CN108486123B - 适于家蚕丝腺表达的改造人乳铁蛋白基因及其表达系统和应用 - Google Patents
适于家蚕丝腺表达的改造人乳铁蛋白基因及其表达系统和应用 Download PDFInfo
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- CN108486123B CN108486123B CN201810214261.1A CN201810214261A CN108486123B CN 108486123 B CN108486123 B CN 108486123B CN 201810214261 A CN201810214261 A CN 201810214261A CN 108486123 B CN108486123 B CN 108486123B
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Abstract
本发明涉及一种适于家蚕丝腺表达的改造人乳铁蛋白基因及其表达系统和应用，改造人乳铁蛋白基因如SEQ ID NO.1所示，其编码的氨基酸如SEQ ID NO.2所示，将改造人乳铁蛋白基因与分泌型丝胶1基因启动子和分泌型丝胶1基因终止子构成表达框，并用增强子hr3增强表达，同时连入piggyBac转座臂和荧光筛选标记基因构建表达系统，该表达系统能够在家蚕丝腺中高效表达重组人乳铁蛋白，并且具有生物活性，能够用于大规模生产重组人乳铁蛋白，具有良好的市场前景。
Description
技术领域
本发明属于生物技术领域，涉及适于家蚕丝腺表达的改造人乳铁蛋白基因，还涉及表达人乳铁蛋白基因的表达系统和应用。
背景技术
进入21世纪以来，随着人类对医用、药用、食用、美容、保健等各种用途的功能性蛋白需求量的日益增大，依靠提取、生产天然来源的蛋白质已无法满足快速增长的市场需求。建立和完善各种高效的原核和真核表达系统，利用大肠杆菌、酵母、昆虫细胞、哺乳动物细胞、昆虫以及哺乳动物等作为宿主生物反应器，是实现低成本、规模化生产具有生物学活性的重组外源蛋白的有效和可持续方法，并成为当今世界研究的热点。利用中国仓鼠卵巢细胞作为生物反应器生产外源蛋白是目前最为标准的表达模式，但其运营成本和对环境的要求极为苛刻，严重限制了其大规模推广应用。为了建立低成本、规模化、安全可持续的高效生产外源蛋白的生物工厂，自2000年以来，科研人员开始尝试利用转基因生物，如哺乳动物、鸟类、昆虫以及植物的器官作为生物反应器来生产重组外源蛋白。
家蚕以泌丝著称，是最早被人类所完全驯化利用的经济动物（昆虫）之一。家蚕的丝腺是合成、分泌蚕丝蛋白的器官，是整个蚕丝业的生物学基础。经过数千年的人工驯化，家蚕的丝腺具备了超强的蛋白质合成与分泌能力，一头体重5g左右家蚕能合成分泌约0.5g蚕丝蛋白，为目前已知昆虫之最。蚕丝蛋白主要由丝素蛋白（fibroin）和包裹在其外层的丝胶蛋白（sericin）构成：丝素蛋白是蚕丝的主体，约占75%，由蚕的后部丝腺合成，包括fib-H链、fib-L链和P25三种主要组分，均不溶于水；其余为丝胶蛋白，约占25%，由蚕的中部丝腺合成，包括丝胶1(Sericin1）、丝胶2(Sericin2）和丝胶3(Sericin3）三种主要组分，其中又以丝胶1蛋白含量最高，均可溶于水。随着现代分子生物学和转基因技术的发展，家蚕绢丝腺的高效合成与分泌丝蛋白这一特征，以及所具备的糖基化、甲基化等对外源蛋白保持活性极其重要的蛋白质翻译后修饰加工能力，饲养成本低，可工厂化生产，对人畜安全等特点，使其成为理想的生物反应器模型，备受各国研究人员关注并竞相开发利用。
2000年，田村等人利用pBac转座子介导显微注射家蚕蚕卵并获得了稳定遗传的转基因家蚕；2003年，夏庆友等人完成了家蚕基因组计划，家蚕丝腺中涉及丝蛋白合成的重要编码基因如丝素重链（FibH chain）基因、丝素轻链（FibL chain）基因、丝胶1（Sericin1）基因、丝胶2（Sericin2）基因、丝胶3（Sericin3）基因以及P25基因等的启动子调控元件被鉴定和克隆，同时，本研究室前期通过对表达系统进行优化，获得了高效的转基因家蚕丝胶1表达系统。这些基础研究结果使得利用转基因家蚕组织特异表达系统，在丝腺中大规模生产重组外源蛋白成为可能。