重组白喉毒素的制备及体外抑瘤效果的研究
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摘要
生长抑素(Somatostatin,简称SST或SS)是一种多肽物质,以14肽和28肽两种形式存在,其广泛分布于脑组织、胰腺、胃肠道,少量分布在甲状腺、颌下腺、肾上腺、前列腺、胎盘、肾脏、肝脏、胆囊等组织中。SS受体在很多肿瘤细胞表面都有过度表达,如神经内分泌瘤、神经系统肿瘤和结肠癌等。SS可以特异的结合过度表达SS受体的肿瘤细胞。
白喉毒素(DT)是白喉杆菌产生的细菌外毒素,有三个不同功能区。N端为催化区( catalytic domain )由193个氨基酸组成(1~193),中间为穿膜区(transmembrance)由173个氨基酸组成(205~378),C端为受体结合区( receptor binding domain)由149个氨基酸组成(386~535)。DTB(390~535)氨基酸残基组成白喉毒素的受体结合区,该区域可以用抗体或细胞因子基因取代。DTA(1~389)为毒素发生毒性作用的部位,是白喉毒素的酶活性区,它通过阻止延长因子-2在核糖体内促进多肽链延长而抑制蛋白质合成造成细胞死亡,具有极强的细胞毒性。
本文利用重叠延伸PCR技术,将DT389基因片断与SS基因片断通过一个柔性肽Linker连接,经特异性酶切后,将基因片段插入到原核表达载体pET-28a中,构建了原核重组表达质粒pET-28a- DT389 -SS,测序结果与设计一致。将重组质粒转化至BL21(DE3),诱导表达后经SDS-PAGE表明,DT389-SS重组蛋白表达量占菌体蛋白总量的31%。免疫印迹分析结果显示,重组蛋白可与anti-6×histag抗体发生特异性免疫反应。
从培养基的选择、诱导剂剂量的选择、诱导条件的优化等方面,研究了重组工程菌BL21(DE3)LysS/pET-28a-DT389-SS的发酵工艺,对重组工程菌进行了发酵,用MTT法测定目的蛋白对细胞生长的抑制作用,结果表明,重组蛋白对多种癌细胞的生长抑制作用明显。
Cancers have been threatening the health of human beings. The chemicals used in clinical now lack selectivity. Toxical effects are common seen in clinical. Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects of anti-tumor medicines.
Somatostatin is a single chain peptide, which has two forms referred to as ss-14 and ss-28, reflecting their amino acid chain length. Somatostatin was found to be sreted by a broad range of tissues, including pancreas, intestinal tract and regions of the central nervous system outside the hypothalamus. The over-expression SSR which can be binded specially with SS was found on many cancer cells surface including neuroendocrine tumour, nervous system tumour and colon carcinomas. DipHtheria toxin (DT) is a single chain bacterial toxin, which is composed of three structural domains. Domain C is responsible for cell binding (amino acids 386~535), which can be replace the gene of antibody and cell factor. Domain T (amino acids 205~378) plays an essential role in the translocation of DT when it across the cell membrane and into the cytosol. Domain N is composed of domain (amino acids 1~193) and is responsible for catalytic domain with ADP-ribosy transferase activity can inactivate elongation factor 2 , and cause cell death.
The gene fragment was amplified from PCDNA3-DTA by overlap extension PCR . The PCR products were digested by EcoRⅠand HindIII,and then inserted into plasmid vector pET28a(+). The positive recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express recombinant protein of DT389-SS by IPTG. The specific protein expressed (about 45.0kDa) was detected by SDS-PAGE. The protein was expressed at high level, amounting to 31% of the total bacterial protein as confirmed by the soft of computer scanning. Western blot analysis showed that the fusion protein may react specifically with anti-6×histag antibody.
High cell density fermentation of the recombinant E.coli strain was studied, for optimizing the cell density and protein production. Several factors were investigated, such as the selection of host bacterium, the selection of medium, the selection of induced dose and optimized condition. In our study, the recombinant plasmid of DT389-SS can be express in four E.coli strain. After trial and error, we choose BL21(DE3)LysS strain. The optimized condition: the medium is LB, the pH is 7.0, the induced temperature is 23℃and time is 16 hours, the dose IPTG is 0.1mmol/L. Under induction of IPTG the recombinant protein was expressed as a soluble protein and was up to 12% of the total protein in E.coli BL21(DE3) LysS.
Using glycerol as carbon sources, IPTG as inducing agent and specific fed-batch mode as feeding method while keeping stiring speed and DO at 20%, E.coli cell density could reach 30g/L and the expression remains at high level. the recombinant protein was expressed as a soluble protein and was up to 20% of the total protein in E. coli BL21(DE3) LysS.
