CN104630172A - 一种血吸虫谷胱甘肽-s-转移酶的突变体及其应用 - Google Patents
一种血吸虫谷胱甘肽-s-转移酶的突变体及其应用 Download PDFInfo
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- CN104630172A CN104630172A CN201510043756.9A CN201510043756A CN104630172A CN 104630172 A CN104630172 A CN 104630172A CN 201510043756 A CN201510043756 A CN 201510043756A CN 104630172 A CN104630172 A CN 104630172A
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Abstract
本发明属于酶的基因工程技术领域，具体涉及一种血吸虫谷胱甘肽-S-转移酶的变体以及其应用。本发明提供了一种改变血吸虫免疫保护性的血吸虫谷胱甘肽-S转移酶(sj26GST)突变酶，将来源于日本血吸虫（Schistosoma japonicum）的26kDa的谷胱甘肽-S转移酶进行突变所得的单突变酶与野生型相比，所述的sj26GST的突变酶的保护性获得提高，突变酶IgG1水平显著高于重组野生型sj26GST蛋白，减虫率由原来的34%提高到40%，减卵率由37%提高到40%。该重组抗原具有良好的免疫原性，适于作为抗血吸虫病候选疫苗。
Description
技术领域
本发明属于酶的基因工程技术领域，具体涉及一种血吸虫谷胱甘肽-S-转移酶的变体以及其应用
背景技术
血吸虫病是一种严重危害人类健康的人畜共患寄生虫病，我国主要流行日本血吸虫病，广泛流行于我国长江沿岸以及四川、云南省等地，急性感染病例时有发生，虽然化学药物对血吸虫病的有治疗作用，但不能阻断血吸虫的反复感染，另外目前唯一治疗血吸虫病的药物吡喹酮已经有了耐药性的报道，因此,研制有效的血吸虫病疫苗和新药物报表的筛选显得十分重要。
目前的血吸虫疫苗所诱导的免疫保护力普遍水平不高，仅能诱导出宿主对血吸虫感染的部分抵抗力。血吸虫谷胱甘肽-S-转移酶(sjGST)是血吸虫生理功能所需的重要解毒酶，对虫体的生理功能起重要作用，是最具发展前途的血吸虫病候选分子疫苗之一。日本血吸虫26kD GST基因能够在大肠杆菌中高效表达(sj26GST),用sj26GST直接免疫小鼠,可以在宿主体内诱导保护性免疫反应，sj26GST抗原分子的研究是日本血吸虫疫苗研究的热点。
由于血吸虫是一种多细胞生物，生活史复杂。感染后宿主保护性免疫机制复杂，因此提高疫苗分子的免疫效果是当前血吸虫病疫苗研究主要方向。
发明内容
本发明要解决的技术问题是提供一种改变血吸虫免疫保护性的血吸虫谷胱甘肽-S转移酶(sj26GST)突变酶，将来源于日本血吸虫(Schistosoma japonicum)的26kDa的谷胱甘肽-S转移酶进行突变所得的单突变酶与野生型相比，所述的sj26GST的突变酶的保护性获得提高，突变酶IgG1水平显著高于重组野生型sj26GST蛋白，减虫率由原来的34％提高到40％，减卵率由37％提高到40％。该重组抗原具有良好的免疫原性，适于作为抗血吸虫病候选疫苗。
具体的，本发明提供了一种谷胱甘肽S-转移酶的突变体，其特征在于，所述突变体谷胱甘肽S-转移酶的第67位的氨基酸进行了突变，且该突变体的免疫原性显著强于其亲本。
进一步的，所述的第67位的谷氨酰胺(Gln)替换为谷氨酸(Glu)。
进一步的，所述突变体是源于日本吸血虫(Schistosoma japonicum)的26kD谷胱甘肽-S转移酶的突变。
进一步的，上述谷胱甘肽S-转移酶的突变体的氨基酸序列如SEQ ID：NO 4所示。
另一方面，本发明还提供了所述的谷胱甘肽S-转移酶的突变体在制备治疗血吸虫病的疫苗中的应用。
另一方面，本发明还提供了一种核酸分子，其编码上述任一项所述的谷胱甘肽S-转移酶的突变体。