近年来，国内外利用pBac转座子介导的转基因技术以及家蚕组织特异启动子元件在丝腺中尝试表达了多个外源蛋白，包括：在后部丝腺表达了丝素重链融合的EGFP(Zhao et al. 2010)、猫干扰素（Kurihara et al. 2007)、蜘蛛丝牵引蛋白（Zhuet al. 2010)、人Ⅲ型胶原蛋白的部分肽段(Tomita et al. 2003)、增强型红色荧光蛋白(Tomita et al. 2003)，丝素轻链融合的羟基脯氨酸胶原部分肽段(Adachi et al.2006)、成纤维细胞生长因子(Hino et al. 2006)、增强型绿色荧光蛋白(Shimizu et al.2007)、部分胶原蛋白肽段（Yanagisawa et al. 2007)，以及P25融合的红色荧光蛋白(Royer et al. 2005)等；在中部丝腺表达了人血清白蛋白（Ogawa et al. 2007)、增强型绿色荧光蛋白(Tomita et al. 2007)、鼠单克隆抗体(Iizuka et al. 2009)、人胶原蛋白α链基因(Adachi et al. 2010)以及可溶性GM-Csf受体α(Urano et al. 2010)等。综合以上国内外的研究结果表明，利用家蚕丝腺作为生物反应器，生产具有高附加值的外源蛋白，不仅具有广阔的市场前景，同时也能打破家蚕只能作为传统产业的壁垒，为家蚕新型产业的开发提供基础技术体系保障。
人乳铁蛋白是由716个氨基酸组成的单链糖蛋白，分子量约为78kDa。人乳铁蛋白可以转运和存储铁，是转铁蛋白家族成员之一。乳铁蛋白具有广谱抗菌、抗病毒、消炎、抑制肿瘤细胞生长和调节机体免疫反应等诸多生物学活性，此外，乳铁蛋白和促进幼体消化道发育、促进铁吸收息息相关。现市场上对乳铁蛋白的应用主要集中于添加至奶粉中作为营养强化剂，用于增强婴幼儿的免疫力。目前人乳铁蛋白的商业化生产主要通过乳清分离纯化途径实现，然而，随着人们对乳铁蛋白旺盛的消费，传统的分离纯化方法难以满足市场需求。传统分离方法不仅价格非常昂贵，同时存在疾病传播的风险，比如肝炎和HIV。监管部门也曾呼吁制药公司使用非动物来源的乳清进行乳铁蛋白的生产，这样可以有效的控制疾病因子感染的风险。因此，利用基因重组技术来生产人乳铁蛋白，不仅可以有效的控制疾病传播的风险，同时也可作为乳清来源的人乳铁蛋白的替代，具有巨大的市场开发前景。2009年，王振兴公开了利用家蚕丝腺生物反应器表达重组人乳铁蛋白的研究，但是该方法仅能在家蚕中部丝腺中有少量蛋白表达，在蚕丝中几乎检测重组人乳铁蛋白的表达。
因此，亟待开发出具有产业价值的高效大规模生产人乳铁蛋白重组蛋白的转基因家蚕素材，探索转基因家蚕丝腺生物反应器实用化的可行性。
发明内容
有鉴于此，本发明的目的在于提供一种适于家蚕丝腺表达的改造人乳铁蛋白基因；本发明的目的之二在于提供含有所述适于家蚕丝腺表达的改造人乳铁蛋白基因的表达系统；本发明的目的之三在于提供所述表达系统在家蚕丝腺表达重组人乳铁蛋白中的应用；本发明的目的之四在于提供利用所述表达系统在家蚕丝腺中表达重组人乳铁蛋白的方法。
为达到上述目的，本发明提供如下技术方案：
1、适于家蚕丝腺表达的改造人乳铁蛋白基因，所述改造人乳铁蛋白基因如SEQ IDNO.1所示。
2、含有权利要求1或2所述适于家蚕丝腺表达的改造人乳铁蛋白基因的表达系统。
优选的，所述表达系统含有顺序连接的增强子hr3，分泌型丝胶1基因启动子、改造人乳铁蛋白基因和丝胶1基因的终止子。
优选的，所述表达系统还含有荧光筛选标记基因表达框，所述荧光筛选标记基因表达框位于所述增强子hr3上游。
3、所述表达系统在家蚕丝腺表达重组人乳铁蛋白中的应用。
4、 利用所述表达系统在家蚕丝腺中表达重组人乳铁蛋白的方法，包括如下步骤：将所述表达系统转化解除滞育家蚕蚕卵，用无毒胶水封口后经甲醛蒸汽消毒，孵化，筛选转基因阳性蛾圈，取阳性转基因家蚕茧壳，在液氮中粉碎成粉末，然后溶解于含50mM Tris-Cl、8M Urea、pH 8.0的缓冲液中，80℃水浴40min，离心收集上清，经纯化，得重组人乳铁蛋白。
优选的，茧壳粉末溶解于缓冲液中茧壳粉末的浓度为20mg/mL。
优选的，所述纯化的具体方法为：将上清液过His TrapTM FF crude亲和层析柱，用咪唑浓度为0~500mM的咪唑洗脱液洗脱，咪唑洗脱液用pH7.0-7.5、浓度为8M Urea溶液配制，收集洗脱液，洗脱液即为纯化后的重组人乳铁蛋白。
本发明的有益效果在于：本发明通过优化人乳铁蛋白的编码序列，获得适合家蚕密码子偏好的人乳铁蛋白基因序列，将优化的人乳铁蛋白基因序列与含有分泌型丝胶1基因启动子和终止子构成表达框并利用增强子hr3增强表达、荧光标记基因筛选和piggyBac转座臂构建高效表达系统，该系统能够在家蚕中部丝腺特异高效表达，获得结构和活性与天然人乳铁蛋白相似的重组人乳铁蛋白，为高效大规模生产人乳铁蛋白重组蛋白提供了可能，生产的重组人乳铁蛋白可以替代血液来源的人乳铁蛋白，能够有效控制疾病传播的风险，具有巨大的市场开发前景。
附图说明
为了使本发明的目的、技术方案和有益效果更加清楚，本发明提供如下附图进行说明：
图1转基因表达载体结构图（3xp3RFP表示转基因荧光筛选标记基因；hr3CQ表示增强子hr3；Ser1表示分泌型丝胶1基因启动子；rhLF表示进过密码子优化设计的人乳铁蛋白基因编码序列；Ser1PA表示丝胶1基因的终止子；ITR表示piggyBac转座臂序列）。
