摘要
目的评价基于载体的靶向乙型肝炎病毒(HBV)C基因的小发卡RNA(shRNA)对耐拉米夫定HBV的抑制作用,探讨新的抗病毒治疗手段。方法从临床耐拉米夫定患儿中提取耐药HBV基因组,经高保真重复延伸及序列拼接后测序,纯化并克隆到pGEM-T Easy TA载体,再经酶切消化,连接入pcDNA3.1载体,构建表达耐药HBV的表达载体pcDNA-HBV-1;根据GenBank收录HepG2细胞系中HBV基因全序列,设计构建针对HBV C基因区的3条siRNA的表达载体pSilencer4.1/HBV-C1,C2,C3。经退火连接入pSilencer-CMV4.1载体。中量超纯提取质粒,定量后与转染试剂siPort XP-1及pcDNA-HBV-1共转染人肝癌细胞系HepG2细胞。分别设立正常HepG2组(未转染组)、载体阴性对照组及共转染组。在共转染后的不同时间点,应用Western blot检测细胞中乙肝病毒表面抗原(HBsAg)蛋白、乙肝病毒e抗原(HBeAg)蛋白的表达;应用实时荧光定量PCR定量检测细胞上清中HBV DNA拷贝的变化。结果构建的耐药HBV表达载体用于转染HepG2细胞,在转染后的第7天至下次传代前该细胞中可检测到稳定表达的HBV DNA,HBsAg、HBeAg可检出。Western blot结果:在转染的第3天载体pSilencer4.1/HBV-C1、pSilencer4.1/HBV-C2、pSilencer4.1/HBV-C3均可抑制HBV的复制与表达,其中pSilencer4.1/HBV-C2抑制效应尤其显著,在转染后第3天对HBsAg、HBeAg的抑制率分别为(83.53±0.48)%、(86.12±0.83)%。定量PCR检测显示特异性siRNA可以显著抑制HBV-DNA,可以使DNA拷贝数降低102个数量级。结论基于载体的特异性siRNA在体外可抑制HepG2细胞中耐药HBV的复制和表达。该特异性siRNA可作为耐拉米夫定HBV的一种有效的基因治疗措施。
Objective To evaluate the inhibition effect of short hairpin RNA（shRNA） which was targeted to C gene of HBV based on vector on the lamivudine - resistant HBV strains, and to explore a new antiviral therapy against hepatitis B virus （HBV）. Methods The complete lamivudine - resistant HBV genome with a YVDD mutation from a clinical patient was cloned as described previously. Gene spliced by overlap extension was used to obtain DNA sequences, and the polymerase chain reaction （PCR） products were purified and cloned into the pGEM -T Easy TA vector,then digested by Hind Ⅲ and Not Ⅰ ,and was cloned into the HindⅢ and Not Ⅰ sites of pcDNA3.1. The expression of vector pcDNA - HBV - 1 which could produce the lamivudine - resistant HBV strains was constructed. Three pairs of siRNA duplexes targeting HBV C gene were designed as pSilencer HBV - C1, C2, C3 according to the full - length HBV sequence of HepG2 cell in GenBank. The duplex were annealed and ligated into the pSilencer - CMV4.1 vector. The recombinant plasmids were purified through ultrapure Midipreps DNA Purification System. Three shRNA expression vectors targeting C gene of HBV were developed, and they were named as pSilencer4.1/HBV - C1, pSileneer4.1/HBV - C2 and pSileneer4.1/HBV - C3. For transfection in vitro, endotoxin - free plasmid DNA was purified by the EndoFree plasmid kit. The shRNA expression vectors were quantitated and mixed with siPort XP - 1, and then the vectors were co - transfected with the peDNA - HBV - 1 to HepG2 cells. Different groups were set,including normal HepG2 group, negative vector group, co - transfected groups. Western blot was carried out to detect the expression of the viral proteins such as HBsAg and HBeAg at the different time - points post - eotransfected. And the HBV DNA was measured by the quantitative real - time PCR. Results The HepG2 cells which were transfected with the peDNA - HBV - 1 could produce the HBV DNA, and the viral markers HBsAg and HBeAg could be detected. The Wes- tern blot showed that shRN
出处
《实用儿科临床杂志》
CAS
CSCD
北大核心
2011年第22期1706-1708,1715,共4页
Journal of Applied Clinical Pediatrics
基金
广东省自然科学基金(8451001002000762)
国家博士后基金(20100470917
201003353)
