2.2.15细胞HBV基因前C和C区PCR测序及其反义寡核苷酸的抗病毒作用

SEQUENCING OF PCR AMPLIFIED HBV GENOME ENCOMPASSING PRE-C AND C REGIONS IN THE 2.2.15 CELLS AND ANTIVIRAL ACTION OF ANTISENSE OLIGONUCLEOTIDES TARGETED TO THE SEQUENCE

目的探讨乙型肝炎病毒(HBV)前 C 和 C 基因区反义寡核苷酸(ASON)对 HBV 表达的序列特异性抑制作用。方法以 HepG_2 2.2.15细胞中转染的 HBV DNA 作为靶基因,通过聚合酶链反应(PCR)产物直接测序法测定前 C和 C 区序列,并根据测序结果在该区设计合成两段硫代磷酸 ASON,用酶联免疫吸咐试验检测作用细胞培养上清液中HBV 表面抗原(HBsAg)和 e 抗原(HBeAg)含量。结果前 C 和 C 区设计的两段 ASON 能特异性抑 HBV 制基因表达,对HBeAg 的抑制率分别为71%和75%,对 HBsAg 的抑制率分别为77%和75%,而无关序列的寡核苷酸未见有效抑制效应;ASON 无细胞毒性作用。结论 ASON 可能成为一种有开发潜力的抗 HBV 基因治疗制剂。

Objective To study the inhibition of HBV gene expression by antisense oligonucleotid- es (ASONs) directed against pre-C and C regions in a sequence-specific manner.Methods We chose the transfected HBV DNA in the 2.2.15 cells as the target gene,which was determined by amplification of PCR and direct sequencing of the genome encompassing pre-C and C regions,and synthesized tow 16- mer ASONs which were directed against the regions.Hepatitis B surface antigen(HBsAg) and e antigen(HBeAg) released by the treated 2.2.15 cells were tested with ELISA.Results We identified that HBV DNA in the 2.2.15 cells was from HBV with surface antigen subtype ayw_1 by sequencing so that ASONs could bind specifically to the target sequence through to base pairing.ASONs directed against the pre-C and C gene could inhibit significantly 75%～77% HBsAg and 71%～75% HBeAg produced at a total final concentration of 10 μmol/L,while an unrelated sequence oligonucleotide showed no effectiveness.All of the oligonucleotides had no cytotoxicity.Conclusion These results demonstrate the application of ASONs in vitro and exemplify their potential as anti-HBV therapeutics.