修回日期: 2005-09-01
接受日期: 2005-09-06
在线出版日期: 2005-10-28
目的: 通过对vacA重组工程菌表达产物包涵体的变性、复性, 提高表达产物的生物学活性, 建立ELISA方法, 讨论VacA抗原规模生产的可行性.
方法: 利用已经构建的表达VacA抗原的工程菌, 用IPTG诱导, 表达的包涵体通过系列条件的变性、复性、透析, 并经活性鉴定.所制抗原用以包被ELISA板, 建立ELISA方法并确定抗原的生物学活性.
结果: 重组工程菌可表达Mr 3 300的目的蛋白, 表达形式为包涵体, 条件优化后的包涵体经SDS-PAGE显示纯度达95%以上, 活性经ELISA法测定正常人126例和blot电泳测定结果符合率为97.1%.
结论: 包涵体变性复性后生物活性大幅度提高, 并且具有良好的重复性和稳定性, 可作为幽门螺杆菌疫苗制备的候选抗原及临床检测的诸多方法的抗原原料.
引文著录: 党双锁, 王宏仓, 贾晓黎, 袁利超, 王宝峰, 张欣, 张正国, 程延安. 幽门螺杆菌vacA基因重组表达的包涵体复性及ELISA方法的建立. 世界华人消化杂志 2005; 13(20): 2500-2503
Revised: September 1, 2005
Accepted: September 6, 2005
Published online: October 28, 2005
AIM: To increase the biological activity of the expressive product through denaturation and renaturation of H. pylori vacA gene recombined expressive inclusion body, and to discuss the feasibility of mass production of VacA antigen.
METHODS: The VacA antigen expressed engineering bacterium was induced by Isopropyl-β-D-thiogalactopy-ranoside (IPTG). The expressed inclusion body was denatured, renatured and dialyzed, and its activity was identified. The plates of ELISA were folded by the produced antigen. Then the ELISA method was constructed to determine the biological activity of the antigen.
RESULTS: The recombined engineering bacterium expressed Mr 3 300 target protein in the form of in-clusion body. SDS-PAGE showed the purity of the optimized inclusion body was above 95%. The ac-cordant rate was 97.1% between ELISA and blot electrophoresis after the activity examination was performed in 126 healthy people.
CONCLUSION: The biological activity of H. pylori vacA gene recombined expressive inclusion body can be significantly and stably improved after denaturation and renaturation. So the inclusion body can be used as a candidate for the production of H. pylori vaccine.
- Citation: Dang SS, Wang HC, Jia XL, Yuan LC, Wang BF, Zhang X, Zhang ZG, Cheng YA. Renaturation of Helicobacter pylori vacA gene recombined expressive inclusion body and construction of ELISA method. Shijie Huaren Xiaohua Zazhi 2005; 13(20): 2500-2503
- URL: https://www.wjgnet.com/1009-3079/full/v13/i20/2500.htm
- DOI: https://dx.doi.org/10.11569/wcjd.v13.i20.2500
幽门螺杆菌(Helicobacter pylori, H. pylori)为革兰氏阴性细菌, 螺杆状, 与多种胃病密切相关, 细胞空泡毒素A(vacuolating cytotoxin A, vacA)存在于所有H. pylori菌株, 但仅在50-60%的菌株中表达[1-9]. 我们以H. pylori菌株11 638总DNA为模板扩增了vacA基因的一个片段, 通过重组技术表达VacA抗原. 该抗原主要以包涵体形式存在, 包涵体一级结构(即氨基酸序列)虽然正确, 但其立体结构是错误的, 所以没有生物活性[10-12]. 因此, 为获得天然状态的目标蛋白, 我们在分离回收包涵体后, 溶解包涵体并使其中的目标蛋白质恢复应有的天然构象和活性. 用复性后的蛋白建立ELISA检测方法.
正常人血清126例取自西安市中心血站; H. pylori菌株11 638为西京医院消化内科惠赠.
1.2.1 构建vacA重组表达载体: vacA引物按照菌株11 638 vacA的基因序列设计, 采用pRSETA His6表达载体, 转化大肠杆菌BL21(DE3), 酶切鉴定重组克隆, IPTG诱导目的蛋白表达; 采用分级分离的方法, 对诱导全菌的外周质, 胞质及沉淀进行分析, 判断目的蛋白表达形式.
