摘要
目的:探讨抗前列腺癌多肽融合蛋白在大肠杆菌中的原核表达,为抗前列腺癌新药的研究奠定基础.方法:通过BH3,K237,DG2结构域和能被PSA特异性水解的短肽序列设计多肽药物的氨基酸序列,并依据大肠杆菌的偏爱密码子反翻译成基因序列.分段合成寡聚脱氧核糖核酸,经磷酸化、退火后,先后克隆至表达载体pGEX4T2的BamHI和EcoRI之间.将经双酶切鉴定获得的阳性重组子进行DNA测序.用IPTG诱导重组菌株表达并进行SDS PAGE分析.结果:pGEX4T2APP216酶切鉴定可见约216bp电泳条带,与设计长度相符.测序结果证实pGEX4T2APP216的基因序列与设计完全一致.SDS PAGE分析表明该基因在原核细胞中获得高效融合表达,所得到蛋白分子质量与预测相符.融合蛋白表达量约占菌体总蛋白的30%.结论:通过抗前列腺癌多肽序列在大肠杆菌中的高效表达,为前列腺癌的治疗提供新的思路和方法.
AIM: To express a novel polypeptide for anti-prostate cancer. METHODS: The polypeptide drug was designed based on the amino sequences of BH3, K237, DG2 domain and peptide that could be digested by PSA. The coding sequence of the drug was obtained by back-translation using preferred codons of E.coli and chemically synthesized in nine oligonucleotides. The whole sequence was then constructed by phosphorylation, annealing and ligating into EcoR I and BamH I site of pGEX-4T-2 expression vector, and was confirmed by restriction enzyme digestion and DNA sequencing. After the recombinant bacteria was induced at 37℃ for 5 h, the expression protein was analyzed by SDS-PAGE. RESULTS: After digested with BamH I and EcoR I, pGEX 4T-2-APP216 yielded a fragment of 216 bp just as we predicted. DNA sequencing result verified that the sequence of pGEX 4T-2-APP216 was consistent with what we had designed. SDS-PAGE analysis demonstrated that protein was expressed in E.coli. The protein band amounted to 30% of total bacteria protein. CONCLUSION: The polypeptide is successfully expressed in E.coli.
出处
《第四军医大学学报》
北大核心
2005年第9期796-798,共3页
Journal of the Fourth Military Medical University
关键词
多肽药物
前列腺癌
设计
基因表达
polypeptide
prostate neoplasms
design
gene expression