图2为转基因荧光筛选图（A：蛾白光图；B：蛾荧光图）。
图3为sgrhLF家蚕茧壳蛋白SDS-Page电泳图（1-65：65个转基因阳性个体茧壳蛋白；WT：正常茧壳蛋白）。
图4为sgrhLF家蚕茧壳蛋白中rhLF的检测（A： SDS-Page电泳图；B：Western blot图）。
图5为rhLF丝胶溶液及纯化定量检测（A： SDS-PAGE电泳图；B： Western blot检测）。
图6为重组rhLF蛋白的二级结构检测。
图7为 rhLF降低LPS诱导细胞产生炎性（A：TNF-α含量；B：NO含量；C：western blot检测细胞产生iNOs结果；D：iNOs相对表达量）。
图8为重组人乳铁蛋白抗菌活性检测结果。
具体实施方式
下面将结合附图，对本发明的优选实施例进行详细的描述。
本发明实施例中使用的材料如下：供试家蚕品种大造(P50)由本实验室保存。幼虫在25℃人工气候箱中以人工饲料饲养。小鼠腹腔巨噬细胞raw264.7由实验室保存，并培养于含有10% (v/v) 胎牛血清（FBS，Gibco）的DMEM培养基中，培养条件为37℃，5% CO2。质粒载体pSLfa1180fa、pBac{3xp3EGFPaf}，pBac{3xp3DsRedaf}由本实验室保存。
主要试剂及溶液的配制：分子克隆过程中所用到的常规培养基，试剂缓冲液等参照《分子克隆实验指南第三版译》以及《TaKaRa商品目录》中实验室常规试剂配制方法章节（S1-S11）进行配置。DNA聚合酶Ex-Taq，LA-Taq Kit，常规限制性内切酶，碱性磷酸酶，测序克隆载体pMD19-T simple载体Kit，DNA Ligation Kit Ver.2.0，荧光定量PCR试剂盒SYBRpremix Ex TaqTM均购自TaKaRa公司。转化用大肠杆菌感受态细胞Trans1-T1，常规质粒DNA提取试剂盒Easypure Plasmid MiniPrep Kit购自全式金公司。琼脂糖凝胶DNA回收试剂盒Gel Extraction Mini Kit (50)购自华舜生物科技公司。转基因注射用超纯质粒提取试剂盒QIA prep Spin Miniprep Kit (50) 购自QIAGEN。Total RNA Kit II (50)试剂盒购自Omega Bio-Tec公司。人乳铁蛋白多克隆抗体 anti-rhLF antibody、重组人乳铁蛋白标准品rhLF-std均购自Sigma公司，一氧化氮合酶（iNOs）多克隆抗体anti-iNOs antibody购自abcam，NO检测试剂盒购自碧云天公司，TNF-α检测试剂盒购自R＆D systems公司。
实施例1、改造人乳铁蛋白基因合成
从NCBI下载人乳铁蛋白（Human lactoferin，hLF，GenBank: M93150.1）成熟肽氨基酸序列，根据家蚕密码子使用偏好型进行优化设计编码序列，具体序列如SEQ ID NO.1第7~2106位所示，氨基酸序列如SEQ ID NO.2第1~700位所示，由公司合成基因序列。人乳铁蛋白（hLF）氨基酸序列的C端融合6个组氨酸氨基酸，形成hLF-his6。合成的两个重组蛋白基因序列的两端分别接上BamHI和NotI酶切位点，如SEQ ID NO.1所示，编码的氨基酸序列如SEQID NO.2。
实施例2、转基因表达载体的构建
将商业合成的hLF-his6基因编码序列通过BamHI和NotI酶切位点构建到psl1180[hr3Pser1spRedSer1PA]中，形成psl1180[hr3CQSer1sphLFSer1]，核苷酸序列如SEQ IDNO.3所示，再通过AscI位点构建到pBac{3xp3EGFPaf}载体的AscI位点中，形成转基因表达载体phShLFSer，载体结构如图1所示。
实施例3、显微注射与荧光筛选
利用QIAGEN Plasimd Mini Kit质粒抽提试剂盒提取转基因表达载体phShLFSer1以及辅助载体pHA3PIG质粒，将各质粒浓度稀释至400ng/μl，并按1∶1摩尔比分别与辅助载体pHA3PIG质粒进行混合。将混合后的质粒注射已解除滞育的大造早期胚胎(产卵后2～5h)，随后用无毒胶水对注射孔进行封口，经35%的甲醛蒸汽消毒5分钟后，置于 25℃，相对湿度85%的环境中孵化，孵化的幼虫(G0代)采用人工饲料饲育，至成虫后进行自交或回交制种，获得的G1代蚕卵（第7天）在宏观体视荧光显微镜(Olypus MVX10, 日本)下检测，红色荧光观察采用波长为510~550 nm的激发光，筛选出在眼睛或神经特异激发红色荧光的转基因阳性蛾圈，并命名为sghLF，如图2所示。转基因家蚕的荧光筛选统计见表1。
表1.转基因筛选统计表
结果显示，从sgrhLF的43个G1代蛾圈中总共筛选到12个阳性蛾圈，阳性率为27.90%.
