CN115247160B - 蛋白核心岩藻糖基化修饰的检测方法 - Google Patents
蛋白核心岩藻糖基化修饰的检测方法 Download PDFInfo
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Abstract
本发明适用于蛋白检测技术领域，提供了蛋白核心岩藻糖基化修饰的检测方法，包括以下步骤：首先运用基因重组的线虫来源的β‑1,4‑半乳糖基转移酶(GALT‑1)催化非天然糖供体UDP‑Gal‑N3上的半乳糖残基转移连接到N‑聚糖最内侧GlcNAc末端的核心岩藻糖上，然后利用点击化学反应将带有炔烃修饰的荧光基团或者亲和(biotin)报告基团连接到修饰的复合物上。本发明基于酶催化的方法对活细胞表面和糖蛋白上的核心岩藻糖基化修饰进行特异性的标记。
Description
技术领域
本发明属于蛋白检测技术领域，尤其涉及蛋白核心岩藻糖基化修饰的检测方法。
背景技术
翻译后修饰是哺乳动物区别于低等生物的一个重要因素。其中，糖基化修饰是一种普遍而复杂的翻译后修饰方式。糖基化修饰的复杂性主要与两方面有关：多样的单糖和各个单糖连接的多样性。糖基化修饰主要分为N-聚糖(N-glycans)、O-聚糖(O-glycans)、糖基磷脂酰肌醇(GPI)锚定蛋白、糖胺聚糖(GAGs)等。
核心岩藻糖基化是岩藻糖以α1,6键的形式与N-聚糖最里面的乙酰葡萄糖胺(以下简称GlcNAc)连接的一种修饰。目前研究表明核心岩藻糖基化只发生在N-聚糖上，并且只有一种糖基转移酶——岩藻糖基转移酶8(FUT8)能催化这一过程。
与正常组织相比，肿瘤组织的核心岩藻糖水平显著升高。而且许多核心岩藻糖修饰的糖蛋白有望作为诊断肿瘤的重要生物标志物。例如，甲胎蛋白(AFP)是肝细胞癌(HCC)的重要生物标志物，而其在肝病中表达量也会升高。但是，甲胎蛋白的核心岩藻糖修饰只在肝细胞癌中升高。因此，核心岩藻糖修饰的AFP是更可靠的肝细胞癌生物标志物。2005年，甲胎蛋白异质体(AFP-L3)被美国食品与药品监督管理局(FDA)批准为肝细胞癌早期诊断的生物标志物。
目前检测核心糖基化修饰的手段主要包括液相色谱-串联质谱联用技术(LC-MS/MS)和凝集素免疫识别。串联质谱方法需要对样品进行较繁琐的前处理，而且数据的获取需要较高的专业技能。凝集素方法中，橙黄网胞盘菌凝集素(Aleuria Aurantia Lectin，AAL)和扁豆凝集素(Lens Culinaris Agglutinin，LCA)主要识别核心岩藻糖基化，但凝集素识别效率不高且信号易丢失，缺乏严谨的特异性，不利于糖蛋白的富集和纯化。因此，亟需开发一种新的方法对核心岩藻糖进行特异性的检测。
发明内容
本发明实施例的目的在于提供蛋白核心岩藻糖基化修饰的检测方法，旨在解决上述背景技术中提出的问题。
为实现上述目的，本发明提供如下技术方案：
蛋白核心岩藻糖基化修饰的检测方法，包括以下步骤：
首先运用基因重组的线虫来源的GALT-1催化非天然糖供体UDP-Gal-N3上的半乳糖残基转移连接到N-聚糖最内侧GlcNAc末端的核心岩藻糖上，然后利用点击化学反应将带有炔烃修饰的荧光基团或亲和报告基团连接到修饰的复合物上。
进一步的，构建GALT-1重组质粒的具体操作为：
首先对目的基因做密码子序列优化之后进行基因合成，该基因全长为1218bp，编码含406个氨基酸的蛋白质，然后以pFastBacTMHT为载体构建重组表达质粒，利用通用引物M13-F和M13-R对重组质粒进行PCR。
进一步的，所述PCR产物的全长为3648bp，所述PCR产物大小的计算方式为：目的基因全长+2430bp。
进一步的，在sf9昆虫细胞中表达所述GALT-1重组蛋白，具体操作为：
将sf9细胞接种于500ml细胞培养瓶中进行体系扩大培养，待细胞生长至对数期，用P2阶段的病毒液感染sf9细胞，直到细胞形态发生明显变化，收集并裂解细胞，对目的蛋白进行检测。
进一步的，纯化所述目的蛋白的具体操作为：
将细胞裂解液上清与镍柱过夜结合后，用不同浓度的咪唑洗脱液对结合在镍柱上的蛋白进行洗脱，先用低浓度咪唑使杂蛋白从镍柱上洗脱下来，再用高浓度咪唑洗脱目的蛋白；对经镍柱纯化并超滤浓缩后的蛋白进行离子交换层析。