进一步的，上述的核苷酸序列如SEQ ID：NO 3所示。
另一方面，本发明还提供了一种重组表达质粒，其含有上述的核苷酸分子。
本发明还提供sj26GST的突变酶Q67E的制备方法，其具体步骤为：
(1)设计定点突变的突变引物，以携带sj26GST酶基因的载体为模板进行定点突变构建突变质粒pET-21d-Q67E；
(2)将突变质粒转化大肠杆菌BL21(DE3)细胞，挑选验证后的阳性单克隆发酵培养；
(3)离心菌体，重悬后超声，亲和层析纯化得到突变体Q67E。
本发明中所述来源Schistosoma japonicum的sj26GST在GenBank的编号为M14654.1,基因全长657个核苷酸，见序列表中SEQ ID No：1，编码218个氨基酸，见序列表中SEQ ID No：2。
所述sj26GST的突变酶是将其67位氨基酸谷氨酰胺Gln突变为谷氨酸Glu，命名为Q67E。Q67E的核苷酸序列见序列表中SEQ ID No.3，氨基酸序列见表中SEQ ID No:4。
本发明在对谷胱甘肽-S转移酶的结构、功能以及对日本血吸虫保护性研究的基础上，通过定点突变的方法，对日本血吸虫来源的sj26GST进行分子改造，发现该突变酶诱导小鼠体内产生抗重组抗原的特异性IgG、IgG1和IgG2a抗体并能达到一个较高的水平，IgG1较重组野生型蛋白有显著提高，动物保护实验诱导了40％的减虫率和40％的减卵率，表明该重组谷胱甘肽-S转移酶突变酶适于作为抗血吸虫病候选疫苗，本发明还表明sj26GST蛋白质的67位氨基酸的突变对血吸虫的免疫性保护反应有重要影响，本研究可为筛选和设计血吸虫疫苗候选分子提供新思路。
附图说明
图1是本发明实施例2重组野生型酶sj26GST与突变体酶Q67E表达纯化结果图。
图2是本发明实施例3重组sj26GST突变酶与野生型酶Q67E酶活，突变酶Q67E的酶活性小于野生型sj26GST。
图3和图4是本发明实施列4突变体酶Q67E与野生型sj26GST相比,突变酶的减虫率较野生酶提高到40％。
图5和图6是本发明实施列4突变体酶Q67E与野生型sj26GST相比,突变酶的减卵率较野生酶提高到40％。
图7和图8是本发明实施列4野生型酶sj26GST与突变体酶Q67E特异性IgG、IgG1和IgG2a抗体水平的检测比较结果图。
具体实施方式
下列实施例中，未注明具体条件的实验方法，按常规条件，如《分子克隆实验指南》第3版(J.莎姆布鲁克、D.W.拉塞尔等主编，黄培堂等译.北京，科学出版社，2005)中所述方法进行。
实施例1 sj26GST及其变体Q67E原核表达载体的构建和蛋白纯化
以质粒pALEX为模板，用PCR的方法扩增野生型GST的编码序列。所用的上游引物为：5’-catgccatgggcatg tcc cct atactaggt tattgg-3’引入NcoI的酶切位点(下划线)，下游引物为：5’-ctgtagcggccgctctagactcgagttt tgg agg atg gtc gcc ac-3’，引入XhoI(下划线)的酶切位点。PCR产物纯化后用NcoI/XhoI双酶切，插入pET21d的相应位点。对重组表达载体进行DNA测序，分析其阅读框架和编码序列的正确性。突变蛋白Q67E的序列和野生型GST构建的N/C端序列一致，仅在特定位点进行定点突变。此构建的GST在C末端额外加上了LEHHHHHH，以便纯化。
突变引物如下所示：
锚定引物和定点突变用引物的磷酸化反应：在两个PCR反应管中分别配制下列两种引物的磷酸化反应液，各反应管在37℃水浴中反应30min，再在70℃水浴中加热10min，置于-20℃保存备用。
(1)锚定引物的磷酸反应液：
(2)定点突变用引物的磷酸化反应液：
退火反应：在PCR反应管中配制下列反应液，99℃加热3min，经30min缓慢降至45℃，在45℃保温10sec后，再降温至4℃
a dsDNA模板为野生型GST的PCR产物，每个反应模板的量为100ng。
b 10×Annealing Buffer:200mM Tris-HCl,pH 7.5,500mM NaCl,100mM MgCl2.