实施例4、SDS-Page和Western blot检测
茧壳总蛋白中重组蛋白的提取和检测方法：将茧壳于液氮中粉碎成粉末，按照20mg/ml的茧壳浓度溶于50mM Tris-Cl，pH 8.0，8M Urea的缓冲液中，80℃水浴40min，之后离心收集上清，离心条件：13500×rpm，25℃，15min。经萃取的茧壳总蛋白进行12% SDS-Page电泳检测，并利用考马斯亮蓝染色。将萃取的总蛋白经12% SDS-Page胶电泳分离后，采用电转膜法将SDS-Page胶中的蛋白转移至PVDF膜上。PVDF膜置于含5%脱脂奶粉的TBST缓冲液中，4℃过夜封闭。免疫杂交前，于室温利用TBST清洗PVDF膜3次，每次5min。利用含5%脱脂奶粉的TBST按1000倍稀释比例配置抗rhLF (Sigma)一抗杂交液，将PVDF膜浸入杂交液中室温振荡孵育2h，TBST洗膜5次，每次10min。利用TBST按20000倍稀释比例配置HRP标记的羊抗兔二抗（购自碧云天公司）杂交液，将TBST清洗后的PVDF膜浸入二抗杂交液中于室温振荡孵育2h，TBST洗膜5次，每次10min。将清洗后的PVDF膜置于干净的保鲜膜上，将ECL显色液（Amersham Biosciences）均匀滴在PDVF膜面上，室温避光孵育5min，利用ChemiscopeSeries（Clinx science instruments）仪器进行曝光和成像。
SDS-Page检测结果如图3所示，结果显示转基因茧壳蛋白泳道在70kDa分子Marker以上出现差异条带，与rhLF蛋白的理论分子量大小一致，并且差异条带在不同的转基因个体来源的茧壳总蛋白中的含量明显不同。为进一步鉴定此特异性条带为rhLF，对编号为53-65茧壳蛋白进行Western blot检测，结果如图4所示。结果显示转基因茧壳蛋白在70kDa分子Marker以上出现的差异条带可以与rhLF的抗体发生特异的反应，此结果证实该差异条带即是转基因家蚕特异表达的rhLF蛋白。
通过与rhLF标准品进行灰度比对，推算出1g蚕丝中rhLF蛋白的含量约为10.27mg，即rhLF转基因蚕茧中rhLF占0.93%（图5）。以上结果表明，本研究建立的Ser1表达系统可以高效的重组生产人乳铁蛋白，并分泌到家蚕蚕丝中。此外，重组蛋白在不同阳性蛾圈来源的个体中的含量具有显著的差异，暗示着转rhLF基因在家蚕丝腺细胞中的表达受到强烈的染色体位置效应的影响。
而使用王振兴的方法仅在丝腺中检测出192μg/ml，推算出1g丝腺中rhLF蛋白的含量约为2.4 mg，而蚕丝中几乎检测不到rhLF蛋白。
实施例5、 从蚕茧分离纯化rhLF及检测
取hLF转基因蚕茧1g剪碎后50mM Tris-Cl，pH 8.0，8M Urea的缓冲液中，80℃水浴40min，13500rpm，25℃离心15min收集上清。上清液首先经过His TrapTM FF crude亲和层析柱（GE healthcare），使rhLF与层析柱中的镍离子结合，然后用不同浓度咪唑洗脱液（8MUrea，pH7.0-7.5配制，咪唑浓度为0、20、50、100、500mM）洗脱收集。样品收集后进行12%SDS-PAGE和western blot检测其纯化和得率。结果显示：转基因蚕茧提取蛋白在70kDa分子Marker以上出现的差异蛋白条带，且该差异蛋白条带可以与rhLF的抗体发生特异的反应，结果与rhLF标准品的结果一致，证实尿素可以将蚕丝中的rhLF溶解出来。进一步对rhLF纯化物的SDS-PAGE和Western blot的结果进行分析，可获得纯度大约90%的rhLF蛋白，1g蚕茧能够纯化得到9.21mg rhLF，rhLF纯化得率高达89.25%（图5）。
（1） 重组蛋白二级结构检测
将纯化的rhLF样品溶解于去离子水，在浓度为0.5mg/mL的条件下，测定rhLF在190-250nm之间的CD图谱，结果如图6所示。结果显示，在CD：190-250nm处，转基因家蚕表达的rhLF与商业化的rhLF标准品蛋白具有相似的吸收峰值，并且通过计算，家蚕表达rhLF与商业化rhLF标准品α-螺旋、β-折叠、β-转角及无规则卷曲等蛋白质二级结构均较为一致（表2）。因此，我们推测利用转基因家蚕表达的rhLF应该具有其生物学功能。该结果标志着本研究建立的Ser1表达系统可以实现外源蛋白的高效重组表达和分离纯化。
表2.重组rhLF蛋白的二级结构分析
| 样品名称 | <i>α</i>-螺旋（%） | <i>β</i>-折叠（%） | <i>β</i>-转角（%） | 无规卷曲（%） |
| rhLF-std | 52.60 | 10.35 | 14.90 | 22.50 |
| rhLF | 51.20 | 10.90 | 15.30 | 22.90 |
（2）重组人乳铁蛋白抗炎活性检测
将纯化的rhLF样品溶于去离子水备用。raw264.7细胞重悬后接种至96孔细胞培养板，密度为5 x 104个/孔，培养12h后细胞贴壁，加入细菌脂多糖（LPS，50ng/μl）和不同浓度rhLF（终浓度为0μg/ml、10μg/ml、20μg/ml、30μg/ml）继续培养12h，rhLFstd为阳性对照；12h后取细胞培养基试剂盒测定NO、TNF-α浓度，冰PBS清洗细胞两次，裂解液裂解细胞，Westernblotting测定细胞iNOs表达情况，结果如图7所示。结果显示，LPS可诱导raw264.7细胞产生NO、TNF-α等炎性因子及iNOs过表达。人乳铁蛋白分子N端的GRRRR和RKVRGPP序列能够结合LPS，从而降低LPS诱导细胞产生炎性因子。将纯化的rhLF进行抗炎活性检测实验结果显示，10μg/ml rhLF即可显著降低LPS诱导raw264.7细胞产生TNF-α和NO，并且随着浓度的升高，抑制率升高；Western blot检测细胞产生iNOs结果显示，当rhLF浓度为20μg/ml时，细胞iNOs的生产与空白对照组相比较显著降低，以商业化rhLF标准品作为阳性对照，家蚕丝腺表达的rhLF与rhLF标准品降低细胞炎性因子产生效果相当。以上实验结果表明，家蚕丝腺表达的重组人乳铁蛋白（rhLF）具有抗炎活性。
（3）重组人乳铁蛋白抗菌活性检测
将纯化的rhLF样品溶于去离子水备用。大肠杆菌用LB培养基活化后接种于96孔培养板，100μl/孔，加入不同浓度rhLF（浓度为0.5mg/ml、1mg/ml、2mg/ml）与大肠杆菌共同孵育，每隔1h于600nm测定吸光度值，连续测定12h，结果如图8所示。结果显示，rhLF对大肠杆菌的生长具有剂量-依赖性和时间-依懒性。人乳铁蛋白能够竞争性结合大肠杆菌生长所需要的铁离子，从而抑制大肠杆菌的生长。纯化的rhLF抑制大肠杆菌的结果显示，从第3h开始rhLF对大肠杆菌的生长有抑制的作用，随着时间的增加，抑制率增强，8h时细菌生长趋于平缓；此外，高浓度rhLF（2mg/ml）抑制效果较低浓度（0.5mg/ml）rhLF抑制作用明显；在12h时，2mg/ml rhLF抑制率达到最高为29.85%。
综上所述，本发明利用建立的Ser1表达系统hSRSE，对人乳铁蛋白（rhLF）进行重组表达。根据家蚕基因组密码子使用的偏好型人工设计并合成了人乳铁蛋白（rhLF）的编码基因，构建转基因表达载体phSrhLFSer1。通过显微注射家蚕胚胎，我们建立转基因家蚕品系sgrhLF，其中sgrhLF筛选获得7个不用阳性蛾圈来源的品系。
