鲑鱼降钙素。因此，利用本发明所提供的方法表达鲑鱼降钙素，具有重要的应用价值。
Description
技术领域
本发明涉及生物技术领域，具体涉及一种表达鲑鱼降钙素的方法及其专用表达盒。
背景技术
鲑鱼降钙素是由鲑鱼后腮体分泌的由32个氨基酸组成的单链多肽。鲑鱼降钙素作为治疗骨质疏松症等代谢性骨病的防治药已被载入多国药典，并已在多国行销近30年。鲑鱼降钙素具有调节体内钙、磷代谢及骨骼代谢的重要生物学功能，可降低血钙水平，还具有抗组胺、抗胆碱、抑制胃酸和胰腺分泌的作用。临床上主要用于治疗骨质疏松症。鲑鱼降钙素的生产方法主要有提取法、化学合成法和基因工程法。提取法是以鲑鱼、鳗鱼的鳃为原料，提取获得天然鲑鱼降钙素(生物学活性达到4500U/mg)，但由于原料有限，因此获得的天然鲑鱼降钙素极少。基因工程法是在大肠杆菌或酵母中表达鲑鱼降钙素，但由于鲑鱼降钙素C端第32位氨基酸不能酰胺化，因此表达出的鲑鱼降钙素的生物学活性，仅为100-200U/mg(天然鲑鱼降钙素为4500U/mg)。迄今为止，商业化销售的鲑鱼降钙素都是采用化学合成法生产的，化学合成法步骤多、得率低且价格昂贵，注射针剂中的鲑鱼降钙素为12000元/mg，原药价格也高达18000-20000元/g。
发明内容
本发明所要解决的技术问题是如何表达鲑鱼降钙素。
为解决上述技术问题，本发明首先提供了表达盒。
本发明所提供的表达盒自上游至下游依次可包括如下元件：植物种子特异表达蛋白基因的启动子、融合基因和终止序列；所述融合基因中可含有区段乙；所述区段乙编码鲑鱼降钙素的前体；所述鲑鱼降钙素的前体可为氨基酸序列是序列表中序列5自N末端起第158至190位所示的蛋白质。
所述融合基因中，5’末端为起始密码子，3’末端为终止密码子，中间为连续的编码区。
所述鲑鱼降钙素的前体在植物体内可自发形成鲑鱼降钙素。
所述区段乙可为如下b1)或b2)或b3)或b4)所示的DNA分子：
b1)具有序列表中序列4自5′末端起第34至132位核苷酸所示的DNA分子；
b2)核苷酸序列是序列表中序列4自5′末端起第34至132位所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有85％或85％以上同一性，且编码所述鲑鱼降钙素的前体的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码所述鲑鱼降钙素的前体的DNA分子。
所述融合基因还可包括区段甲；所述区段甲编码筛选标记蛋白。
所述融合基因还可包括一个以上区段丙；所述区段丙编码蛋白标签。所述蛋白标签具体可为6×His标签。
当融合基因中包括2个以上区段丙时，每个区段丙可以编码不同的蛋白标签。
所述融合基因还可包括区段丁；所述区段丁可为切割物质的识别序列。
所述融合基因自上游至下游依次可包括如下区段：所述区段丙、所述区段甲、所述区段丁和所述区段乙。
上述任一所述植物种子特异表达蛋白基因的启动子可为菜豆储存蛋白(GenBank号为X52626.1)基因的启动子。所述菜豆储存蛋白(GenBank号为X52626.1)基因的启动子的核苷酸序列具体可如序列表中序列5自5′末端起第1至1619位所示。
上述任一所述筛选标记蛋白可为GUS蛋白。所述GUS蛋白的核苷酸序列如序列表中序列3自5′末端起第34至1839位所示。
上述任一所述终止序列可为植物种子特异表达蛋白基因的终止子。所述植物种子特异表达蛋白基因的终止子可为菜豆储存蛋白(GenBank号为X52626.1)基因的终止子。所述菜豆储存蛋白(GenBank号为X52626.1)基因的终止子的核苷酸序列具体可如序列表中序列5自5′末端起第3609至4213位所示。
上述任一所述切割物质可为满足如下条件的切割物质：所述切割物质对于所述融合基因的表达产物的酶切位置为特定氨基酸残基与其前一位氨基酸残基之间；所述特定氨基酸残基为所述鲑鱼降钙素的前体的第一个氨基酸残基。
上述任一所述切割物质具体可为甲酸。
上述任一所述区段丁的核苷酸序列可为序列表中序列5自5′末端起第3465至3491位所示。
所述表达盒的核苷酸序列具体可如序列表中序列5所示。
含有上述任一所述表达盒的重组载体也属于本发明的保护范围。
所述重组载体Ⅰ具体可为重组质粒pPha(p/gc/t)2300。重组质粒pPha(p/gc/t)2300为将载体pCAMBIA2300的限制性内切酶SacⅠ和HindⅢ识别序列间的小片段替换为序列表中序列5所示的DNA分子，得到的重组质粒。重组质粒pPha(p/gc/t)2300表达序列表中序列6所示的蛋白质。
所述重组载体Ⅰ具体可为重组质粒pPha(p/gc/t)2300-DZ。重组质粒pPha(p/gc/t)2300和重组质粒pPha(p/gc/t)2300-DZ的唯一不同在于：重组质粒pPha(p/gc/t)2300的鲑鱼降钙素的前体的编码基因(即sCT基因)为序列表中序列4自5′末端起第34至132位所示，重组质粒pPha(p/gc/t)2300-DZ的鲑鱼降钙素的前体的编码基因(即sCT-DZ基因)为序列表中序列8自5′末端起第34至132位所示。重组质粒pPha(p/gc/t)2300-DZ表达序列表中序列6所示的蛋白质。
为解决上述技术问题，本发明还提供了表达鲑鱼降钙素的方法。
本发明所提供的表达鲑鱼降钙素的方法，依次可包括如下步骤：
(1)将上述任一所述重组载体导入受体植物，得到转基因植物；
(2)通过培养所述转基因植物得到鲑鱼降钙素。
上述方法中，所述鲑鱼降钙素存在或主要存在于转基因植物的种子中。
上述方法中，所述受体植物可为油菜品种中双4号。
上述方法中，所述“培养所述转基因植物得到鲑鱼降钙素”还依次可包括如下步骤：
(4)取转基因植物的种子，脱脂后提取粗蛋白；
(5)利用所述蛋白标签从所述粗蛋白中纯化所述融合蛋白；
(6)取步骤(5)得到的融合蛋白，用切割物质进行切割；
(7)从步骤(6)的产物中分离鲑鱼降钙素；
(8)醋酸化；
(9)脱盐。
上述方法中，所述步骤(4)中，所述“脱脂”的步骤可为：先粉碎转基因植物的种子，然后用正己烷脱脂。所述粉碎的程度可为200目。
上述方法中，所述步骤(4)中，所述“提取粗蛋白”可为用含8-12mg/mL蔗糖和1.5-2.5mg/mL SDS的pH6.5-7.0、0.1-0.3M Tris-Hcl缓冲液进行提取。
上述方法中，所述步骤(4)中，所述“提取粗蛋白”具体可为用含10mg/mL蔗糖和2.0mg/mL SDS的pH6.8、0.2M Tris-Hcl缓冲液进行提取。
上述方法中，所述步骤(5)中，所述蛋白标签可为6×His标签。
上述方法中，所述步骤(6)中，所述切割物质可为甲酸。
上述方法中，所述步骤(7)中，所述“分离鲑鱼降钙素”可采用阳离子交换层析进行分离。
上述方法中，所述步骤(8)中，所述醋酸化的步骤可为：调节步骤(7)分离的鲑鱼降钙素的pH值至1.0-3.0，然后加入醋酸钠处理40-80min。
上述方法中，所述步骤(8)中，所述醋酸化的步骤具体可为：取步骤(7)分离的鲑鱼降钙素，用磷酸调节pH值至2.0，得到溶液1；取1体积份溶液1，加入3体积份浓度为333mM的醋酸钠水溶液，得到溶液2；取所述溶液2，室温放置60min。
上述方法中，所述步骤(9)中，所述脱盐可为反相色谱脱盐。
上述方法中，完成步骤(9)后，还可包括冻干的步骤。
实验证明，将重组质粒pPha(p/gc/t)2300或重组质粒pPha(p/gc/t)2300-DZ导入油菜品种中双4号，得到转基因植物，然后培养该转基因植物，进一步得到生物学活性较高的鲑鱼降钙素。可见，利用本发明所提供的方法可以表达鲑鱼降钙素，具有重要的应用价值。
附图说明
图1为实施例1步骤二8中(1)的实验结果。
图2为实施例1步骤二8中(2)的实验结果。
图3为实施例1中步骤三的实验结果。
图4为实施例1中步骤四的实验结果。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。
下述实施例中的实验方法，如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
油菜品种中双4号记载于如下文献中：罗晓辉，叶明海.“中双4号”育苗移栽高产栽培[J].上海农业科技，1999年05期.在下文中，油菜品种中双4号简称中双4号。
下述实施例中的光暗交替培养(即光照培养和暗培养交替)的条件为：25℃。光照培养时的光照强度为15000Lx。光暗交替培养的周期具体为：14h光照培养/10h黑暗培养。
MS0培养基：将NH4NO3 1650mg、KNO3 1900mg、KH2PO4 170mg、MgSO4·7H2O 370mg、CaCl2·2H2O 440mg、FeSO4·7H2O 27.80mg、Na2EDTA 37.30mg、MnSO4·4H2O 22.30mg、ZnSO4·7H2O 8.60mg、H3BO3 6.20mg、KI 0.83mg、Na2MOO4·2H2O 0.25mg、CuSO4·5H2O0.025mg、COCl2·6H2O 0.025mg、肌醇100.00mg、VB10.1mg、VB60.5mg、烟酸0.5mg、甘氨酸2.0mg和琼脂7g溶于1L蒸馏水，调节pH值至5.8。