与现有技术相比，本发明的有益效果是：
该蛋白核心岩藻糖基化修饰的检测方法，首先运用基因重组的线虫来源的β-1,4-半乳糖基转移酶(GALT-1)催化非天然糖供体UDP-Gal-N3上的半乳糖残基转移连接到N-聚糖最内侧GlcNAc末端的核心岩藻糖上，然后利用点击化学反应将带有炔烃修饰的荧光基团或者亲和(biotin)报告基团连接到修饰的复合物上。本发明基于酶催化的方法对活细胞表面和糖蛋白上的核心岩藻糖基化修饰进行特异性的标记。
附图说明
图1为本发明的示意图。
图2为本发明中琼脂糖凝胶电泳结果图。
图3为本发明中序列比对结果图。
图4为本发明中目的蛋白检测结果图。(M：标准分子量蛋白Marker；P：His阳性对照；Lane 1：sf9细胞裂解沉淀；Lane 2:sf9细胞裂解上清)
图5为本发明中SDS-PAGE和Western Blot检测离子交换层析洗脱曲线的各洗脱峰对应的蛋白溶液，根据分子量大小确定目的蛋白GALT-1所在泳道位置。(M：标准分子量蛋白Marker；E：镍柱纯化并超滤浓缩后的总蛋白；Lane 1-6：离子交换层析洗脱曲线的各洗脱峰下收集的蛋白)
图6为本发明中SDS-PAGE和Western Blot检测过表达FUT8对GALT-1化学酶标记法标记细胞裂解液的影响结果图。
图7为本发明中Western Blot检测敲减FUT8对GALT-1化学酶标记法标记细胞裂解液的影响结果图。
图8为本发明中Western Blot检测GALT-1化学酶标记法特异性标记核心岩藻糖基化。
图9为本发明中不同浓度梯度的GALT-1和糖供体的标记效果结果图。
图10为本发明中免疫荧光实验检测过表达FUT8对GALT-1化学酶标记法标记活细胞表面糖链的影响结果图。
图11为本发明中免疫荧光实验检测敲减FUT8对GALT-1化学酶标记法标记活细胞表面糖链的影响结果图。
图12为本发明中使用PNGase F对CHO细胞上的N-糖链进行水解验证GALT-1化学酶法在CHO细胞表面的标记位点结果图。
图13为本发明中采用酶联免疫吸附测定法，通过生物素-链霉亲和素-TMB显色检测标记效果结果图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行详细描述。
本发明一个实施例提供的蛋白核心岩藻糖基化修饰的检测方法，包括以下步骤：
首先运用基因重组的线虫来源的β-1,4-半乳糖基转移酶(GALT-1)催化非天然糖供体UDP-Gal-N3上的半乳糖残基转移连接到N-聚糖最内侧GlcNAc末端的核心岩藻糖上，然后利用点击化学反应将带有炔烃修饰的荧光基团或亲和报告基团连接到修饰的复合物上。
作为本发明的一种优选实施例，构建GALT-1重组质粒的具体操作为：
首先对目的基因做密码子序列优化之后进行基因合成，该基因全长为1218bp，编码含406个氨基酸的蛋白质，然后以pFastBacTMHT为载体构建重组表达质粒，利用通用引物M13-F和M13-R对重组质粒进行PCR。
在本发明实施例中，由于β-1,4-半乳糖基转移酶(GALT-1)在大肠杆菌E.coli原核表达系统中容易产生大量没有活性的包涵体蛋白，因此尝试在杆状病毒-昆虫表达系统中表达具有活性的目的蛋白。
作为本发明的一种优选实施例，所述PCR产物的全长为3648bp，所述PCR产物大小的计算方式为：目的基因全长+2430bp。
在本发明实施例中，参见图2，琼脂糖凝胶电泳结果显示PCR产物大小在3000到4000bp之间，验证了重组载体的正确性。接着重组载体经测序后，参见图3，序列比对结果显示目的基因GALT-1的序列一致率为100％。
作为本发明的一种优选实施例，在sf9昆虫细胞中表达所述GALT-1重组蛋白，具体操作为：
将sf9细胞接种于500ml细胞培养瓶中进行体系扩大培养，待细胞生长至对数期，用P2阶段的病毒液感染sf9细胞，直到细胞形态发生明显变化，收集并裂解细胞，对目的蛋白进行检测。
在本发明实施例中，参见图4，检测结果表明，当扩大细胞体系对蛋白进行放大表达时，GALT-1在沉淀中的蛋白含量明显高于上清，且在细胞裂解沉淀和上清中均有不同分子量但可以被His抗体所识别的杂蛋白，推测造成该现象的原因可能是：(1)His抗体特异性不高，可以与杂蛋白结合；(2)细胞培养体系扩大之后导致杂蛋白含量的提高。