利用SDS-PAGE和Western blot检测方法，在sgrhLF家蚕的茧壳中检测到rhLF蛋白有高效表达。与rhLF标准品进行灰度比对，推算出sgrhLF茧丝中rhLF蛋白含量为10.27mg/g。结果说明经过系统性优化建立的hSRSE表达系统，其表达效率达到了国外目前所使用表达系统的水平。在此基础之上，我们利用尿素对sgrhLF家蚕蚕丝蛋白进行分离纯化，现阶段通过His TrapTM FF crude亲和层析柱一步纯化即可获得纯度为90%的rhLF蛋白，相信通过对纯化步骤的进一步优化，重组蛋白的纯度会进一步提高。随后，我们对纯化获得重组蛋白的二级空间结构进行CD光谱扫描，结果显示rhLF蛋白与商品化rhLF蛋白具有一致的二级结构，进一步抗炎活性检测实验结果证明，通过家蚕丝腺表达的rhLF具有与商品化rhLF一致的生物学活性。
因此，通过对rhLF靶标外源蛋白的重组表达，分离纯化和活性鉴定，初步证实前面所建立的Ser1表达系统hSRSE可实现外源蛋白在转基因家蚕丝腺中的高效分泌表达，并生产具有生物学活性的重组蛋白。结果表明hSRSE表达系统可为家蚕丝腺生物反应的实用化进程的基础技术体系。
最后说明的是，以上优选实施例仅用以说明本发明的技术方案而非限制，尽管通过上述优选实施例已经对本发明进行了详细的描述，但本领域技术人员应当理解，可以在形式上和细节上对其作出各种各样的改变，而不偏离本发明权利要求书所限定的范围。
序列表
<110> 西南大学
<120> 适于家蚕丝腺表达的改造人乳铁蛋白基因及其表达系统和应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 2121
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
atgggcagaa gaagaagatc tgtccaatgg tgcgctgtga gccagccgga agccaccaaa 60
tgtttccaat ggcagagaaa tatgagaaag gttagaggtc ctccagtctc ttgcataaaa 120
agagatagcc ccatccaatg tattcaggct atagccgaaa acagagctga cgccgtgaca 180
ttggatggtg gattcatcta cgaagctggt ctcgccccgt acaaactgag acccgttgct 240
gccgaagtct acggaaccga aagacaaccg agaacacact actacgctgt ggccgtggtt 300
aaaaagggcg gttctttcca attgaatgaa ctccagggtc tgaagagctg ccacaccggt 360
ctgagaagaa cagctggatg gaatgttcct actggcacct tgagaccatt cctcaactgg 420
acaggaccgc ccgaacctat tgaagctgcc gttgctagat tcttctcagc ctcttgcgtc 480
cctggagctg acaagggcca attcccaaat ttgtgcagac tctgtgctgg tactggagaa 540
aacaaatgcg ccttctcatc tcaggaacca tacttcagct actccggagc tttcaagtgt 600
ttgagagacg gcgctggtga cgtcgccttc ataagagaat ccaccgtgtt cgaagacctc 660
tcggatgaag ctgaaagaga cgaatacgaa ctgttgtgcc cggataacac aagaaaaccc 720
gttgacaaat tcaaggattg tcacctcgct agagtgccaa gtcacgctgt cgtggccaga 780
tcagttaatg gtaaagaaga tgctatctgg aacctcctga gacaagccca ggaaaaattc 840
ggaaaagaca agagcccgaa gttccaactg ttcggttcgc ccagtggaca gaaagacttg 900
ctcttcaagg atagtgctat cggattctca agagtccctc caagaattga ttccggcttg 960
tacctcggat cgggctactt cacagccatc cagaatttga gaaaatcaga agaagaagtt 1020
gctgccagaa gagctagagt tgtctggtgc gccgtcggtg aacaagaact gagaaagtgt 1080
aaccagtggt cgggcttgag tgaaggttca gtgacttgca gctccgcttc tacaactgaa 1140
gactgtattg ccctggtttt gaaaggcgaa gctgacgcca tgagcctgga tggaggctac 1200
gtctacaccg cttgcaagtg tggtctggtt cctgtcttgg ccgaaaatta caaatctcaa 1260
cagtcgtcag accctgaccc taactgcgtg gatagaccag ttgaaggata cttggctgtg 1320
gccgtggtta gaagatctga cacaagcctc acttggaaca gtgtcaaagg caaaaagtca 1380
tgtcacactg ctgtggacag aaccgccggt tggaatattc cgatgggact gttgttcaac 1440
caaactggct cctgcaagtt cgacgaatac ttctcacagt cttgtgctcc tggttccgat 1500
ccaagatcga atctgtgcgc cttgtgtata ggagacgaac aaggcgaaaa caaatgcgtc 1560
cccaattcga acgaaagata ctacggttac accggagctt tcagatgtct cgctgaaaat 1620
gccggagacg tcgccttcgt gaaggatgtg acagttctgc agaacactga tggcaacaat 1680
aacgaagctt gggccaaaga cctcaagctg gctgatttcg ccctcctgtg cctggacgga 1740
aaaagaaagc ctgtgactga agctagaagt tgtcacttgg ctatggcccc aaaccacgcc 1800
gtcgtgtcaa gaatggataa agtggaaaga ctgaagcaag ttttgctcca ccaacaggct 1860
aaattcggca gaaatggttc cgactgccct gataaattct gtttgttcca gtcggaaaca 1920