延伸、连接反应：将下列组分混合后加入到退火反应液中，37℃水浴锅中反应2hr。
PCR扩增反应：按下列组分配制PCR反应液，总体积为50μl。PCR反应条件为：94℃预变性4min；随后94℃变性40sec，55℃退火50sec，72℃延伸1min，进行28个循环，最后72℃保温7min
经琼脂糖电泳胶回收纯化后的PCR扩增产物经限制性内切酶Nco I和Xho I消化后连接至载体pET-21d，并转至大肠杆菌TOP10感受态细胞中，在含50μg/ml氨苄青霉素的LB固体培养基培养过夜。挑取单菌落，应用菌落PCR方法筛选阳性克隆，对阳性克隆进行酶切鉴定和DNA测序，鉴定为正确的序列。
实施例2 Schistosoma japonicum的sj26GST及突变酶Q67E的表达纯化
将测序验证后的GST质粒pET-21d-GST及其变体表达质粒pET-21d-Q67E分别转化大肠杆菌BL21(DE3)宿主菌中诱导表达。挑取阳性转化子于LB培养基37℃、200rpm摇培过夜，后接种于LB培养基(含100μg/ml氨苄青霉素)28℃、250rpm下培养至OD600为0.8左右，加入终浓度为0.2mM的IPTG，28℃诱导表达6hr。
发酵液6000rpm、4℃离心10min，取菌体加缓冲液20mL(50mM Na2HPO4-NaH2PO4，pH8.0，150mM NaCl，1mM PMSF)重悬，经超声破碎后离心回收可溶性蛋白。
蛋白样品上样于预先用缓冲液B(50mM Na2HPO4-NaH2PO4，pH8.0，150mM NaCl)平衡好的镍金属螯合亲和层析柱(1.6cm×5cm)。非特异性结合的蛋白使用缓冲液C(50mMNa2HPO4-NaH2PO4，pH 8.0，150mM NaCl，20mM咪唑)进行洗脱，含组氨酸标签的融合蛋白使用缓冲液(50mM Na2HPO4-NaH2PO4，pH 8.0，150mM NaCl，250mM咪唑)洗脱，再用Sephadex G-25凝胶过滤层析去除高浓度的咪唑，得到目的蛋白。纯化后的野生型酶sj26GST和突变体酶Q67E达到电泳纯(如图1)。
实施例3 日本血吸虫sj26GST及变体的酶活性检测
将酶液加入含有1mM 2,4-二硝基氯苯(CDNB)和1mM还原型谷胱甘肽(GSH)的0.1M pH 6.5磷酸钾溶液中，测定其340nm下析光值变化，实验重复三次(Habig,W.H.and Jakoby,W.B.Assays for differentiation of glutathione S-transferases.Meth.Enzymol.1981,77:398-405)。以野生型sj26GST最高活性为100％，计算突变酶的活性相对野生型酶活的比值。(图2)
GST对GSH和CDNB表观米氏常数测定：反应体系固定为1.0ml,体系中CDNB终浓度为1.0mmol/L,GSH的终浓度范围为40、50、66、100、200μmol/L；固定反应体系中GSH终浓度为1.0mmol/L，CDNB的终浓度为400、500、660、1000μmol/L；分别采用初速法测定，双倒数作图法求出表观Km。
Sj26GST突变体酶活测定结果：实验结果表明突变酶Q67E的酶活性只有野生型sj26GST的60％(图2)表明反应速率较野生型低。Q67E相对于底物GSH的表观米氏常数KmGSH为0.88mmol/L，明显小于野生型sj26GST的KmGSH1.48mmol/L(表1)，表明突变酶Q67E对GSH的较野生型的sj26GST的亲和性高。
表1重组sj26GST与变体酶Q67E表观米氏常数
实施例4日本血吸虫重组sj26GST变体酶Q67E的免疫预防实验
1方法步骤
1.1动物免疫保护实验
将4-5周龄雌性BALB/c小鼠随机分为4组，每组16只，即具体为自然感染组(PBS)、佐剂对照组(FCA)、野生型GST组(Wild)、突变酶组(Mutant)。无菌PBS调整蛋白浓度至1mg/ml，实验组(C-H组)每只小鼠分别于第0、2、4周经背部皮下多点注射100μl蛋白+100μl佐剂(初次免疫用福氏完全佐剂，后两次加强免疫用福氏不完全佐剂)，而对照组(A、B组)每只小鼠分别于0、2、4周经背部皮下多点注射等体积的无菌PBS及佐剂。末次免疫后2周，所有小鼠同时经腹部皮肤感染(40±1)条尾蚴。攻击感染后42d剖杀小鼠，用枸橼酸生理盐水灌冲，在门静脉收集成虫并计数，取出肝脏称重后剪碎置5ml 5％KOH中，37℃消化过夜，计数虫卵，每份样本计数3次取平均值。按下列公式计算减虫率及减卵率：