载体pUC57为百奥迈科生物技术有限公司的产品，产品目录号为BK0033。λDNA/EcoRI+HindⅢ Marker为北京康润生物科技有限公司的产品，产品目录号为M125-01。粉碎机为绍兴市科宏仪器有限公司的产品，产品型号为EW-100。鲑鱼降钙素标准品为北京华大蛋白质研发中心有限公司合成。密钙息为Novartis Pharma Stein AG公司的产品。载体pCAMBIA2301为华越洋生物(北京)科技有限公司的产品，产品目录号为VECT0310。
菜豆储存蛋白的GenBank号为X52626.1。
实施例1、利用转基因油菜种子表达系统表达鲑鱼降钙素
一、重组质粒pPha(p/gc/t)2300的构建
1、人工合成序列表中序列1所示的双链DNA分子。序列表中序列1中，自5′末端起第1至6位为限制性内切酶SacⅠ的识别序列，第7至1625位为菜豆储存蛋白的启动子的核苷酸序列，第1626至1631位为限制性内切酶KpnⅠ的识别序列。
2、用限制性内切酶SacⅠ和KpnⅠ双酶切步骤1合成的双链DNA分子，回收1631bp的DNA片段甲。
3、用限制性内切酶SacⅠ和KpnⅠ双酶切载体pCAMBIA2301，回收约11.6bp的载体骨架甲。
4、将DNA片段甲和载体骨架甲进行连接，得到中间载体甲。
5、人工合成序列表中序列2所示的双链DNA分子。序列表中序列2中，自5′末端起第1至6位为限制性内切酶PstI的识别序列，第7至611位为菜豆储存蛋白的终止子的核苷酸序列，第612至617位为限制性内切酶HindⅢ的识别序列。
6、用限制性内切酶PstI和HindⅢ双酶切步骤5合成的双链DNA分子，回收617bp的DNA片段乙。
7、用限制性内切酶PstI和HindⅢ双酶切中间载体甲，回收约13.2Kb的载体骨架乙。
8、将DNA片段乙和载体骨架乙进行连接，得到中间载体乙。
9、人工合成序列表中序列3所示的双链DNA分子。序列表中序列3中，自5′末端起第1至6位为限制性内切酶KpnⅠ的识别序列，第7至9位为起始密码子ATG(编码甲硫氨酸)，第10至33位为编码8×His标签的核苷酸序列，第34至1839位为编码GUS蛋白的核苷酸序列(已按油菜偏爱密码子对GUS蛋白的核苷酸序列进行优化)，第1840至1845位为限制性内切酶BamHI的识别序列。
10、用限制性内切酶Kpn Ⅰ和BamHI双酶切步骤9合成的双链DNA分子，回收1845bp的DNA片段丙。
11、用限制性内切酶Kpn Ⅰ和BamHI双酶切中间载体乙，回收约13.8kb的载体骨架丙。
12、将DNA片段丙和载体骨架丙进行连接，得到中间载体丙。
13、人工合成序列表中序列4所示的双链DNA分子。序列表中序列4中，自5′末端起第1至6位为限制性内切酶BamHⅠ的识别序列，第7至33位为甲酸切割的识别序列，第34至132位为鲑鱼降钙素的前体的编码基因(以下简称sCT基因)，第133至144位为四个终止密码子，第145至150位为限制性内切酶PstI的识别序列。
14、将步骤13合成的双链DNA分子连接至载体pUC57，得到重组质粒psCT。
15、用限制性内切酶BamHⅠ和PstI双酶切重组质粒psCT，回收150bp的DNA片段丁。
16、用限制性内切酶BamHⅠ和PstI双酶切中间载体丙，回收约15.6kb的载体骨架丁。
17、将DNA片段丁和载体骨架丁进行连接，得到重组质粒pPha(p/gc/t)2300。
根据测序结果，对重组质粒pPha(p/gc/t)2300进行结构描述如下：将载体pCAMBIA2300的限制性内切酶SacⅠ和HindⅢ识别序列间的小片段替换为序列表中序列5所示的DNA分子。重组质粒pPha(p/gc/t)2300表达序列表中序列6所示的融合蛋白甲。
序列表中序列5中，自5′末端起第1至1619位菜豆储存蛋白的启动子的核苷酸序列，第1620至1625位为限制性内切酶KpnⅠ的识别序列，第1626至1628位为起始密码子ATG(编码甲硫氨酸)，第1629至1652位为编码8×His标签的核苷酸序列，第1653至3458位为编码GUS蛋白的核苷酸序列，第3459至3464位为限制性内切酶BamHI的识别序列，第3465至3491位为甲酸的识别序列，第3492至3590位为鲑鱼降钙素的前体的编码基因(以下简称sCT基因)，第3591至3602位为四个终止密码子，第3603至3608位为限制性内切酶PstI的识别序列，第3609至4213位为菜豆储存蛋白的终止子的核苷酸序列。
序列表中序列6中，自N末端起第1位为甲硫氨酸，第2至9位为8×His标签，第10至611位为GUS蛋白，第614至622位为甲酸的识别位点，第623至655位为鲑鱼降钙素的前体。
二、油菜遗传转化
1、农杆菌侵染液的制备
(1)采用电击法将重组质粒pPha(p/gc/t)2300导入根癌农杆菌LBA4404，得到重组农杆菌，命名为LBA4404/pPha(p/gc/t)2300。
(2)将LBA4404/pPha(p/gc/t)2300的单克隆接种至5mL含100mg/L卡那霉素(Kanamycin，Kan)和50mg/L利福平(Rifampicin，Rif)的YEB液体培养基，28℃、200rpm振荡培养12h，得到培养菌液1。
(3)将1mL培养菌液1接种至50mL含100mg/L Kan和50mg/L Rif的YEB液体培养基，28℃、200rpm振荡培养至OD600nm值为0.3～0.4，得到培养菌液2。
(4)取步骤(3)得到的培养菌液2，4℃、5000rpm离心5min，收集菌体1。
(5)取步骤(4)收集的菌体1，用含1.0mg/L 6-BA的MS0培养基重悬，然后4℃、5000rpm离心5min，收集菌体2。
(6)取步骤(5)收集的菌体2，用含100mg/L乙酰丁香酮(Acetosyringone，As)的MS0培养基重悬，得到农杆菌侵染液。以含100mg/L As的MS0培养基为参照，调整农杆菌侵染液的OD600nm值至0.15～0.25。
2、中双4号的子叶的获得
取中双4号的种子，先用75％(v/v)的乙醇水溶液浸泡2min，然后用次氯酸钠水溶液(由1体积份次氯酸钠溶液(有效氯≥10.0)和4体积份水混合而成)浸泡10min，最后用无菌水反复冲洗，沥干后接种于MS0培养基上，光暗交替培养5d，得到中双4号的材料。
取中双4号的材料，剪取子叶(尽量靠近生长点)，得到中双4号的子叶。
3、侵染
取步骤2获得的中双4号的子叶，置于离心管，然后加入农杆菌侵染液(OD600nm值为0.2)浸泡10min。
4、共培养
完成步骤3后，将所述中双4号的子叶转移至共培养培养基上(子叶需正面朝上)共培养4d。
共培养培养基：含1.25mg/L NAA、5.0mg/L 6-BA和5.0mg/L AgNO3的MS0培养基。
共培养条件：25℃，暗培养。
5、选择培养
完成步骤4后，将所述中双4号的子叶转移至选择培养基上(子叶需正面朝上)，光暗交替培养4d。
选择培养基：含1.0mg/L NAA、3.0mg/L 6-BA和100mg/L头孢噻吩(cefalotin，Cf)的MS0培养基。
6、分化培养
完成步骤5后，将所述中双4号的子叶转移至分化培养基上(子叶需正面朝上)，光暗交替培养4周，得到长约1cm的分化芽。
分化培养基：含0.2mg/L NAA、2.0mg/L 6-BA、40mg/L Kan、100mg/L Cf的MS0培养基。
7、生根培养
完成步骤6后，将所述分化芽转移至生根培养基上，光暗交替培养2周，获得拟转sCT基因油菜。
生根培养基：含100mg/L Kan和100mg/L Cf的MS0培养基。
随机选择7株拟转sCT基因油菜，依次命名为GCt1至GCt7。
8、分子检测
(1)PCR检测
分别提取步骤7获得的7株拟转sCT基因油菜叶片的基因组DNA并作为模板，以GC5：5'-ACCGATACCATCAGCGATC-3'和GC3：5'-GTTCCAGATCCGGTGTTAG-3'为引物进行PCR扩增，得到PCR扩增产物。用等体积超纯水代替拟转sCT基因油菜的叶片的基因组DNA，作为阴性对照。用等体积重组质粒pPha(p/gc/t)2300的DNA代替拟转sCT基因油菜的叶片的基因组DNA，作为阳性对照。
PCR反应程序为：94℃预变性5min；94℃变性30s，55℃退火1min，72℃延伸1min，35个循环；72℃延伸10min，4℃保持。
PCR扩增反应完成后，取10μL PCR扩增产物进行1％琼脂糖凝胶电泳。实验结果见图1(泳道1为λDNA/EcoRI+HindⅢMarker，泳道2为GCt1，泳道3为GCt2，泳道4为GCt3，泳道5为GCt4，泳道6为GCt5，泳道7为GCt6，泳道8为GCt7)。结果表明，如果以拟转sCT基因油菜的叶片的基因组DNA为模板，能够扩增出约570bp的条带，则该拟转sCT基因油菜即为转sCT基因油菜；阴性对照不能获得570bp的条带；阳性对照可以获得570bp的条带。
(2)RT-PCR检测
菜豆储存蛋白是种子中特异表达的蛋白，一般在开花后4-6周的种子中开始转录，9-11周的种子中mRNA的含量达到最高水平，种子的成熟后期的mRNA含量急剧降低。
