摘要
目的 获得一种特异性抗人乳头瘤病毒(HPV)16型E6基因的核酶,用以在基因调控水平预防、治疗HPV相关性肿瘤。方法 以核酶(ribozyme,Rz)的锤头结构为模型,采用计算机软件针对HPV16E6基因,设计相应的Rz;体外合成其基因后,克隆于原核表达质粒中。将HPV16E6基因片段也克隆于原核表达质粒中,通过体外转录而得到核酶和病毒mRNA,进行体外切割试验以鉴定核酶的活性。结果 抗HPV16E6核酶(简称抗16HRz)被装入pRG523中而构成pRG16HRz,体外转录后能将核酶独立地释放出来。HPV16E6mRNA转录质粒pTZ16E6含有HPV16E6基因片段(+94位～+296位),体外切割实验证明抗16HRz具备切割活性,在体外能准确、有效地识别和切割HPV16E6mRNA片段,得到82nt和132nt的切割产物。结论 所获得的抗HPV16E6核酶能特异性切割HPV16E6mRNA,可作为对HPV相关性肿瘤进行基因治疗的工具。
Objective To acquire a ribozyme against the E 6 gene of human papillomaviruses type 16 as a potential gene therapy tool on HPV related tumors. Methods According to Symon′s hammerhead structure, anti HPV16E 6 ribozyme (anti 16HRz) was designed by using computer programs. The DNA sequence encoding the ribozyme was synthesized, and incorporated into a pRG523 expressing vector to construct plasmid pRG16HRz. Ribozyme and HPV16E 6 mRNA were acquired by transcription in vitro , and ribozyme′s activity was identified by cleavage experiment in vitro. Results In vitro transcription of the pRG16HRz which had a cis ribozyme gene on both sides of the anti 16HRz gene showed that anti 16HRz could be trimmed to definite length by the 3′ cis Rz and 5′ cis Rz. The HPV16E 6 gene (+94-+296) was got by PCR amplification, then incorporated into pTZ19 to construct pTZ16E 6, which could generate HPV16E 6 mRNA fragment by transcription in vitro. In vitro cleavage reaction demonstrated that anti 16HRz could site specifically cleave the target HPV16E 6 mRNA fragment of 214nt to yield two products (82nt and 132nt). The temperature and time of reaction affected cleavage activity, which was maximum when reacting at 40℃ for 100 minutes. Conclusion Anti 16HRz may become a gene therapy tool on HPV16 related tumors as it can site specifically cleave the target HPV16E 6 mRNA.
出处
《中华微生物学和免疫学杂志》
CAS
CSCD
北大核心
2000年第1期79-82,共4页
Chinese Journal of Microbiology and Immunology
基金
广东省自然科学基金
关键词
核酶
乳头瘤病毒16型
体外转录
肿瘤
Ribozyme
Human Papillomaviruses type16
In vitro transcription
In vitro cleavage