aagaacctgt tgttcaatga caacactgaa tgcctcgcta gactgcacgg caaaaccaca 1980
tacgaaaagt acctcggccc gcaatacgtt gctggtataa ctaatctgaa aaagtgcagc 2040
acctcccccc tcctggaagc ctgtgaattc ttgagaaaag aaaacctcta cttccagtca 2100
caccaccacc accaccacta a 2121
<210> 2
<211> 706
<212> PRT
<213>人工序列(Artificial Sequence)
<400> 2
Met Gly Arg Arg Arg Arg Ser Val Gln Trp Cys Ala Val Ser Gln Pro
1 5 10 15
Glu Ala Thr Lys Cys Phe Gln Trp Gln Arg Asn Met Arg Lys Val Arg
20 25 30
Gly Pro Pro Val Ser Cys Ile Lys Arg Asp Ser Pro Ile Gln Cys Ile
35 40 45
Gln Ala Ile Ala Glu Asn Arg Ala Asp Ala Val Thr Leu Asp Gly Gly
50 55 60
Phe Ile Tyr Glu Ala Gly Leu Ala Pro Tyr Lys Leu Arg Pro Val Ala
65 70 75 80
Ala Glu Val Tyr Gly Thr Glu Arg Gln Pro Arg Thr His Tyr Tyr Ala
85 90 95
Val Ala Val Val Lys Lys Gly Gly Ser Phe Gln Leu Asn Glu Leu Gln
100 105 110
Gly Leu Lys Ser Cys His Thr Gly Leu Arg Arg Thr Ala Gly Trp Asn
115 120 125
Val Pro Thr Gly Thr Leu Arg Pro Phe Leu Asn Trp Thr Gly Pro Pro
130 135 140
Glu Pro Ile Glu Ala Ala Val Ala Arg Phe Phe Ser Ala Ser Cys Val
145 150 155 160
Pro Gly Ala Asp Lys Gly Gln Phe Pro Asn Leu Cys Arg Leu Cys Ala
165 170 175
Gly Thr Gly Glu Asn Lys Cys Ala Phe Ser Ser Gln Glu Pro Tyr Phe
180 185 190
Ser Tyr Ser Gly Ala Phe Lys Cys Leu Arg Asp Gly Ala Gly Asp Val
195 200 205
Ala Phe Ile Arg Glu Ser Thr Val Phe Glu Asp Leu Ser Asp Glu Ala
210 215 220
Glu Arg Asp Glu Tyr Glu Leu Leu Cys Pro Asp Asn Thr Arg Lys Pro
225 230 235 240
Val Asp Lys Phe Lys Asp Cys His Leu Ala Arg Val Pro Ser His Ala
245 250 255
Val Val Ala Arg Ser Val Asn Gly Lys Glu Asp Ala Ile Trp Asn Leu
260 265 270
Leu Arg Gln Ala Gln Glu Lys Phe Gly Lys Asp Lys Ser Pro Lys Phe
275 280 285
Gln Leu Phe Gly Ser Pro Ser Gly Gln Lys Asp Leu Leu Phe Lys Asp
290 295 300
Ser Ala Ile Gly Phe Ser Arg Val Pro Pro Arg Ile Asp Ser Gly Leu
305 310 315 320
Tyr Leu Gly Ser Gly Tyr Phe Thr Ala Ile Gln Asn Leu Arg Lys Ser
325 330 335
Glu Glu Glu Val Ala Ala Arg Arg Ala Arg Val Val Trp Cys Ala Val
340 345 350
Gly Glu Gln Glu Leu Arg Lys Cys Asn Gln Trp Ser Gly Leu Ser Glu
355 360 365
Gly Ser Val Thr Cys Ser Ser Ala Ser Thr Thr Glu Asp Cys Ile Ala
370 375 380
Leu Val Leu Lys Gly Glu Ala Asp Ala Met Ser Leu Asp Gly Gly Tyr
385 390 395 400
Val Tyr Thr Ala Cys Lys Cys Gly Leu Val Pro Val Leu Ala Glu Asn
405 410 415
Tyr Lys Ser Gln Gln Ser Ser Asp Pro Asp Pro Asn Cys Val Asp Arg
420 425 430
Pro Val Glu Gly Tyr Leu Ala Val Ala Val Val Arg Arg Ser Asp Thr
435 440 445
Ser Leu Thr Trp Asn Ser Val Lys Gly Lys Lys Ser Cys His Thr Ala
450 455 460
Val Asp Arg Thr Ala Gly Trp Asn Ile Pro Met Gly Leu Leu Phe Asn
465 470 475 480
Gln Thr Gly Ser Cys Lys Phe Asp Glu Tyr Phe Ser Gln Ser Cys Ala
485 490 495
Pro Gly Ser Asp Pro Arg Ser Asn Leu Cys Ala Leu Cys Ile Gly Asp
500 505 510
Glu Gln Gly Glu Asn Lys Cys Val Pro Asn Ser Asn Glu Arg Tyr Tyr
515 520 525
Gly Tyr Thr Gly Ala Phe Arg Cys Leu Ala Glu Asn Ala Gly Asp Val
530 535 540
Ala Phe Val Lys Asp Val Thr Val Leu Gln Asn Thr Asp Gly Asn Asn
545 550 555 560
Asn Glu Ala Trp Ala Lys Asp Leu Lys Leu Ala Asp Phe Ala Leu Leu
565 570 575
Cys Leu Asp Gly Lys Arg Lys Pro Val Thr Glu Ala Arg Ser Cys His
580 585 590
