摘要
目的建立快速获得丙型肝炎(HCV)基因组5非编码区(5 UTR)真末端序列的分子生物学方法。方法逆转录后利用末端聚合酶(TDT)进行加尾反应,再利用套式聚合酶链反应(PCR)扩增出目的末端基因的cDNA片段,A-T克隆,用限制性内切酶片段长度多态性分析(RFLP)与PCR鉴定重组子,全自动序列分析仪测定插入子序列。结果cD-NA末端快速扩增技术(RACE)获得5株HCV5 UTR克隆,包括3株全长克隆和2株缺失克隆。2株缺失克隆,一条在5末端缺失53个碱基,另一条缺失144个碱基。结论RACE技术快速、有效、实用,可有效获得丙型肝炎病毒基因组的5非编码区末端序列。
Objective To obtain full-length cDNA of hepatitis C virus （HCV）5＇untranslated region （5＇UTR） by rapid amplication of cDNA ends （RACE）. Methods Total RNA extracted from the serum of a patients infected genotype 2a HCV as template, reverse transcription and ligated poly（A）-tail by terminal deoxynucleotidyl transferase（TDT）, the cDNA of 5＇noncoding region was amplified us- ing polymerase chain reaction（PCR）. The fragments were recombinanted by A-T clone strategy, the recombinants were confirmed by restriction fragment length polymorphism（RFLP） and the PCR products was then sequenced. Results Very end fulUength cDNA of 2a genotype HCV 5＇UTR was obtained by RACE. In five clones obtained, three contained full-length 5＇UTR cDNA. 2 out of 5 clones had deletions of 53bp and 144bp at 5＇terminal of HCV 5＇UTR, respectively. Conclusion RACE is rapid and effective to obtain very end of virus genome. We obtain full-length cDNA of 2a genotype HCV 5＇UTR.
出处
《实用肝脏病杂志》
CAS
2005年第6期321-323,共3页
Journal of Practical Hepatology
基金
国家自然科学基金(39770684
30170844)资助
