摘要
目的应用伴侣分子在大肠杆菌表达系统中高效表达家蝇天蚕素,研究伴侣分子对家蝇天蚕素基因在大肠杆菌中表达的影响。方法RT-PCR克隆家蝇的天蚕素成熟肽(mature cecropin,MC)与泛素(ubiquitin,UBI)基因,利用生物信息学方法分析硫氧还蛋白(thioredoxin,Trx)-MC和MC分子结构及Trx-MC分子和Trx-UBI-MC两种融合蛋白mRNA5'端的二级结构的异同。分别构建pET32a-MC和pET32a-UBI-MC两种重组质粒,转化入大肠杆菌(E.coli)BL21(DE3)中。检测Trx-MC融合蛋白的表达和pET32a-MC和pET32a-UBI-MC在相同诱导时间及诱导剂浓度的条件下,融合蛋白表达量的差异。结果生物信息学分析结果显示MC分子被Trx分子包裹在其内部,MC分子的细胞毒性被去除,使其可以在大肠杆菌中正常表达。分子生物学实验结果进一步验证了生物信息学分析结果的正确性。同时,也显示UBI分子的加入,未影响融合蛋白质Trx-UBI-MC与Trx-MC的mRNA5'端二级结构。并且Trx-UBI-MC融合蛋白在全菌蛋白中的含量明显高于Trx-MC。结论Trx作为伴侣分子包裹了MC的天然结构,使其对宿主菌无毒性。UBI作为伴侣分子,在相同转录水平、翻译水平条件下,明显提高了蛋白的表达量。
Objective To investigate of the effectiveness of utilizinge molecular partner for high level expression of Musca domestica cecropin in E.coli. Method Gene of mature cecropin and ubiquitin of M.domestica were amplified by RT-PCR. Using bioinformatics methods, we analyzed the three-dimensional structure of the fusion protein of thioredoxin-MC （mature cecropin） and MC, and the secondary structure of the 5＇ sequences of thioredoxin-MC and thioredoxin-ubiquitin mRNA. Two expression plasmids, pET32a-MC and pET32a-Ubiquitin-MC, were cloned. The expression plasmids were transformed into E.coli and were induced with IPTG. Protein expression of pET32a-MC and pET32a-Ubiquitin-MC was analyzed by SDS-PAGE. Result The result of Bioinformatics analysis showed that Trx MC is wrapped up by Trx thus to reduce the cytotoxicity. Thioredoxin-MC was expressed in E.coli. The result of Bioinformatics analysis was proved by experiment of molecular biology. The result of Bioinformatics analysis showed that adding UBI does not change the structure of the fusion protein and the secondary structure of mRNA 5＇-terminal. The expression level of Trx-Ubiquitin-MC was higher than Trx-MC in E.coli. Conclusion Trx as a molecular partner can reduce the toxicity of MC and UBI and increase the expression level of the fusion protein in E.coli.
出处
《热带医学杂志》
CAS
2009年第2期128-131,139,共5页
Journal of Tropical Medicine
基金
国家自然科学基金(No.30471504)
广东省科技攻关基金(No.2005B33701004)
关键词
家蝇
天蚕素
伴侣分子
融合表达
生物信息学
Musca domestica
cecropin
molecular partner
fusion expression
Ubiquitin