Leu Ala Met Ala Pro Asn His Ala Val Val Ser Arg Met Asp Lys Val
595 600 605
Glu Arg Leu Lys Gln Val Leu Leu His Gln Gln Ala Lys Phe Gly Arg
610 615 620
Asn Gly Ser Asp Cys Pro Asp Lys Phe Cys Leu Phe Gln Ser Glu Thr
625 630 635 640
Lys Asn Leu Leu Phe Asn Asp Asn Thr Glu Cys Leu Ala Arg Leu His
645 650 655
Gly Lys Thr Thr Tyr Glu Lys Tyr Leu Gly Pro Gln Tyr Val Ala Gly
660 665 670
Ile Thr Asn Leu Lys Lys Cys Ser Thr Ser Pro Leu Leu Glu Ala Cys
675 680 685
Glu Phe Leu Arg Lys Glu Asn Leu Tyr Phe Gln Ser His His His His
690 695 700
His His
705
<210> 3
<211> 4157
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
ccatggcagc gtcgtgaaaa gaggcaatga caaatacaaa acgacgtatg agcagacccg 60
tcgccaagac gggtctacct ctaagatgat gtcatttgtt ttttaaaact aactcgcttt 120
acgagtagaa ttctacgtgt aaaacataat caagagatga tgtcatttgt ttttcaaaac 180
caaactcgct ttacgagtag aattctacgt gtaaaacaca atcaaaagat gatgtcattc 240
gtttttcaaa accgaattta agaaatgatg tcatttgttt ttcaaaacca aactcgcttt 300
acgagcagaa ttctacgtgt aaaacacaat caagagatga tgtcatttgt ttttcaaaac 360
tgaatgatgt catttgtttt tcaaaactaa acttgctttg cgagtagaat tctacgtgta 420
aaacacagtc aagagatgat gtcatttgtt tttcaaaact gaaccggctt tacgagtaga 480
attctacttg taaaacataa tcaagagatg atgtcatttg tttttcaaaa ctgaactggc 540
tttacgagta gaattctacg tgtaaaacat aatcaagaga tgatgtcatc attaaactga 600
tgtcatttta tacacgattg ttaacatgtt taataatgac taatttgttt ttccaaatta 660
aactcgcttt acgagtagaa ttctacttgt aacgcacgat taagtatgaa tcataagctg 720
atgtcatttg ttttcgacat aaaatgttta tacaatggaa tcttcttgta aattatccaa 780
ataatataat ttatccgatt ctacgttaca tttaaattcg ttgttatcgt acaattcttc 840
aggacacgcc atgtattggt catttttagc gtgcaaccaa cgattgtatt tgacgccgtc 900
gttggattgc gtgttcaggt tggcgtacac gtgactgggc acggcttctt tttccatggg 960
acgtcgacga aaacagcaca cacactacat accatgtatt tgacgcacac acgcatgtat 1020
actatttatt gtcaaacttt tgttcttgac gtctgtgttc aaactgagaa tagattaaat 1080
attgtttgtc tttattaata ttttttaata gtgtagtctt ggcgaaattt gtgattataa 1140
aagtataaaa tacaatcata atagtgtacg aacttacaat tccaattaat tatagtcgaa 1200
tttcgactac tgcgggacct ctagtattaa taattctctt taaaaaaaaa cagagcatca 1260
aatactgcac aaatgtcaag cgggtctcaa cgagccatga ataaattaga aatcaattaa 1320
taacataaaa taggcaaaca aaataaaacc atttacatag agaacgtttg ttgaacaaaa 1380
acaataactt gtatacattg tttgcacaaa tgtttgaagc gaaaatttat tactctctac 1440
gtaagcttga tcaaacttcg ttttcgtata aaacgcgttg gcccaaccac tttggcatag 1500
tcgtcttatc atcgggtctc taaggatcaa gcgatccaaa gaccgccaac atgcgtttcg 1560
ttctgtgctg cactttgatt gcgttggctg cgctcagcgt aaaagccttc ggtcaccacc 1620
ccggcaatcg agatacagga tccatgggca gaagaagaag atctgtccaa tggtgcgctg 1680
tgagccagcc ggaagccacc aaatgtttcc aatggcagag aaatatgaga aaggttagag 1740
gtcctccagt ctcttgcata aaaagagata gccccatcca atgtattcag gctatagccg 1800
aaaacagagc tgacgccgtg acattggatg gtggattcat ctacgaagct ggtctcgccc 1860
cgtacaaact gagacccgtt gctgccgaag tctacggaac cgaaagacaa ccgagaacac 1920
actactacgc tgtggccgtg gttaaaaagg gcggttcttt ccaattgaat gaactccagg 1980
gtctgaagag ctgccacacc ggtctgagaa gaacagctgg atggaatgtt cctactggca 2040
ccttgagacc attcctcaac tggacaggac cgcccgaacc tattgaagct gccgttgcta 2100
gattcttctc agcctcttgc gtccctggag ctgacaaggg ccaattccca aatttgtgca 2160
