Objective: To study the biological activity of Myco-plasma penetrans 35kDa lipoprotein(P35) in vitro, prokaryotic expression vector pQE31//p35 was constructed and recombinant fusion protein P35 (rP35) was expressed in...Objective: To study the biological activity of Myco-plasma penetrans 35kDa lipoprotein(P35) in vitro, prokaryotic expression vector pQE31//p35 was constructed and recombinant fusion protein P35 (rP35) was expressed in E.coli. Methods: The p35 gene was amplified by polymerase chain reaction(PCR), cloned to pQE31, and a positive clone was screened. PCR-mediated mutagenesis was used to change the two "TGA" triplets to "TGG" triplets within the p35 gene. Production of the recombinant protein was induced by the addition of IPTG to the E.coli culture. rP35 was purified with a Ni-NTA Spin Kit and rP35 purification was analyzed by Western blot. Results: About 1Kb PCR amplification was cloned into pQE31. The two "TGA" triplets within the p35 gene were successfully changed to "TGG" triplets. The pQE31/p35 vector expressed a protein with a calculated molecular mass of 37.4kDa in E.coli. Western blot indicated the 37.4kDa protein was rP35 . Conclusion: PQE31/p35, a prokaryotic expression vector containing p35 gene, was successfully constructed and expressed in E.coli.展开更多
口咽癌(oropharyngeal carcinoma)是一种高度异质性疾病,其主要病因是烟草、酒精的滥用或高危人类乳头瘤病毒(human papillomavirus,HPV)感染的结果。HPV阳性口咽癌与HPV阴性口咽癌存在明显的病因、流行病学、预后等方面的差异,因此治...口咽癌(oropharyngeal carcinoma)是一种高度异质性疾病,其主要病因是烟草、酒精的滥用或高危人类乳头瘤病毒(human papillomavirus,HPV)感染的结果。HPV阳性口咽癌与HPV阴性口咽癌存在明显的病因、流行病学、预后等方面的差异,因此治疗上应采取不同的方法。目前已知HPV阳性口咽癌对放射治疗敏感,认为其对放疗敏感可能通过多种机制共同完成。HPV阳性口咽癌存在低表达的野生肿瘤蛋白p35(tumor protein p53,TP53)基因,放射治疗可通过DNA双链断裂损伤方式激活p53并诱导细胞发生凋亡;细胞对DNA损伤存在常见的非同源末端连接(non⁃homologous end joining,NHEJ)修复路径,HPV癌蛋白抑制该路径可使肿瘤对放疗更为敏感;此外,免疫应答在放疗作用下进一步激活也参与对肿瘤的消除作用。本文就HPV阳性口咽癌对放疗敏感的研究进行综述,为未来临床上针对口咽癌不同致病因素及临床分期采取针对性的治疗手段提供科学依据。展开更多
基金Supported by the Nature Science Fund of Hunan Province(02JJY2025) Health Office of Hunun Province(Y02-066).
文摘Objective: To study the biological activity of Myco-plasma penetrans 35kDa lipoprotein(P35) in vitro, prokaryotic expression vector pQE31//p35 was constructed and recombinant fusion protein P35 (rP35) was expressed in E.coli. Methods: The p35 gene was amplified by polymerase chain reaction(PCR), cloned to pQE31, and a positive clone was screened. PCR-mediated mutagenesis was used to change the two "TGA" triplets to "TGG" triplets within the p35 gene. Production of the recombinant protein was induced by the addition of IPTG to the E.coli culture. rP35 was purified with a Ni-NTA Spin Kit and rP35 purification was analyzed by Western blot. Results: About 1Kb PCR amplification was cloned into pQE31. The two "TGA" triplets within the p35 gene were successfully changed to "TGG" triplets. The pQE31/p35 vector expressed a protein with a calculated molecular mass of 37.4kDa in E.coli. Western blot indicated the 37.4kDa protein was rP35 . Conclusion: PQE31/p35, a prokaryotic expression vector containing p35 gene, was successfully constructed and expressed in E.coli.
文摘口咽癌(oropharyngeal carcinoma)是一种高度异质性疾病,其主要病因是烟草、酒精的滥用或高危人类乳头瘤病毒(human papillomavirus,HPV)感染的结果。HPV阳性口咽癌与HPV阴性口咽癌存在明显的病因、流行病学、预后等方面的差异,因此治疗上应采取不同的方法。目前已知HPV阳性口咽癌对放射治疗敏感,认为其对放疗敏感可能通过多种机制共同完成。HPV阳性口咽癌存在低表达的野生肿瘤蛋白p35(tumor protein p53,TP53)基因,放射治疗可通过DNA双链断裂损伤方式激活p53并诱导细胞发生凋亡;细胞对DNA损伤存在常见的非同源末端连接(non⁃homologous end joining,NHEJ)修复路径,HPV癌蛋白抑制该路径可使肿瘤对放疗更为敏感;此外,免疫应答在放疗作用下进一步激活也参与对肿瘤的消除作用。本文就HPV阳性口咽癌对放疗敏感的研究进行综述,为未来临床上针对口咽癌不同致病因素及临床分期采取针对性的治疗手段提供科学依据。
