| | | 丙型肝炎病毒1b型5′端5223 bp半基因组扩增
| | 超声医学论文 作者：王小红，熊瑜琳，李俊刚，谭朝霞，胡亚君
【关键词】 丙型肝炎病毒
Amplification of 5′ terminal 5223 bp fragment of hepatitis C virus genotype 1b
【Abstract】 AIM: To amplify 5′ terminal 5223 bp fragment of hepatitis C virus (HCV) genotype 1b. METHODS: Five human serum specimens infected with HCV 1b were obtained from HCV RNA positive repository. Total RNA was extracted by using 3 different methods including TRIzol LS Reagent, QIAamp viral RNA mini kit and Magnetic bead. The extracted RNA was reversely transcribed with 3 reverse transcriptases under different reaction conditions. Optimization for long PCR amplification was performed by comparing different Taq enzymes and cycle systems. The 5′ half of the HCV1b genome was amplified with the refined long RTPCR technique. RESULTS: The integrity of long RNA templates was achieved with TRIzol LS Reagent. Using ReverTra AceaTM and SuperScriptTM ⅡRNase H- reverse transcriptase, the complete cDNA molecules were made with incubation at 42℃ for 10 min followed by 30 cycles of 42℃ for 2 min and 48℃ for 2 min and then 75℃ for 10 min. Long PCR amplification of HCV fragment was established by using TaKaLa LA TaqTM or Platinum Taq DNA polymerase High Fidelity as well as the cycling condition of denaturation for 2 min at 94℃ followed by 30 cycles of 30 s at 94℃, 30 s at 62℃, and 4.5 min at 72℃, or 5 cycles of 15 s at 94℃, and 5 min at 68℃ followed by 20 cycles of 15 s at 94℃, and 5 min with the increment of 10 s per cycle at 68℃ and finally 14.5 min at 72℃. Both cycling conditions were feasible, but amplification efficiency of TaKaLa LA TaqTM is superior to Platinum Taq DNA polymerase High Fidelity under this reaction condition. 5223 bp HCV fragment was obtained from all 5 specimens by utilizing the optimized RTPCR protocol and their genotypes were confirmed by sequence analysis. CONCLUSION: The 5′ terminal 5223 bp fragment of HCV was successfully achieved, which should provide a basis for further construction of fulllength cDNA clone and replicon of HCV 1b.
【Keywords】 HCV; long RTPCR; hemigenome
【摘要】 目的超声医学论文：扩增丙型肝炎病毒(HCV) 1b型5′端5223 bp半基因组. 方法：取5份HCV 1b血清标本，采用3种RNA提取方法，比较几种逆转录酶及逆转录条件，选取不同Taq酶进行长链RTNested PCR反应体系及循环条件的超声医学论文筛选和优化，利用最佳的模板提取方法及优化的RTPCR反应条件扩增出5′端长度为5223 bp(5223 bp)半基因组. 结果：Trizol法可以获得完整的RNA模板，适合长链的扩增；应用ReverTra AceaTM酶和SuperScriptTMⅡ，反应条件为42℃ 10 min后，42℃ 2 min,48℃ 2 min，30个循环，然后75℃,10 min；可获得完整的cDNA模板；TaKaLa LA TaqTM和Platinum Taq DNA polymerase High Fidelity采用两种扩增条件：①94℃变性2 min,94℃ 30 s，62℃ 30 s，72℃ 4.5 min,扩增30个循环；②94℃变性2 min，94℃ 15 s，68℃ 5 min，扩增5个循环，然后94℃ 15 s，68℃ 5 min(每个循环延长10 s)， 扩增20个循环,最后72℃延伸10 min，均可获得HCV 1b型5′端5223 bp半基因组的阳性结果，TaKaLa LA TaqTM扩增效果最好；PCR产物经核苷酸序列测定证实为HCV 1b型. 结论：通过各种条件的优化，成功地实现了5′端半基因组的扩增，为下一步全长质粒及复制子的构建打下了基础.
【关键词】 丙型肝炎病毒；长链RTPCR；半基因组
0引言
丙型肝炎病毒(hepatitis C virus, HCV)基因组是一单股正链RNA，全长大约由9600核苷酸组成，是引起丙型肝炎的病原体. HCV基因组具有明显的异质性，根据其核苷酸序列的差异，分为6个主要的基因型，50多个亚型及不同的HCV分离株［1］. 同时，HCV以一群不同的但密切相关的变异株存在于患者体内，称为准种（quasispecies）. 传统的HCV全基因组质粒的构建通常采用短片段拼接而成［2］，国内有报道采用4个片段扩增构建全长质粒［3］，最近国外文献报道的全长基因组质粒的构建也多是采用3个片段拼接而成［4］. 这种短片段的拼接很容易造成准种间的不同变异株的不真实连接，使所构建的HCV全基因组并不是实际存在的基因，限制了HCV分子水平的研究. 长链扩增可以最大限度地避免这种准种间不真实连接，使所构建的HCV全基因组质粒更为真实可靠. 我们选取中国HCV主要流行株1b型［5］为研究对象，建立了HCV长链RTPCR扩增方法，并获得了HCV 1b型5′端5223 bp半基因组，为下一步构建病毒全基因组质粒及中国人HCV复制子奠定了基础.
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