作为本发明的一种优选实施例，纯化所述目的蛋白的具体操作为：
将细胞裂解液上清与镍柱过夜结合后，用不同浓度的咪唑洗脱液对结合在镍柱上的蛋白进行洗脱，先用低浓度咪唑使杂蛋白从镍柱上洗脱下来，再用高浓度咪唑洗脱目的蛋白；对经镍柱纯化并超滤浓缩后的蛋白进行离子交换层析。
在本发明实施例中，目的蛋白的序列如SEQ ID NO:1所示；参见图5，将层析曲线中各个洗脱峰对应的蛋白溶液收集下来进行SDS-PAGE和Western Blot分析，根据分子量可知层析得到的目的蛋白位于6号泳道，根据目的蛋白与杂蛋白条带的比例可知，目的蛋白GALT-1纯度在50％左右，BCA检测蛋白浓度为0.2mg/mL。
研究与分析
1、GALT1标记细胞裂解液蛋白的核心岩藻糖修饰
1.1过表达FUT8对GALT-1化学酶标记法标记细胞裂解液的影响
由于FUT8是目前发现的唯一参与催化核心岩藻糖基化的糖基转移酶，且CHO细胞系被广泛应用于FUT8的过表达与敲低。因此，通过在CHO细胞系中过表达FUT8来提高核心岩藻糖基化修饰水平，从而研究GALT-1酶的活性以及其标记效果是否受FUT8介导的核心岩藻糖基化修饰的影响。
参见图6，SDS-PAGE和Western Blot结果表明，GALT-1能标记细胞裂解液的核心岩藻糖，且过表达FUT8能显著增强GALT-1化学酶法对细胞裂解液的标记效果。
1.2敲减FUT8对GALT-1化学酶标记法标记细胞裂解液的影响
在CHOK1细胞系中利用shRNA敲减了FUT8，从而进一步验证GALT-1化学酶标记法的裂解液标记效果与FUT8介导的核心岩藻糖基化修饰之间的相关性。
参见图7，Western Blot结果表明，FUT8表达水平降低后，GALT-1化学酶法对细胞裂解液的标记效果显著减弱。因此，通过过表达和shRNA改变CHOK1的FUT8表达水平可以得出结论：GALT-1化学酶标记法能够在细胞裂解液水平上对核心岩藻糖基化修饰进行标记，且GALT-1化学酶标记法的标记效果与FUT8介导的核心岩藻糖基化修饰水平之间呈正相关。
1.3GALT-1化学酶标记法对核心岩藻糖的标记特异性
为了进一步研究GALT-1化学酶标记法除了标记细胞裂解液核心岩藻糖基化修饰以外，是否会对其他类型的岩藻糖基化修饰进行标记。使用α-1-2岩藻糖苷酶和α-1-3/4岩藻糖苷酶分别处理细胞裂解液，接着标记去验证GALT-1化学酶法的标记特异性。
参见图8，Western Blot结果表明，在其他条件一致的情况下，水解α-1-2和α1-3/4岩藻糖基的实验组与不去糖基的实验组相比，标记效果均无明显差异，说明GALT-1化学酶标记法只会特异性标记核心岩藻糖基化，而不会对α-1-2和α-1-3/4连接类型的岩藻糖基化修饰进行标记。
2、GALT1标记活细胞表面岩藻糖基化
2.1GALT-1化学酶标记法标记活细胞表面的条件优化
在活细胞水平上进行免疫荧光实验验证了GALT-1化学酶标记法的标记效果。首先，研究不同浓度梯度的GALT-1和糖供体的标记效果。
参见图9，结果表明，当糖供体与GALT-1的终浓度分别在125-500μM和0.1-0.4mg/mL范围时，标记强度随着二者浓度的增加呈现倍数式增长，且当糖供体终浓度为250μM、GALT-1终浓度为0.4mg/mL时，可以实现细胞表面的完整标记，使荧光在细胞表面的分布具有相对连续性。因此在后续的实验中，将基于250μM糖供体和0.4mg/mL GALT-1这一条件进行研究。
2.2过表达FUT8对GALT-1化学酶标记法标记活细胞表面糖链的影响
在优化了活细胞表面的标记条件后，利用过表达FUT8的CHO细胞系，探究FUT8表达水平是否会对GALT1在活细胞表面的标记效果产生影响。
参见图10，荧光共聚焦结果表明，FUT8表达水平上调能显著增强GALT-1化学酶标记法对活细胞表面的标记效果。
2.3敲减FUT8对GALT-1化学酶标记法标记活细胞表面糖链的影响