1.2.2 包涵体变性和复性: 变性液为20 mmol/L Tris-HCl, pH8.0; 6 mol/L盐酸胍; 20 mmol/L 2-巯基乙醇(pH8.5); 1 mmol/L EDTA·2Na; 10 mL/g包涵体, 室温过夜充分溶解包涵体. 复性液为20 mmol/L Tris-HCl, pH8.0; 0.4 mol/L L-精氨酸; 1 mmol/L还原型谷胱甘肽; 0.1 mmol/L氧化型谷胱甘肽, 透析液为0.1 mol/L盐酸胍; 20 mmol/L Tris-HCl, pH8.0; 25 mmol/L NaCl, 4℃下透析, 离心, 取上清浓缩.
1.2.3 复性蛋白检测: 活性鉴定用ELISA方法检测, 以复性蛋白为包被抗原, 0.05-15 μg/孔, 血清按1∶100稀释, 博士德酶标羊抗人IgG 1∶10 000稀释, TMB显色; 深圳伯劳特公司的H. pylori blot电泳测定临界阳性血清, PBS为空白对照.
1.2.4 血清抗体检测: 用复性蛋白为包被抗原, 1 μg/孔, 血清按1∶100稀释, 博士德酶标羊抗人IgG 1∶10 000稀释, TMB显色, 以A450nm>0.5为阳性, 设立阳性对照和阴性对照, 确定ELISA方法, 检测血清的抗vacA抗体, 结果和blot电泳测定结果相比较.
重组质粒可切出903 bp的外源片断.
表达vacA相对分子质量为Mr 3 300, 主要以包涵体形式存在.
经纯化的vacA倍比稀释后包被ELISA板, 用抗vacA抗体阳性血清(深圳伯劳特H. pylori抗原筛出)测定其活性, 可见复性的vacA具有良好的抗原活性.
幽门螺杆菌为定植于人胃部的革兰氏阴性螺杆状细菌, 和慢性胃炎、消化性溃疡、黏膜相关淋巴样组织性淋巴瘤和胃癌等疾病的发生密切相关. 它是世界性分布的病菌, 在人群中约有半数受到H. pylori的感染. 细胞空泡毒素A基因存在于所有的H. pylori菌株中, 编码可损伤上皮细胞的空泡毒素A, 但仅在50%-60%的菌株中表达[13].
vacA基因编码的蛋白质, 是H. pylori所特有的分泌性毒素, 能使哺乳动物的上皮细胞发生空泡变化, 最终导致细胞死亡. 是一种重要致病因子[16], 也是H. pylori毒力分型的标志[14-17], 感染H. pylori的个体是否发病, 与宿主基因易感性、环境因素及所感染H. pylori菌株的型别有关, 在感染了H. pylori的人群中约有15%的人发病[18-21], 因而对H. pylori的菌株型别的检测就尤为重要.
vacA作为H. pylori的一种与疾病发生密切相关的重要抗原, 在制备H. pylori的疫苗方面有很大的应用价值. 我们以H. pylori菌株11 638总DNA为模板扩增了vacA基因的一个片段, 并选择pRSETA His6表达载体, 高水平的表达His6-vacA. 由于许多外源蛋白质在大肠杆菌细胞内表达时, 不能自发折叠生成一定的空间结构和特定生物功能的蛋白质, 而是以包涵体形式存在于细胞内, 虽然包涵体具有富集目标蛋白、抗蛋白酶及对宿主毒性小等特点, 但由于没有生物活性, 不能直接应用, 目前对包涵体形成和复性过程中发生聚集的机制尚不清楚, 许多已建立的高效复性方法是在反复实验和优化的基础上建立的, 且没有普遍性[22-25]. 本研究中所表达的外源蛋白以包涵体形式存在, 上清中可溶性目的蛋白极少, 因此, 解决包涵体的复性成为获得具有天然构象的目的蛋白的关键.
我们根据His6与金属离子Ni2+高亲和力结合的特性[26], 通过镍柱直接一步纯化并进行重折叠, 纯度可直接达到98%, 并具有较高的生物学活性.
但由于亲和柱的特点, 获得大量的抗原比较困难, 我们通过更换不同的变性剂(脲素、盐酸胍、SDS等)使包涵体彻底溶解, 改变pH值、温度、蛋白浓度、变性剂浓度等, 确立复性条件, 采用稀释复性的方法, 获得大量的目的蛋白, 和阳性对照抗原比较具有良好的活性和抗原性.
利用该抗原来建立检测血清抗体的ELISA方法, 和blot电泳测定结果相比, 特异性为97.6%, 敏感性为96.5%, 符合率97.1%. 可作为筛查幽门螺杆菌vacA感染的有效的方法. 该抗原可以大批量生产, 满足临床检测的需要.
电编: 张敏 编辑: 张海宁
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