(a)提取待测油菜(GCt1、GCt2、GCt3、GCt4、GCt5、GCt6或GCt7)开花后9-11周的种子的总RNA，然后该总RNA用M-MLV反转录出第一链cDNA，得到待测油菜的cDNA。待测油菜的cDNA中，DNA浓度为500ng/μL。
(b)以步骤(a)得到的待测油菜的cDNA为模板，以GC5：5'-ACCGATACCATCAGCGATC-3'和GC3：5'-GTTCCAGATCCGGTGTTAG-3'为引物进行RT-PCR扩增，得到PCR扩增产物。用等体积超纯水代替待测油菜的cDNA，作为阴性对照。
PCR反应程序为：94℃预变性5min；94℃变性30s，55℃退火1min，72℃延伸1min，35个循环；72℃延伸10min，4℃保持。
PCR扩增反应完成后，取10μL PCR扩增产物进行1％琼脂糖凝胶电泳。实验结果见图2(泳道1为λDNA/EcoRI+HindⅢMarker，泳道2为GCt1，泳道3为GCt2，泳道4为GCt3，泳道5为GCt4，泳道6为GCt5，泳道7为GCt6)。结果表明，如果以拟转sCT基因油菜的cDNA为模板，能够扩增出约570bp的条带，则该拟转sCT基因油菜即为转sCT基因油菜，说明sCT基因已经整合到中双4号的基因组，并转录表达；阴性对照不能获得570bp的条带。
PCR检测和RT-PCR检测的结果完全一致。因此，GCt1、GCt2、GCt3、GCt4、GCt5、GCt6和GCt7均为转sCT基因油菜。
待GCt1成熟后收集种子，然后进行繁育和PCR检测，直至获得纯合种子。将该纯合种子命名为GCt1种子，进行后续实验。
三、鲑鱼降钙素的提取和纯化
1、脱脂
(1)取步骤二收集的GCt1种子，用粉碎机(粉碎程度200目)粉碎，得到油菜籽粉。
(2)将油菜籽粉和正己烷按照1:10(1g/10mL)的比例混匀，得到混合物甲；将混合物甲置于密封玻璃的容器中室温搅拌8h，然后弃上清，收集残渣甲。
(3)将残渣甲和正己烷按照1:10(1g/10mL)的比例混匀，得到混合物乙；将混合物乙置于密封玻璃的容器中室温搅拌8h，然后弃上清，收集残渣乙。
(4)将残渣乙和正己烷按照1:10(1g/10mL)的比例混匀，得到混合物丙；将混合物丙置于密封玻璃的容器中室温搅拌8h，然后弃上清，收集残渣丙。
(5)将残渣丙冷冻风干，即为脱脂的油菜籽粉。
2、提取
(1)取离心管(规格为50mL)，加入100mg脱脂的油菜籽粉和20mL含10％(10mg/mL)蔗糖和2％(2mg/mL)SDS的pH6.8、0.2M Tris-Hcl缓冲液，充分混匀，4℃搅拌9h。
(2)完成步骤(1)后，取所述离心管，4℃、20000rpm离心60min。收集上清液。上清液即为GCt1种子蛋白的粗提物。
3、His-tag初步纯化
镍离子螯合磁珠的商品名称为BeaverBeadsTM IDA-Nickel，具体为苏州海狸生物医学工程有限公司的产品，产品编号为70501-5。
Buffer A为含500mM NaCl和10mM咪唑的pH7.4、20mM磷酸钠缓冲液。
Buffer B为含500mM NaCl和50mM咪唑的pH7.4、20mM磷酸钠缓冲液。
Buffer C为含500mM NaCl和500mM咪唑的pH7.4、20mM磷酸钠缓冲液。
(1)取10mL步骤2收集的上清液和10mL Buffer A，充分混匀；然后加入2mL磁珠悬浮液，充分悬浮后置于旋转混合仪上温育30min；最后通过磁性分离，将镍离子螯合磁珠转移到新的离心管中。
磁珠悬浮液的制备方法为：取2mL镍离子螯合磁珠，加入5mL Buffer A，用移液器轻轻吹打数次得到的悬浮液。
(2)完成步骤(1)后，取装有镍离子螯合磁珠的离心管，加入10mL Buffer B，用移液器轻轻吹打数次，然后通过磁性分离，将镍离子螯合磁珠转移到新的离心管中。
(3)重复步骤(2)两次。
(4)完成步骤(3)后，取装有镍离子螯合磁珠的离心管，加入5mL Buffer C，用移液器轻轻吹打数次，然后通过磁性分离，收集上清液。
(5)将步骤(4)收集的上清液利用分子量为10kD MW的超滤管进行超滤浓缩，获得融合蛋白乙的浓缩液。
鲑鱼降钙素主要用于治疗骨质疏松症。通过基因工程法生产鲑鱼降钙素，主要是在大肠杆菌或酵母中表达鲑鱼降钙素，但由于鲑鱼降钙素C端第32位氨基酸不能酰胺化，因此表达出的鲑鱼降钙素的生物学活性仅为100-200U/mg；而天然的鲑鱼降钙素的生物学活性为4500U/mg。已有研究结果表明，鲑鱼降钙素的前体(氨基酸序列如序列表中序列6自N末端起第623至655位所示)通过植物自身的酰胺化系统，可转化为鲑鱼降钙素。
融合蛋白乙氨基酸序列如序列表中序列7所示，且其C末端酰胺化。融合蛋白乙在转sCT基因油菜体内经过如下转化得到：转sCT基因油菜中首先表达融合蛋白甲，之后通过自身的酰胺化系统，将融合蛋白甲转化为C末端酰胺化的融合蛋白乙。
4、甲酸切割
(1)取融合蛋白乙的浓缩液，加入无菌水和甲酸，得到反应体系；反应体系中，融合蛋白乙的浓度为1mg/mL，甲酸的浓度为37％(v/v)。
(2)完成步骤(1)后，取所述反应体系，45℃切割2.5h。
5、阳离子交换纯化
将完成步骤4的体系在AKTA蛋白纯化系统上经阳离子交换层析纯化，得到鲑鱼降钙素溶液。具体步骤如下：
(1)将1体积份完成步骤4的体系和3体积份pH3.0、10mM柠檬酸钠缓冲液混合，得到混合液(电导率为7mS/cm)；然后将混合液用0.22μM的滤膜过滤，得到上样液。
(2)取SP-Sepharose fast flow(5×1mL)(美国GE公司的产品)，先用10个柱体积的pH3.0、10mM柠檬酸钠缓冲液预平衡(流速为1mL/min)。
(3)完成步骤(2)后，取步骤(1)得到的上样液，进行上样(流速为0.5mL/min)。
(4)完成步骤(3)后，用10个柱体积的pH3.0、10mM柠檬酸钠缓冲液洗脱(流速为1mL/min)至紫外检测基线平稳(检测波长为220nm)。
(5)完成步骤(4)后，用含400mM NaCl的pH3.0、10mM柠檬酸钠缓冲液进行洗脱(流速为0.5mL/min)，收集洗脱峰约5-10mL的洗脱溶液。
6、醋酸化
(1)取步骤5收集的洗脱溶液，先加入磷酸调节pH值至2.0，得到溶液1。
(2)取1体积份溶液1，加入3体积份浓度为333mM的醋酸钠水溶液，混合均匀，得到溶液2。
(3)取所述溶液2，室温放置60min，得到溶液3。
7、反相色谱脱盐及冻干
Amberchrom CG300md resin为北京慧德易科技有限责任公司的产品。
(1)取Amberchrom CG300md resin，用10个柱体积的0.1％(v/v)醋酸水溶液洗脱(流速为5mL/min)至电导率基线平稳。
(2)完成步骤(1)后，用10个柱体积的浓度为250mM醋酸钠水溶液洗脱(流速为5mL/min)至pH值基线平稳。
(3)完成步骤(2)后，取步骤6得到的溶液3，进行上样(流速2mL/min)。
(4)完成步骤(3)后，用10个柱体积的浓度为250mM醋酸钠水溶液洗脱(流速为5mL/min)至pH值基线平稳。
(5)完成步骤(4)后，用10个柱体积的0.1％(v/v)醋酸水溶液洗脱(流速为5mL/min)至电导率基线平稳。
(6)完成步骤(5)后，用40％(v/v)乙醇水溶液洗脱(流速为2mL/min)，收集洗脱峰约5-10mL的洗脱溶液。
(7)用冷冻干燥机将步骤(6)收集的洗脱溶液冻干至粉末，即鲑鱼降钙素粉末。-20℃备用。
取1mg鲑鱼降钙素粉末，加入1mL超纯水溶解，得到鲑鱼降钙素溶液。
将鲑鱼降钙素溶液和浓度为1mg/mL鲑鱼降钙素标准品水溶液进行SDS-PAGE。
实验结果见图3(1为蛋白Marker，2为鲑鱼降钙素溶液，3为鲑鱼降钙素标准品水溶液)。结果表明，鲑鱼降钙素溶液中含有鲑鱼降钙素，纯度约90％。每10g步骤二收集的GCt1种子产生0.2mg鲑鱼降钙素(以100％纯度计)。
四、鲑鱼降钙素的HPLC检测
使用配有Agilent Zorbax SB C18柱(5.0μm，150mm×2.1mm)的Agilent 1200液相色谱仪分析步骤三中的鲑鱼降钙素溶液和浓度为1mg/mL的鲑鱼降钙素标准品水溶液。进样量20μL。流动相由0.1％(v/v)三氟乙酸(trifluoroacetic acid，TFA)水溶液(A液)和0.1％(v/v)TFA乙腈溶液(B液)组成，流速为0.2mL/min，使用流动相中B液递增、A液递减的梯度洗脱条件进行梯度洗脱，具体如下(％均为体积百分比)：0-8min，流动相中B液的体积百分含量由10％匀速升至30％；8-50min，流动相中B液的体积百分含量由30％匀速升至55％；50-55min，流动相中B液的体积百分含量由55％匀速升至100％。检测波长为210nm。
实验结果见图4(A为鲑鱼降钙素标准品水溶液，B为步骤三中的鲑鱼降钙素溶液)：步骤三中的鲑鱼降钙素溶液与鲑鱼降钙素标准品水溶液的目的峰出峰时间基本一致(见图4中箭头标注，出峰时间为5.8min)。结果表明，步骤三中的鲑鱼降钙素溶液中含有鲑鱼降钙素。
五、检测鲑鱼降钙素的生物学活性