减虫率＝(对照组平均每鼠的成虫数-实验组平均每鼠成虫数)/对照组平均每鼠的成虫数×100％
减卵率＝(对照组平均每鼠肝脏虫卵数-实验组平均每鼠肝脏虫卵数)/对照组平均每鼠肝脏虫卵数×100％
1.2特异性抗体水平检测
检测特异性IgG抗体初次免疫前、感染前(各组小鼠)及感染后1w(自然感染组和野生型GST组)分别经尾静脉采血，分离血清-20℃保存备用。将野生型GST调整蛋白浓度至5μg/ml后取100μl/孔包被酶标板，用间接酶联免疫吸附试验(ELISA)法检测小鼠血清中的特异性IgG抗体。羊抗鼠IgG-HRP为1∶5000稀释，待测血清1∶100稀释，免疫前小鼠血清为阴性参考，重感染小鼠血清作为阳性参考。反应底物为TMB，室温显色5～10min后，每孔加50μl 2mol/L H2SO4终止反应，酶标仪测定A450值。
检测抗体亚类IgG1及IgG2a用间接酶联免疫吸附试验(ELISA)法检测小鼠血清中IgG1及IgG2a亚类水平。包被酶标板的抗原及浓度同上述IgG检测，血清1∶100稀释，羊抗鼠IgG1-HRP及IgG2a-HRP为1∶5000稀释，其他操作同IgG检测
2结果
2.1重组蛋白sj26GST突变酶Q67E的免疫保护效果
动物保护试验表明免疫重组蛋白Q67E在小鼠体内诱导部分免疫保护效果，与PBS空白对照组相比，成虫数和虫卵数显著减少(P<0.05)。Q67E在BALB/c小鼠中的减虫率和减卵率都达到了40％，高于野生型sj26GST的减虫率(34％)和减卵率(37％)。
2.2特异性抗体水平的检测
应用ELISA方法检测PBS对照组(简称PBS)、佐剂对照组(简称FCA)、重组野生型sj26GST和突变酶Q67E小鼠血清中特异性IgG、IgG1和IgG2a水平的变化，结果如图5所示。与野生型sj26GST一样，突变酶Q67E可诱导产生特异性的IgG抗体，与野生型的IgG抗体水平无显著差异，Q67E也能诱导小鼠产生较高水平的IgG1和IgG2a，并且突变酶Q67E的IgG1水平显著高于重组野生型sj26GST蛋白。
以上所述实例仅表达了本发明的实施方式，其描述较为具体和详细，但不能因此理解为对本发明权利范围的限制。对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，可以做若干变形和改进，例如将本体蛋白sj26GST的67位谷氨酸突变成其他氨基酸探索血吸虫的免疫保护研究，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准。
序列表
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Claims (4)
1.一种吸血虫谷胱甘肽S-转移酶的突变体，其特征在于，所述突变体谷胱甘肽S-转移酶的第67位的氨基酸进行了突变，且其氨基酸序列如SEQ ID NO：4所示。
2.一种核酸分子，其编码权利要求1所述的吸血虫谷胱甘肽S-转移酶的突变体 。
3.含有权利要求2或3的吸血虫谷胱甘肽S-转移酶的突变体的重组表达质粒 。
4.上述权利要求1-3所述的吸血虫谷胱甘肽S-转移酶的突变体在制备治疗血吸虫病的疫苗中的应用。
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| CN105153290B (zh) * | 2015-08-21 | 2019-03-15 | 复旦大学 | 日本血吸虫SjCTRL重组抗原蛋白及其制备方法和用途 |
| CN114317474A (zh) * | 2021-11-12 | 2022-04-12 | 广东省农业科学院农业质量标准与监测技术研究所 | 一种酶活提高的谷胱甘肽s-转移酶突变体及其应用 |
| CN114317474B (zh) * | 2021-11-12 | 2022-10-11 | 广东省农业科学院农业质量标准与监测技术研究所 | 一种酶活提高的谷胱甘肽s-转移酶突变体及其应用 |
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