gactctgtgc tggtactgga gaaaacaaat gcgccttctc atctcaggaa ccatacttca 2220
gctactccgg agctttcaag tgtttgagag acggcgctgg tgacgtcgcc ttcataagag 2280
aatccaccgt gttcgaagac ctctcggatg aagctgaaag agacgaatac gaactgttgt 2340
gcccggataa cacaagaaaa cccgttgaca aattcaagga ttgtcacctc gctagagtgc 2400
caagtcacgc tgtcgtggcc agatcagtta atggtaaaga agatgctatc tggaacctcc 2460
tgagacaagc ccaggaaaaa ttcggaaaag acaagagccc gaagttccaa ctgttcggtt 2520
cgcccagtgg acagaaagac ttgctcttca aggatagtgc tatcggattc tcaagagtcc 2580
ctccaagaat tgattccggc ttgtacctcg gatcgggcta cttcacagcc atccagaatt 2640
tgagaaaatc agaagaagaa gttgctgcca gaagagctag agttgtctgg tgcgccgtcg 2700
gtgaacaaga actgagaaag tgtaaccagt ggtcgggctt gagtgaaggt tcagtgactt 2760
gcagctccgc ttctacaact gaagactgta ttgccctggt tttgaaaggc gaagctgacg 2820
ccatgagcct ggatggaggc tacgtctaca ccgcttgcaa gtgtggtctg gttcctgtct 2880
tggccgaaaa ttacaaatct caacagtcgt cagaccctga ccctaactgc gtggatagac 2940
cagttgaagg atacttggct gtggccgtgg ttagaagatc tgacacaagc ctcacttgga 3000
acagtgtcaa aggcaaaaag tcatgtcaca ctgctgtgga cagaaccgcc ggttggaata 3060
ttccgatggg actgttgttc aaccaaactg gctcctgcaa gttcgacgaa tacttctcac 3120
agtcttgtgc tcctggttcc gatccaagat cgaatctgtg cgccttgtgt ataggagacg 3180
aacaaggcga aaacaaatgc gtccccaatt cgaacgaaag atactacggt tacaccggag 3240
ctttcagatg tctcgctgaa aatgccggag acgtcgcctt cgtgaaggat gtgacagttc 3300
tgcagaacac tgatggcaac aataacgaag cttgggccaa agacctcaag ctggctgatt 3360
tcgccctcct gtgcctggac ggaaaaagaa agcctgtgac tgaagctaga agttgtcact 3420
tggctatggc cccaaaccac gccgtcgtgt caagaatgga taaagtggaa agactgaagc 3480
aagttttgct ccaccaacag gctaaattcg gcagaaatgg ttccgactgc cctgataaat 3540
tctgtttgtt ccagtcggaa acaaagaacc tgttgttcaa tgacaacact gaatgcctcg 3600
ctagactgca cggcaaaacc acatacgaaa agtacctcgg cccgcaatac gttgctggta 3660
taactaatct gaaaaagtgc agcacctccc ccctcctgga agcctgtgaa ttcttgagaa 3720
aagaaaacct ctacttccag tcacaccacc accaccacca ctaagcggcc gctacaacta 3780
aacacgactt ggagtattcc ttgtagtgtt taagatttta aatcttactt aatgacttcg 3840
aacgatttta acgataactt tctctttgtt taactttaat cagcatacat aaaaagcccc 3900
ggttttgtat cgggaagaaa aaaaatgtaa ttgtgttgcc tagataataa acgtattatc 3960
aaagtgtgtg gttttccttt accaaagacc cctttaagat gggcctaatg ggcttaagtc 4020
gagtcctttc cgatgtgtta aatacacatt tattacactg atgcgtcgaa tgtacacttt 4080
taataggata gctccactaa aaattatttt atttatttaa tttgttgcac caaaactgat 4140
acattgacga aaagctt 4157
Claims (8)
1.适于家蚕丝腺表达的改造人乳铁蛋白基因，其特征在于：所述改造人乳铁蛋白基因如SEQ ID NO.1所示。
2.含有权利要求1所述适于家蚕丝腺表达的改造人乳铁蛋白基因的表达载体。
3.根据权利要求2所述的表达载体，其特征在于：所述表达载体含有顺序连接的增强子hr3，分泌型丝胶1基因启动子、改造人乳铁蛋白基因和丝胶1基因的终止子。
4.根据权利要求2所述的表达载体，其特征在于：所述表达载体还含有荧光筛选标记基因表达框，所述荧光筛选标记基因表达框位于所述增强子hr3上游。
5.权利要求3或4所述表达载体在家蚕丝腺中表达重组人乳铁蛋白中的应用。
6.利用权利要求3或4所述表达载体在家蚕丝腺中表达重组人乳铁蛋白的方法，其特征在于，包括如下步骤：将所述表达载体转化解除滞育家蚕蚕卵，用无毒胶水封口后经甲醛蒸汽消毒，孵化，筛选转基因阳性蛾圈，取阳性转基因家蚕茧壳，在液氮中粉碎成粉末，然后溶解于含50mM Tris-Cl、8M Urea、pH 8.0的缓冲液中，80℃水浴40min，离心收集上清，经纯化，得重组人乳铁蛋白。
7.根据权利要求6所述的方法，其特征在于：茧壳粉末溶解于缓冲液中茧壳粉末的浓度为20mg/mL。
8.根据权利要求6所述的方法，其特征在于，所述纯化的具体方法为：将上清液过HisTrapTM FF crude亲和层析柱，用咪唑浓度为0~500mM的咪唑洗脱液洗脱，咪唑洗脱液用pH7.0-7.5、浓度为8M Urea溶液配制，收集洗脱液，洗脱液即为纯化后的重组人乳铁蛋白。
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