Vr:CD1(ICR)小鼠为北京维通利华实验动物技术有限公司的产品。钙测定试剂盒为北京中生北控生物科技股份有限公司的产品。
1、取30只体重为24-26g的健康Vr:CD1(ICR)小鼠，随机分成鲑鱼降钙素组、密钙息组和生理盐水组(每组10只，雌雄各半)，禁食10-12h(仅给蒸馏水)后，分别进行如下处理：
鲑鱼降钙素组：尾静脉注射步骤三中的鲑鱼降钙素溶液(注射剂量为0.1mL/只)；
密钙息组：尾静脉注射密钙息(注射剂量为0.5IU/只)；
生理盐水组：尾静脉注射0.9％(0.9mg/100mL)生理盐水(注射剂量为0.1mL/只)。
2、完成步骤1 60min后，每只小鼠先腹腔注射10％(10mg/100mL)水合氯醛水溶液(注射剂量为0.15mL/只)，再分别从眼眶取血，室温静置2h；最后3000g离心15min，上清液即为血清。
3、完成步骤2后，取血清，按照钙测定试剂盒的操作步骤，用全自动生化分析测定每只小鼠的血钙浓度。
实验结果见表1。结果表明，密钙息组小鼠的血钙浓度比生理盐水组降低26.09％，鲑鱼降钙素组小鼠的血钙浓度比生理盐水组降低25.45％，方差分析差异极显著。因此，使用步骤一至步骤三的方法获得的鲑鱼降钙素溶液具有降低血钙的生物学活性。
表1
实施例2、利用转基因油菜种子表达系统表达鲑鱼降钙素
按照实施例1步骤一至步骤三的方法，将步骤一中的重组质粒pPha(p/gc/t)2300替换为重组质粒pPha(p/gc/t)2300-DZ，其它步骤均不变，得到鲑鱼降钙素溶液；每10g油菜种子产生0.15mg鲑鱼降钙素(以100％纯度计)。
重组质粒pPha(p/gc/t)2300-DZ的构建方法为：按照实施例1步骤一的方法，将步骤13中“人工合成序列表中序列4所示的双链DNA分子”替换为“人工合成序列表中序列8所示的双链DNA分子”，其它步骤均不变，得到重组质粒pPha(p/gc/t)2300-DZ。重组质粒pPha(p/gc/t)2300-DZ也表达序列表中序列6所示的融合蛋白甲。
序列表中序列8中，自5′末端起第1至6位为限制性内切酶BamHⅠ的识别序列，第7至33位为甲酸的识别序列，第34至132位为鲑鱼降钙素的前体的编码基因(以下简称sCT-DZ基因)，第133至144位为四个终止密码子，第145至150位为限制性内切酶PstI的识别序列。重组质粒pPha(p/gc/t)2300-DZ和重组质粒pPha(p/gc/t)2300的唯一不同在于：重组质粒pPha(p/gc/t)2300的鲑鱼降钙素的前体的编码基因(即sCT基因)为序列表中序列4自5′末端起第34至132位所示，重组质粒pPha(p/gc/t)2300-DZ的鲑鱼降钙素的前体的编码基因(即sCT-DZ基因)为序列表中序列8自5′末端起第34至132位所示。
<110> 中国农业科学院生物技术研究所
<120> 一种表达鲑鱼降钙素的方法及其专用表达盒
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gagctcgaat tcattgtact cccagtatca ttatagtgaa agttttggct ctctcgccgg 60
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ttacttgtta ctttaatttc tcataatctt tggttgaaat tatcacgctt ccgcacacga 180
tatccctaca aatttattat ttgttaaaca ttttcaaacc gcataaaatt ttatgaagtc 240
ccgtctatct ttaatgtagt ctaacatttt catattgaaa tatataattt acttaatttt 300
agcgttggta gaaagcataa agatttattc ttattcttct tcatataaat gtttaatata 360
caatataaac aaattcttta ccttaagaag gatttcccat tttatatttt aaaaatatat 420
ttatcaaata tttttcaacc acgtaaatct cataataata agttgtttca aaagtaataa 480
aatttaactc cataattttt ttattcgact gatcttaaag caacacccag tgacacaact 540
agccattttt ttctttgaat aaaaaaatcc aattatcatt gtattttttt tatacaatga 600
aaatttcacc aaacaatcat ttgtggtatt tctgaagcaa gtcatgttat gcaaaattct 660
ataattccca tttgacacta cggaagtaac tgaagatctg cttttacatg cgagacacat 720
cttctaaagt aattttaata atagttacta tattcaagat ttcatatatc aaatactcaa 780
tattacttct aaaaaattaa ttagatataa ttaaaatatt acttttttaa ttttaagttt 840
aattgttgaa tttgtgacta ttgatttatt attctactat gtttaaattg ttttatagat 900
agtttaaagt aaatataagt aatgtagtag agtgttagag tgttacccta aaccataaac 960
tataacattt atggtggact aattttcata tatttcttat tgcttttacc ttttcttggt 1020
atgtaagtcc gtaactagaa ttacagtggg ttgccatggc actctgtggt cttttggttc 1080
atgcatgggt cttgcgcaag aaaaagacaa agaacaaaga aaaaagacaa aacagagaga 1140
caaaacgcaa tcacacaacc aactcaaatt agtcactggc tgatcaagat cgccgcgtcc 1200
atgtatgtct aaatgccatg caaagcaaca cgtgcttaac atgcacttta aatggctcac 1260
ccatctcaac ccacacacaa acacattgcc tttttcttca tcatcaccac aaccacctgt 1320
atatattcat tctcttccgc cacctcaatt tcttcacttc aacacacgtc aacctgcata 1380
tgcgtgtcat cccatgccca aatctccatg catgttccaa ccaccttctc tcttatataa 1440
tacctataaa tacctctaat atcactcact tctttcatca tccatccatc cagagtacta 1500
ctactctact actataatac cccaacccaa ctcatattca atactactct actatgatga 1560
gagcaagggt tccactcctg ttgctgggaa ttcttttcct ggcatcactt tctgcctcat 1620
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ctgcagataa gtatgaacta aaatgcatgt atggtgtaag agcacatgga gagcatggaa 60
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aaacaaagga tgttatgata tattaacact atatgcacct tcacaagtaa tacattaata 180
tttaatactt tttattttaa ctttttagtt taaaatatta ttatattatt aactttttag 240
tttaaaatat ttatattatt ataaagagaa ataaacaaag gatgttatga tatattaaca 300
ctatatgtac cttacatagt aatatattaa tatttaatac tttttatttt aactttttaa 360
tttaaaatat tattataaat gacgcttgtg ttttatgtgt tggcatgctt gtattttatg 420
tgttgacttt ctgtgtgaag gtaatgtgat atggtgagct ggtggtaaca attgtgtttt 480
atgtgttggc tttctgtgaa gctaatttga tatggttagc tgatgtgaac aaaatattaa 540
aggaagctaa tttgatatgg ttagccgata gtaacaaaat atcaaaataa atttcttctt 600
actttaataa aaagctt 617
