CHARACTERIZATION OF HEPATITIS C VIRUS IN PLASMA CE OF ANTI-PRES1

抗 PRES1 血浆中丙型肝炎病毒的特征

• 批准号: 3748301
• 负责人: Z P GUO
• 金额: --
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/3748301
• 关键词: complementary DNA; drug quality /standard; hepatitis C virus; method development; molecular cloning; nucleic acid sequence; plasma; plasmids; polymerase chain reaction; synthetic nucleic acid; virus RNA

In order to facilitate the characterizaton of HCV in plasma, we attempted to establish a quantitative competitive PCR (QC-PCR) for HCV RNA. For this purpose, we synthesized a mutant cDNA as a competitive template which shared the primers' sequences but differed from the wide type cDNA in having an internal deletion. Both mutant and wild type cDNAs contained 217 and 291 nucleotide sequences, respectively, from the 5' non-coding region of HCV. Both cDNAs were derived from a RNA extracted from a plasma sample by reverse transcription and DNA amplification with the same set of outer primers, and subseqently the first PCR product was amplified with inner primers within which either a mutant sense primer containing a sequence with an internal deletion of 74 nucleotides, or a wild type sense primer was used. Both cDNAs were cloned into the vector pCR II (TA-cloning kit, Invitrogen) and transformed in E. coli. The cloned HCV cDNA sequences were confirmed by DNA sequencing. Both wild type and mutant DNA plasmids were purified and quantitated by measuring absorbance at 260 nm. A known amount of the wild type plasmid was co- amplified with the serial diluted mutant plasmid solutions, and the resulting two bands corresponding to the expected sizes of the two plasmids can be demonstrated in a agarose gel. By measuring the incorporation of a labeled nucleotide or quantitating with a densitometer, we were able to obtain ratio of the two bands and found that the amount of wide type was indeed equivalent to that of the mutant plasmid when the ratio was 1. Thus, the number of HCV RNA molecules in an unknown sample can be calculated at the cDNA level if it is assumed that the efficiency of cDNA synthesis is 100%. We are in the process of applying this QA-PCR procedure to measure HCV RNA in plasma and its derived products. we also have synthesized RNAs from both wild type and mutant plasmids by Sp6 RNA polymeraseare in attempt to set up a similar QA-PCR procedure to quantitate the HCV RNA directly.

为了便于血浆中HCV的特征，我们尝试 建立丙型肝炎病毒RNA的定量竞争PCR（QC-PCR）。 为 为此，我们合成了一个突变体cDNA作为竞争性模板， 与野生型cDNA序列相同，但不同 有一个内部删除。 突变型和野生型cDNA 分别含有217和291个核苷酸序列， HCV的非编码区 这两种cDNA均来源于提取的RNA 通过逆转录和DNA扩增， 使用相同的外部引物组，随后扩增第一个PCR产物。 用内部引物扩增，其中突变体正义引物 含有具有74个核苷酸的内部缺失的序列，或 使用野生型有义引物。 将两个cDNA克隆到载体中， pCR II（TA-克隆试剂盒，Invitrogen）并转化到E.杆菌 的 通过DNA测序确认克隆的HCV cDNA序列。 野生 型和突变体DNA质粒进行纯化，并通过测量定量 在260 nm处的吸光度。 将已知量的野生型质粒共转染至细胞中。 用系列稀释的突变质粒溶液扩增， 得到两个条带，其对应于两个条带的预期尺寸， 质粒可以在琼脂糖凝胶中显示。 通过测量 掺入标记的核苷酸或用 密度计，我们能够获得两个波段的比率，并发现 野生型的数量确实与突变型的数量相当 质粒的比例为1. 因此，HCV RNA分子的数量在 如果假设未知样品是在cDNA水平上计算的， cDNA合成效率为100%。 我们正在 应用该QA-PCR方法检测血浆中HCV RNA， 制品. 我们还合成了野生型和 通过Sp 6 RNA聚合酶突变质粒，试图建立一个类似的 QA-PCR程序直接定量HCV RNA。