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原发性肝癌为一种严重威胁人们健康的恶性肿瘤。甲胎蛋白(AFP)mRNA在监测原发性肝癌的肝外转移以及术后复发方面是一个很好的标志物,也可用于肝癌治疗效果的评价。通常AFPmRNA的检测方法为逆转录巢式聚合酶链反应(RT-PCR)法进行定性检测,或利用凝胶电泳扫描的方法进行半定量检测。近来随着实时荧光PCR技术的发展使得准确定量检测AFPmRNA成为可能。实时荧光PCR主要采用提取肝癌细胞系HepG2等细胞的RNA或将RNA逆转录为cDNA作为外标来进行AFPmRNA定量检测,RNA极易被广泛存在的RNase所降解,而cDNA作为外标并不能反映待测mRNA的逆转录过程,从而影响测定的准确性,而且这些标准的制备过程相当繁琐,将极大的影响以后的临床应用。所以研制一种适用于AFPmRNA检测的标准品对于肝细胞癌的诊断及研究都有非常重要的意义。本研究在构建完成耐RNase病毒样颗粒的基础上,将AFPmRNA的部分序列克隆到相应的表达载体上,再进行原核表达,得到了可用于上述目的的内含部分AFPmRNA序列的耐RNase病毒样颗粒。
Primary liver cancer is a serious threat to people’s health and malignant tumors. Alpha-fetoprotein (AFP) mRNA is a good marker for monitoring extrahepatic metastases and postoperative recurrence of primary liver cancer and can be used for the evaluation of the therapeutic effect of liver cancer. AFP mRNA is usually detected by reverse transcription nested polymerase chain reaction (RT-PCR) for qualitative detection, or by gel electrophoresis scanning for semi-quantitative detection. Recently, with the development of real-time fluorescence PCR technology, it is possible to accurately and quantitatively detect AFP mRNA. Real-time fluorescent PCR is mainly used to extract RNA from HepG2 cell lines or reverse transcripts of RNA into cDNA for quantitative detection of AFP mRNA. RNA is easily degraded by the widespread RNase, and cDNA as an external standard does not reflect Measurement of mRNA reverse transcription process, thereby affecting the accuracy of the determination, and the preparation of these standards is quite complicated, will greatly affect the future clinical application. Therefore, the development of a standard AFP mRNA for the diagnosis and research of hepatocellular carcinoma are of great significance. In this study, based on the construction of RNase-like particles, a partial sequence of AFP mRNA was cloned into the corresponding expression vector, and then prokaryotic expression was carried out to obtain the RNase-like virus-like particle containing partial AFP mRNA sequence .