<210>3
<211>1845
<212>DNA
<213>人工序列
<220>
<223>
<400>3
ggtaccatgc atcatcacca ccatcaccat cacttacgtc ctgtagaaac cccaacccgt 60
gaaatcaaaa aactcgacgg cctgtgggca ttcagtctgg atcgcgaaaa ctgtggaatt 120
gatcagcgtt ggtgggaaag cgcgttacaa gaaagccggg caattgctgt gccaggcagt 180
tttaacgatc agttcgccga tgcagatatt cgtaattatg cgggcaacgt ctggtatcag 240
cgcgaagtct ttataccgaa aggttgggca ggccagcgta tcgtgctgcg tttcgatgcg 300
gtcactcatt acggcaaagt gtgggtcaat aatcaggaag tgatcgagca tcagggcggc 360
tatacgccat ttgaagccga tgtcacgccg tatgttattg ccgggaaaag tgtacgtatc 420
accgtttgtg tgaacaacga actgaactgg cagactatcc cgccgggagc tgtgattacc 480
gacgaaaacg gcaagaaaaa gcagtcttac ttccatgatt tctttaacta tgccggaatc 540
catcgcagcg taatcctcta caccacgccg aacacctggg tggacgatat caccgtggtg 600
acgcatgtcg cgcaagactg taaccacgcg tctgttgact ggcaggtggt ggccaatggt 660
gatgtcagcg ttgaactgcg tgatgcggat caacaggtgg ttgcaactgg acaaggcact 720
agcgggactt tgcaagtggt gaatccgcac ctctggcaac cgggtgaagg ttatctctat 780
gaactgtgcg tcacagccaa aagccagaca gagtgtgata tctacccgct tcgcgtcggc 840
atccggtcag tggcagtgaa gggcgaacag ttcctgatta accacaaacc gttctacttt 900
actggctttg gtcgtcatga agatgcggac ttgcgtggca aaggattcga taacgtgctg 960
attgtgcacg accacgcatt agctgactgg attggggcca actcctaccg tacctcgcat 1020
tacccttacg ctgaagagat cctcgactgg gcagatgaac atggcatcgt ggtgattgat 1080
gaaactgctg ctgtcggctt taacctctct ttaggcattg gtttcgaagc gggcaacaag 1140
ccgaaagaac tgtacagcga agaggcagtc aacggggaaa ctcagcaagc gcacttacag 1200
gcgattaaag agctgatagc gcgtgacaaa aaccacccaa gcgtggtgat ttggagtatt 1260
gccaacgaac cggatacccg tccgcaaggt gcacgggaat atttcgcgcc actggcggaa 1320
gcaacgcgta aactcgaccc gacgcgtccg atcacctgcg tcaatgtagc attctgcgac 1380
gctcacaccg ataccatcag cgatctcttt gatgtgctgt gcctgaaccg ttattacgga 1440
tggtatgtcc aaagcggcga tttggaaacg gcagagaagg tactggaaaa agaacttctg 1500
gcctggcagg agaaactgca tcagccgatt atcatcaccg aatacggcgt ggatacgtta 1560
gccgggctgc actcagcata caccgacatt tggagtgaag agtatcagtg tgcatggctg 1620
gatacctatc accgcgtctt tgatcgcgtc agcgccgtcg tcggtgaaca ggtatggaat 1680
ttcgccgatt ttgcgacctc gcaaggcata ttgcgcgttg gcggtaacaa gaaagggatc 1740
ttcactcgcg accgcaaacc gaagtcggcg gcttttctgc tgcaaaaacg ctggactggc 1800
ataaacttcg gtgaaaaacc gcagcaggga ggcaaacaag gatcc 1845
<210>4
<211>150
<212>DNA
<213>人工序列
<220>
<223>
<400>4
ggatccgacc cacctgatcc acctgatcca atgtgctcta acctttctac ttgcgttctt 60
ggaaagttgt ctcaagagct tcataaactt caaacttacc caagaactaa caccggatct 120
ggaactccag gataatgata atgactgcag 150
<210>5
<211>4213
<212>DNA
<213>人工序列
<220>
<223>
<400>5
gaattcattg tactcccagt atcattatag tgaaagtttt ggctctctcg ccggtggttt 60
tttacctcta tttaaagggg ttttccacct aaaaattctg gtatcattct cactttactt 120
gttactttaa tttctcataa tctttggttg aaattatcac gcttccgcac acgatatccc 180
tacaaattta ttatttgtta aacattttca aaccgcataa aattttatga agtcccgtct 240
atctttaatg tagtctaaca ttttcatatt gaaatatata atttacttaa ttttagcgtt 300
ggtagaaagc ataaagattt attcttattc ttcttcatat aaatgtttaa tatacaatat 360
aaacaaattc tttaccttaa gaaggatttc ccattttata ttttaaaaat atatttatca 420
aatatttttc aaccacgtaa atctcataat aataagttgt ttcaaaagta ataaaattta 480
actccataat ttttttattc gactgatctt aaagcaacac ccagtgacac aactagccat 540
ttttttcttt gaataaaaaa atccaattat cattgtattt tttttataca atgaaaattt 600
caccaaacaa tcatttgtgg tatttctgaa gcaagtcatg ttatgcaaaa ttctataatt 660
cccatttgac actacggaag taactgaaga tctgctttta catgcgagac acatcttcta 720
aagtaatttt aataatagtt actatattca agatttcata tatcaaatac tcaatattac 780
ttctaaaaaa ttaattagat ataattaaaa tattactttt ttaattttaa gtttaattgt 840
tgaatttgtg actattgatt tattattcta ctatgtttaa attgttttat agatagttta 900
aagtaaatat aagtaatgta gtagagtgtt agagtgttac cctaaaccat aaactataac 960
atttatggtg gactaatttt catatatttc ttattgcttt taccttttct tggtatgtaa 1020
gtccgtaact agaattacag tgggttgcca tggcactctg tggtcttttg gttcatgcat 1080
gggtcttgcg caagaaaaag acaaagaaca aagaaaaaag acaaaacaga gagacaaaac 1140
gcaatcacac aaccaactca aattagtcac tggctgatca agatcgccgc gtccatgtat 1200