对应上面的过表达，为了进一步验证GALT-1化学酶标记法的标记效果与FUT8介导的核心岩藻糖基化修饰之间的相关性，利用FUT8敲减的CHO细胞系进行免疫荧光实验。
参见图11，结果表明，FUT8表达水平下降会显著减弱GALT-1对CHO细胞表面的标记效果。因此，通过过表达和敲减FUT8可以得出结论：GALT-1能够对活细胞表面由FUT8介导的核心岩藻糖基化修饰进行标记，且标记效果与FUT8的表达水平呈正相关。
2.4去N-糖基化对GALT-1化学酶标记法标记细胞表面糖链的影响
为了进一步验证GALT-1化学酶法在CHO细胞表面的标记位点，使用PNGase F对CHO细胞上的N-糖链进行水解。
参见图12，结果表明，PNGase F处理的实验组与不加PNGase F的实验组相比标记强度显著减弱，证明了GALT-1化学酶标记法在细胞表面的标记位点位于N-糖链上，该实验在细胞裂解液的水平上也得到了同样的结果。
3、GALT-1在ELISA平板标记抗体糖链
在动物细胞分泌的免疫球蛋白中N-糖基化是最常见的糖基化修饰，同时也是研究最多的一种糖基化修饰。以IgG1为例，其糖基化修饰以岩藻糖(Fuc)为核心，再由N-乙酰氨基葡萄糖(N-GlcNAc)分出两条等长的分支，其分支上伴以甘露糖(Man)、半乳糖(Gal)和唾液酸(Sia)，由此构成IgG1的Fc端N位糖基化。由于其含有核心岩藻糖基化修饰，可以用来检测GALT-1的标记效果。
而本实验采用酶联免疫吸附测定法(Enzyme Linked Immunosorbent Assay，ELISA)的方法，利用固相载体聚苯乙烯对蛋白质较强的吸附作用，在96孔板上吸附抗体蛋白，并且吸附后仍保留原来活性。对吸附完成的96孔板进行封闭以占满未结合的位置，封闭完成后在孔中进行GALT-1标记反应，最后通过生物素-链霉亲和素-TMB显色检测标记效果。
参见图13，从结果可以看出，与对照组相比，随着包被抗体量的增加，显色反应显著增强，表明GALT-1在96孔板上能成功标记抗体的核心岩藻糖基化修饰。
以上仅是本发明的优选实施方式，应当指出，对于本领域的技术人员来说，在不脱离本发明构思的前提下，还可以作出若干变形和改进，这些也应该视为本发明的保护范围，这些均不会影响本发明实施的效果和专利的实用性。
Claims (2)
1.蛋白核心岩藻糖基化修饰的检测方法，其特征在于，包括以下步骤：
首先运用基因重组的线虫来源的GALT-1催化非天然糖供体UDP-Gal-N3上的半乳糖残基转移连接到N-聚糖最内侧GlcNAc末端的核心岩藻糖上，然后利用点击化学反应将带有炔烃修饰的荧光基团或亲和报告基团连接到修饰的复合物上；
构建GALT-1重组质粒的具体操作为：
首先对目的基因做密码子序列优化之后进行基因合成，该基因全长为1218 bp，编码含406个氨基酸的蛋白质，然后以pFastBacTMHT 为载体构建重组表达质粒，利用通用引物M13-F和M13-R对重组质粒进行 PCR；
所述PCR产物的全长为3648 bp，所述PCR产物大小的计算方式为：目的基因全长+2430bp；
所述目的基因序列如SEQ ID NO:1所示；
在sf9昆虫细胞中表达所述GALT-1重组蛋白，具体操作为：
将sf9细胞接种于500 ml细胞培养瓶中进行体系扩大培养，待细胞生长至对数期，用P2阶段的病毒液感染sf9细胞，直到细胞形态发生明显变化，收集并裂解细胞，对目的蛋白进行检测；
所述蛋白核心岩藻糖基化修饰的检测方法是采用酶联免疫吸附测定法，利用固相载体聚苯乙烯对蛋白质较强的吸附作用，在96孔板上吸附抗体蛋白，并且吸附后仍保留原来活性；对吸附完成的96孔板进行封闭以占满未结合的位置，封闭完成后在孔中进行GALT-1标记反应，最后通过生物素-链霉亲和素-TMB显色检测标记效果。
2.根据权利要求1所述的蛋白核心岩藻糖基化修饰的检测方法，其特征在于，纯化所述目的蛋白的具体操作为：
将细胞裂解液上清与镍柱过夜结合后，用不同浓度的咪唑洗脱液对结合在镍柱上的蛋白进行洗脱，先用低浓度咪唑使杂蛋白从镍柱上洗脱下来，再用高浓度咪唑洗脱目的蛋白；对经镍柱纯化并超滤浓缩后的蛋白进行离子交换层析。
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