Cells was collected by centrifugation and lysed by sonication. Protein was purified by Ni+ chromatography. With the above purification, the purity of DT389-SS was about 95%. The cytotoxity of DT389-SS on SWWC and SPC-A1 cell growth was analyzed with MTT. The ID50 of DT389-SS on two cell were 16μg/mL and 15μg/mL.
白喉毒素(DT)是白喉杆菌产生的细菌外毒素,有三个不同功能区。N端为催化区( catalytic domain )由193个氨基酸组成(1~193),中间为穿膜区(transmembrance)由173个氨基酸组成(205~378),C端为受体结合区( receptor binding domain)由149个氨基酸组成(386~535)。DTB(390~535)氨基酸残基组成白喉毒素的受体结合区,该区域可以用抗体或细胞因子基因取代。DTA(1~389)为毒素发生毒性作用的部位,是白喉毒素的酶活性区,它通过阻止延长因子-2在核糖体内促进多肽链延长而抑制蛋白质合成造成细胞死亡,具有极强的细胞毒性。
本文利用重叠延伸PCR技术,将DT389基因片断与SS基因片断通过一个柔性肽Linker连接,经特异性酶切后,将基因片段插入到原核表达载体pET-28a中,构建了原核重组表达质粒pET-28a- DT389 -SS,测序结果与设计一致。将重组质粒转化至BL21(DE3),诱导表达后经SDS-PAGE表明,DT389-SS重组蛋白表达量占菌体蛋白总量的31%。免疫印迹分析结果显示,重组蛋白可与anti-6×histag抗体发生特异性免疫反应。
从培养基的选择、诱导剂剂量的选择、诱导条件的优化等方面,研究了重组工程菌BL21(DE3)LysS/pET-28a-DT389-SS的发酵工艺,对重组工程菌进行了发酵,用MTT法测定目的蛋白对细胞生长的抑制作用,结果表明,重组蛋白对多种癌细胞的生长抑制作用明显。
Cancers have been threatening the health of human beings. The chemicals used in clinical now lack selectivity. Toxical effects are common seen in clinical. Tumor-targeting therapy will be satisfactory for enhancing therapeutic effects of anti-tumor medicines.
Somatostatin is a single chain peptide, which has two forms referred to as ss-14 and ss-28, reflecting their amino acid chain length. Somatostatin was found to be sreted by a broad range of tissues, including pancreas, intestinal tract and regions of the central nervous system outside the hypothalamus. The over-expression SSR which can be binded specially with SS was found on many cancer cells surface including neuroendocrine tumour, nervous system tumour and colon carcinomas. DipHtheria toxin (DT) is a single chain bacterial toxin, which is composed of three structural domains. Domain C is responsible for cell binding (amino acids 386~535), which can be replace the gene of antibody and cell factor. Domain T (amino acids 205~378) plays an essential role in the translocation of DT when it across the cell membrane and into the cytosol. Domain N is composed of domain (amino acids 1~193) and is responsible for catalytic domain with ADP-ribosy transferase activity can inactivate elongation factor 2 , and cause cell death.
The gene fragment was amplified from PCDNA3-DTA by overlap extension PCR . The PCR products were digested by EcoRⅠand HindIII,and then inserted into plasmid vector pET28a(+). The positive recombinant plasmids were transformed into host strain E.coli BL21(DE3) and induced to express recombinant protein of DT389-SS by IPTG. The specific protein expressed (about 45.0kDa) was detected by SDS-PAGE. The protein was expressed at high level, amounting to 31% of the total bacterial protein as confirmed by the soft of computer scanning. Western blot analysis showed that the fusion protein may react specifically with anti-6×histag antibody.
High cell density fermentation of the recombinant E.coli strain was studied, for optimizing the cell density and protein production. Several factors were investigated, such as the selection of host bacterium, the selection of medium, the selection of induced dose and optimized condition. In our study, the recombinant plasmid of DT389-SS can be express in four E.coli strain. After trial and error, we choose BL21(DE3)LysS strain. The optimized condition: the medium is LB, the pH is 7.0, the induced temperature is 23℃and time is 16 hours, the dose IPTG is 0.1mmol/L. Under induction of IPTG the recombinant protein was expressed as a soluble protein and was up to 12% of the total protein in E.coli BL21(DE3) LysS.
Using glycerol as carbon sources, IPTG as inducing agent and specific fed-batch mode as feeding method while keeping stiring speed and DO at 20%, E.coli cell density could reach 30g/L and the expression remains at high level. the recombinant protein was expressed as a soluble protein and was up to 20% of the total protein in E. coli BL21(DE3) LysS.
Cells was collected by centrifugation and lysed by sonication. Protein was purified by Ni+ chromatography. With the above purification, the purity of DT389-SS was about 95%. The cytotoxity of DT389-SS on SWWC and SPC-A1 cell growth was analyzed with MTT. The ID50 of DT389-SS on two cell were 16μg/mL and 15μg/mL.
引文
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