gtctaaatgc catgcaaagc aacacgtgct taacatgcac tttaaatggc tcacccatct 1260
caacccacac acaaacacat tgcctttttc ttcatcatca ccacaaccac ctgtatatat 1320
tcattctctt ccgccacctc aatttcttca cttcaacaca cgtcaacctg catatgcgtg 1380
tcatcccatg cccaaatctc catgcatgtt ccaaccacct tctctcttat ataataccta 1440
taaatacctc taatatcact cacttctttc atcatccatc catccagagt actactactc 1500
tactactata ataccccaac ccaactcata ttcaatacta ctctactatg atgagagcaa 1560
gggttccact cctgttgctg ggaattcttt tcctggcatc actttctgcc tcatttgccg 1620
gtaccatgca tcatcaccac catcaccatc acttacgtcc tgtagaaacc ccaacccgtg 1680
aaatcaaaaa actcgacggc ctgtgggcat tcagtctgga tcgcgaaaac tgtggaattg 1740
atcagcgttg gtgggaaagc gcgttacaag aaagccgggc aattgctgtg ccaggcagtt 1800
ttaacgatca gttcgccgat gcagatattc gtaattatgc gggcaacgtc tggtatcagc 1860
gcgaagtctt tataccgaaa ggttgggcag gccagcgtat cgtgctgcgt ttcgatgcgg 1920
tcactcatta cggcaaagtg tgggtcaata atcaggaagt gatcgagcat cagggcggct 1980
atacgccatt tgaagccgat gtcacgccgt atgttattgc cgggaaaagt gtacgtatca 2040
ccgtttgtgt gaacaacgaa ctgaactggc agactatccc gccgggagct gtgattaccg 2100
acgaaaacgg caagaaaaag cagtcttact tccatgattt ctttaactat gccggaatcc 2160
atcgcagcgt aatcctctac accacgccga acacctgggt ggacgatatc accgtggtga 2220
cgcatgtcgc gcaagactgt aaccacgcgt ctgttgactg gcaggtggtg gccaatggtg 2280
atgtcagcgt tgaactgcgt gatgcggatc aacaggtggt tgcaactgga caaggcacta 2340
gcgggacttt gcaagtggtg aatccgcacc tctggcaacc gggtgaaggt tatctctatg 2400
aactgtgcgt cacagccaaa agccagacag agtgtgatat ctacccgctt cgcgtcggca 2460
tccggtcagt ggcagtgaag ggcgaacagt tcctgattaa ccacaaaccg ttctacttta 2520
ctggctttgg tcgtcatgaa gatgcggact tgcgtggcaa aggattcgat aacgtgctga 2580
ttgtgcacga ccacgcatta gctgactgga ttggggccaa ctcctaccgt acctcgcatt 2640
acccttacgc tgaagagatc ctcgactggg cagatgaaca tggcatcgtg gtgattgatg 2700
aaactgctgc tgtcggcttt aacctctctt taggcattgg tttcgaagcg ggcaacaagc 2760
cgaaagaact gtacagcgaa gaggcagtca acggggaaac tcagcaagcg cacttacagg 2820
cgattaaaga gctgatagcg cgtgacaaaa accacccaag cgtggtgatt tggagtattg 2880
ccaacgaacc ggatacccgt ccgcaaggtg cacgggaata tttcgcgcca ctggcggaag 2940
caacgcgtaa actcgacccg acgcgtccga tcacctgcgt caatgtagca ttctgcgacg 3000
ctcacaccga taccatcagc gatctctttg atgtgctgtg cctgaaccgt tattacggat 3060
ggtatgtcca aagcggcgat ttggaaacgg cagagaaggt actggaaaaa gaacttctgg 3120
cctggcagga gaaactgcat cagccgatta tcatcaccga atacggcgtg gatacgttag 3180
ccgggctgca ctcagcatac accgacattt ggagtgaaga gtatcagtgt gcatggctgg 3240
atacctatca ccgcgtcttt gatcgcgtca gcgccgtcgt cggtgaacag gtatggaatt 3300
tcgccgattt tgcgacctcg caaggcatat tgcgcgttgg cggtaacaag aaagggatct 3360
tcactcgcga ccgcaaaccg aagtcggcgg cttttctgct gcaaaaacgc tggactggca 3420
taaacttcgg tgaaaaaccg cagcagggag gcaaacaagg atccgaccca cctgatccac 3480
ctgatccaat gtgctctaac ctttctactt gcgttcttgg aaagttgtct caagagcttc 3540
ataaacttca aacttaccca agaactaaca ccggatctgg aactccagga taatgataat 3600
gactgcagat aagtatgaac taaaatgcat gtatggtgta agagcacatg gagagcatgg 3660
aaatatgtat ccgaccatgt aacactataa taactgtgct ccatctcact tcttctatga 3720
ataaacaaag gatgttatga tatattaaca ctatatgcac cttcacaagt aatacattaa 3780
tatttaatac tttttatttt aactttttag tttaaaatat tattatatta ttaacttttt 3840
agtttaaaat atttatatta ttataaagag aaataaacaa aggatgttat gatatattaa 3900
cactatatgt accttacata gtaatatatt aatatttaat actttttatt ttaacttttt 3960
aatttaaaat attattataa atgacgcttg tgttttatgt gttggcatgc ttgtatttta 4020
tgtgttgact ttctgtgtga aggtaatgtg atatggtgag ctggtggtaa caattgtgtt 4080
ttatgtgttg gctttctgtg aagctaattt gatatggtta gctgatgtga acaaaatatt 4140
aaaggaagct aatttgatat ggttagccga tagtaacaaa atatcaaaat aaatttcttc 4200
ttactttaat aaa 4213
<210>6
<211>655
<212>PRT
<213>人工序列
<220>
<223>
<400>6
Met His His His His His His His His Leu Arg Pro Val Glu Thr Pro
1 5 10 15
Thr Arg Glu Ile Lys Lys Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp
20 25 30
Arg Glu Asn Cys Gly Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln
35 40 45
Glu Ser Arg Ala Ile Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala
50 55 60
Asp Ala Asp Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu
65 70 75 80
Val Phe Ile Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe
85 90 95
Asp Ala Val Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu Val
100 105 110
Ile Glu His Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro
115 120 125
Tyr Val Ile Ala Gly Lys Ser Val Arg Ile Thr Val Cys Val Asn Asn
130 135 140
Glu Leu Asn Trp Gln Thr Ile Pro Pro Gly Ala Val Ile Thr Asp Glu
145 150 155 160
Asn Gly Lys Lys Lys Gln Ser Tyr Phe His Asp Phe Phe Asn Tyr Ala
165 170 175
Gly Ile His Arg Ser Val Ile Leu Tyr Thr Thr Pro Asn Thr Trp Val
180 185 190
Asp Asp Ile Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala
195 200 205
Ser Val Asp Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu
210 215 220
Arg Asp Ala Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly
225 230 235 240
Thr Leu Gln Val Val Asn Pro His Leu Trp Gln Pro Gly Glu Gly Tyr
245 250 255
Leu Tyr Glu Leu Cys Val Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile
260 265 270
Tyr Pro Leu Arg Val Gly Ile Arg Ser Val Ala Val Lys Gly Glu Gln
275 280 285
Phe Leu Ile Asn His Lys Pro Phe Tyr Phe Thr Gly Phe Gly Arg His
290 295 300
Glu Asp Ala Asp Leu Arg Gly Lys Gly Phe Asp Asn Val Leu Ile Val
305 310 315 320
His Asp His Ala Leu Ala Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr
325 330 335
Ser His Tyr Pro Tyr Ala Glu Glu Ile Leu Asp Trp Ala Asp Glu His
340 345 350
Gly Ile Val Val Ile Asp Glu Thr Ala Ala Val Gly Phe Asn Leu Ser
355 360 365
Leu Gly Ile Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser
370 375 380
Glu Glu Ala Val Asn Gly Glu Thr Gln Gln Ala His Leu Gln Ala Ile
385 390 395 400
Lys Glu Leu Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Ile Trp
405 410 415
Ser Ile Ala Asn Glu Pro Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr
420 425 430
Phe Ala Pro Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro
435 440 445
Ile Thr Cys Val Asn Val Ala Phe Cys Asp Ala His Thr Asp Thr Ile
450 455 460
Ser Asp Leu Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr
465 470 475 480
Val Gln Ser Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu Lys Glu
485 490 495
Leu Leu Ala Trp Gln Glu Lys Leu His Gln Pro Ile Ile Ile Thr Glu
500 505 510
Tyr Gly Val Asp Thr Leu Ala Gly Leu His Ser Ala Tyr Thr Asp Ile
515 520 525
Trp Ser Glu Glu Tyr Gln Cys Ala Trp Leu Asp Thr Tyr His Arg Val
530 535 540
Phe Asp Arg Val Ser Ala Val Val Gly Glu Gln Val Trp Asn Phe Ala
545 550 555 560
Asp Phe Ala Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys
565 570 575
Gly Ile Phe Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu
580 585 590
Gln Lys Arg Trp Thr Gly Ile Asn Phe Gly Glu Lys Pro Gln Gln Gly
595 600 605
Gly Lys Gln Gly Ser Asp Pro Pro Asp Pro Pro Asp Pro Met Cys Ser
610 615 620
Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu His Lys
625 630 635 640
Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro Gly
645 650 655
<210>7
<211>654
<212>PRT
<213>人工序列
<220>
<223>
<400>7
Met His His His His His His His His Leu Arg Pro Val Glu Thr Pro
1 5 10 15
Thr Arg Glu Ile Lys Lys Leu Asp Gly Leu Trp Ala Phe Ser Leu Asp
20 25 30
Arg Glu Asn Cys Gly Ile Asp Gln Arg Trp Trp Glu Ser Ala Leu Gln
35 40 45
Glu Ser Arg Ala Ile Ala Val Pro Gly Ser Phe Asn Asp Gln Phe Ala
50 55 60
Asp Ala Asp Ile Arg Asn Tyr Ala Gly Asn Val Trp Tyr Gln Arg Glu
65 70 75 80
Val Phe Ile Pro Lys Gly Trp Ala Gly Gln Arg Ile Val Leu Arg Phe
85 90 95
Asp Ala Val Thr His Tyr Gly Lys Val Trp Val Asn Asn Gln Glu Val
100 105 110
Ile Glu His Gln Gly Gly Tyr Thr Pro Phe Glu Ala Asp Val Thr Pro
115 120 125
Tyr Val Ile Ala Gly Lys Ser Val Arg Ile Thr Val Cys Val Asn Asn
130 135 140
Glu Leu Asn Trp Gln Thr Ile Pro Pro Gly Ala Val Ile Thr Asp Glu
145 150 155 160
Asn Gly Lys Lys Lys Gln Ser Tyr Phe His Asp Phe Phe Asn Tyr Ala
165 170 175
Gly Ile His Arg Ser Val Ile Leu Tyr Thr Thr Pro Asn Thr Trp Val
180 185 190
Asp Asp Ile Thr Val Val Thr His Val Ala Gln Asp Cys Asn His Ala
195 200 205
Ser Val Asp Trp Gln Val Val Ala Asn Gly Asp Val Ser Val Glu Leu
210 215 220
Arg Asp Ala Asp Gln Gln Val Val Ala Thr Gly Gln Gly Thr Ser Gly
225 230 235 240
Thr Leu Gln Val Val Asn Pro His Leu Trp Gln Pro Gly Glu Gly Tyr
245 250 255
Leu Tyr Glu Leu Cys Val Thr Ala Lys Ser Gln Thr Glu Cys Asp Ile
260 265 270
Tyr Pro Leu Arg Val Gly Ile Arg Ser Val Ala Val Lys Gly Glu Gln
275 280 285
Phe Leu Ile Asn His Lys Pro Phe Tyr Phe Thr Gly Phe Gly Arg His
290 295 300
Glu Asp Ala Asp Leu Arg Gly Lys Gly Phe Asp Asn Val Leu Ile Val
305 310 315 320
His Asp His Ala Leu Ala Asp Trp Ile Gly Ala Asn Ser Tyr Arg Thr
325 330 335
Ser His Tyr Pro Tyr Ala Glu Glu Ile Leu Asp Trp Ala Asp Glu His
340 345 350
Gly Ile Val Val Ile Asp Glu Thr Ala Ala Val Gly Phe Asn Leu Ser
355 360 365
Leu Gly Ile Gly Phe Glu Ala Gly Asn Lys Pro Lys Glu Leu Tyr Ser
370 375 380
Glu Glu Ala Val Asn Gly Glu Thr Gln Gln Ala His Leu Gln Ala Ile
385 390 395 400
Lys Glu Leu Ile Ala Arg Asp Lys Asn His Pro Ser Val Val Ile Trp
405 410 415
Ser Ile Ala Asn Glu Pro Asp Thr Arg Pro Gln Gly Ala Arg Glu Tyr
420 425 430
Phe Ala Pro Leu Ala Glu Ala Thr Arg Lys Leu Asp Pro Thr Arg Pro
435 440 445
Ile Thr Cys Val Asn Val Ala Phe Cys Asp Ala His Thr Asp Thr Ile
450 455 460
Ser Asp Leu Phe Asp Val Leu Cys Leu Asn Arg Tyr Tyr Gly Trp Tyr
465 470 475 480
Val Gln Ser Gly Asp Leu Glu Thr Ala Glu Lys Val Leu Glu Lys Glu
485 490 495
Leu Leu Ala Trp Gln Glu Lys Leu His Gln Pro Ile Ile Ile Thr Glu
500 505 510
Tyr Gly Val Asp Thr Leu Ala Gly Leu His Ser Ala Tyr Thr Asp Ile
515 520 525
Trp Ser Glu Glu Tyr Gln Cys Ala Trp Leu Asp Thr Tyr His Arg Val
530 535 540
Phe Asp Arg Val Ser Ala Val Val Gly Glu Gln Val Trp Asn Phe Ala
545 550 555 560
Asp Phe Ala Thr Ser Gln Gly Ile Leu Arg Val Gly Gly Asn Lys Lys
565 570 575
Gly Ile Phe Thr Arg Asp Arg Lys Pro Lys Ser Ala Ala Phe Leu Leu
580 585 590
Gln Lys Arg Trp Thr Gly Ile Asn Phe Gly Glu Lys Pro Gln Gln Gly
595 600 605
Gly Lys Gln Gly Ser Asp Pro Pro Asp Pro Pro Asp Pro Met Cys Ser
610 615 620
Asn Leu Ser Thr Cys Val Leu Gly Lys Leu Ser Gln Glu Leu His Lys
625 630 635 640
Leu Gln Thr Tyr Pro Arg Thr Asn Thr Gly Ser Gly Thr Pro
645 650
<210>8
<211>150
<212>DNA
<213>人工序列
<220>
<223>
<400>8
ggatccgacc cacctgatcc acctgatcca atgtgctcaa acttgtctac ctgtgttctt 60
ggtaagttgt ctcaggaact tcacaagttg caaacctacc caaggactaa cactggttct 120
ggaaccccag gataatgata atgactgcag 150
Claims (10)
1.表达盒；所述表达盒自上游至下游依次包括如下元件：植物种子特异表达蛋白基因的启动子、融合基因和终止序列；所述融合基因中含有区段乙；所述区段乙编码鲑鱼降钙素的前体；所述鲑鱼降钙素的前体为氨基酸序列是序列表中序列5自N末端起第158至190位所示的蛋白质。
2.如权利要求1所述表达盒，其特征在于：所述区段乙为如下b1)或b2)或b3)或b4)所示的DNA分子：
b1)具有序列表中序列1自5′末端起第22至120位核苷酸所示的DNA分子；
b2)核苷酸序列是序列表中序列4自5′末端起第34至132位所示的DNA分子；
b3)与b1)或b2)限定的核苷酸序列具有85％或85％以上同一性，且编码权利要求1中所述鲑鱼降钙素的前体的DNA分子；
b4)在严格条件下与b1)或b2)限定的核苷酸序列杂交，且编码权利要求1中所述鲑鱼降钙素的前体的DNA分子。
3.如权利要求1或2所述表达盒，其特征在于：所述融合基因还包括区段甲；所述区段甲编码筛选标记蛋白。
4.如权利要求1至3任一所述表达盒，其特征在于：所述融合基因还包括一个以上区段丙；所述区段丙编码蛋白标签。
5.如权利要求1至4任一所述表达盒，其特征在于：所述融合基因还包括区段丁；所述区段丁为切割物质的识别序列。
6.如权利要求5所述表达盒，其特征在于：所述切割物质为满足如下条件的切割物质：所述切割物质对于所述融合基因的表达产物的酶切位置为特定氨基酸残基与其前一位氨基酸残基之间；所述特定氨基酸残基为所述鲑鱼降钙素的前体的第一个氨基酸残基。
7.如权利要求1至6任一所述表达盒，其特征在于：所述融合基因自上游至下游依次包括如下区段：所述区段丙、所述区段甲、所述区段丁和所述区段乙。
8.含有权利要求1至7任一所述表达盒的重组载体。
9.一种表达鲑鱼降钙素的方法，依次包括如下步骤：
(1)将权利要求8所述重组载体导入受体植物，得到转基因植物；
(2)通过培养所述转基因植物得到鲑鱼降钙素。
10.如权利要求9所述的方法，其特征在于：所述受体植物为油菜品种中双4号。
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| CN1424399A (zh) * | 2001-12-14 | 2003-06-18 | 中国农业科学院生物技术研究所 | 一种新型鲑鱼降钙素类似物及其在植物油体中表达的方法 |
| CN102373196A (zh) * | 2010-08-09 | 2012-03-14 | 中国农业科学院生物技术研究所 | 利用染色体替换技术使鲑鱼降钙素蛋白在转基因油菜油体中超高量、靶向表达的方法 |
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| CN1424399A (zh) * | 2001-12-14 | 2003-06-18 | 中国农业科学院生物技术研究所 | 一种新型鲑鱼降钙素类似物及其在植物油体中表达的方法 |
| CN102373196A (zh) * | 2010-08-09 | 2012-03-14 | 中国农业科学院生物技术研究所 | 利用染色体替换技术使鲑鱼降钙素蛋白在转基因油菜油体中超高量、靶向表达的方法 |
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