表达HBsAg特异性siRNA的重组腺相关病毒载体的构建

目的构建表达针对HBsAg小发卡RNA(shRNA)的重组腺相关病毒载体,以观察该病毒在体外对HBsAg和HBeAg的抑制作用。方法将表达针对HBsAg的shRNA定向克隆到pAAV/U6-hrGFP质粒中,获得重组表达质粒(pAAV-shHBs-hrGFP);采用磷酸钙转染法将该质粒与包装质粒pAAV-RC和辅助质粒pHelper共同转染AAV293细胞,进行rAAV-shHBs-hrGFP重组病毒包装。收获病毒感染HepG2.215细胞;采用ELISA法检测HBsAg和HBeAg水平。结果酶切鉴定、测序结果表明,pAAV-shHBs-hrGFP载体成功构建。包装收获的rAAV-shHBs-hrGFP病毒液可以感染HepG2.215细胞,并且可以抑制HBsAg和HBeAg的表达。结论制备的rAAV-shHBs-hrGFP病毒载体能够抑制HBV在体外的抗原表达。     

针对HBV X基因的siRNA抑制HepG2细胞HBV的复制和表达

目的构建能转录产生针对乙型肝炎病毒（HBV）X基因转录体的小干扰RNA（siRNA）转录载体pSIH—BV／X,研究其在体外对HBV复制和抗原表达的抑制作用。方法将构建成功的pSIHBV／X与HBV1．3倍体真核表达质粒pHBV1．3共转染HepG2细胞，转染后24、48、72h检测上清液中HBsAg、HBeAg的变化，并检测72h时HBV DNA的变化。结果成功构建了针对HBVX基因转录体的siRNA表达载体pSIHBV／X，并发现它能抑制HBsAg、HBeAg的分泌，抑制高峰在72h．抑制率分别是64％和61％，而无关对照序列的siRNA无此作用。荧光定量PCR证实HBV DNA的复制亦受到抑制，抑制率31％。结论针对HBVX基因转录体的siRNA在体外具有抑制HBV复制和抗原表达的作用。     

针对HBV—P基因的RNA干扰抑制乙型肝炎病毒的研究

目的构建针对乙型肝炎病毒P基因编码区、能在体内转录产生发夹状小干扰RNA（siRNA）的表达载体psiHBV／p,观察RNA干扰对HBsAg、HBeAg及乙型肝炎病毒DNA复制的抑制作用。方法针对HBV—P基因区特异序列,构建siRNA的表达载体psiHBV／p。采用脂质体介导方法将其与1．3倍HBV真核表达质粒pHBV1．3共转染HepG2细胞。分别于转染后24小时、48小时、72小时用ELISA法对HepG2细胞培养上清液进行HBsAg、HBeAg的检测;于转染后72小时通过FQ-PCR法分析RNA干扰作用对HBVDNA的抑制效果。结果成功构建了针对HBV—P基因区的siRNA的真核表达重组体psiHBV／p,并发现它能明显抑制HBsAg及HBeAg的分泌,转染后第二天抑制率达高峰,分别为84％、65％。FQ—PCR结果也证实了转染72小时后,随psiHBV／p比例的升高,其对HBV DNA的抑制作用也随之增加。结论成功构建的psiHBV／p,它能在体内持续转录产生针对P基因转录体的发夹状siRNA;在细胞水平上,体内转录产生的、针对乙型肝炎病毒P基因区特异序列的siRNA对共转染的重组载体pHBV1．3有显著和特异的抑制作用。     

基因重组HBV的构建及其表达反义RNA抗-HBV作用的研究

目的　探索利用乙型肝炎病毒 (HBV)作为基因治疗载体的可能性及检验其表达反义RNA抗HBV的作用。方法　在表达完整HBV颗粒的质粒上 ,经基因修饰后分别表达S或S启动子区的反义RNA ,整合于具有HBV复制的 2 .2 .15细胞 ,形成细胞克隆 ,酶联免疫吸附 (ELISA)法检测细胞培养上清液中HBsAg和HBeAg ,斑点杂交法检测细胞内HBV核壳中HBVDNA ,聚合酶链反应(PCR)法检测上清液中重组HBV颗粒。结果　 2 .2 .15 pMEP4组、2 .2 .15 SAS组和 2 .2 .15 PAS组对HBsAg平均抑制率分别为 (2 .74± 3.83) %、(6 6 .5 4± 4 .4 5 ) % (P <0 .0 1)和 (5 5 .18± 3.2 7) % (P <0 .0 1) ;对HBeAg平均抑制率分别为 (4 .4 6± 4 .2 5 ) %、(2 6 .36± 1.6 9) % (P <0 .0 1)和 (6 5 .5 4± 3.2 2 ) % (P <0 .0 1) ;对HBV复制的抑制率分别为 17.0 %、5 9.9%和 72 .8%。 2 .2 .15 SAS组及 2 .2 .15 PAS组培养上清液中能检测出突变型HBV颗粒。结论　经过对HBV基因组的两处改造 ,分别在细胞内表达S区及S启动子区反义RNA具有干扰HBV复制及抑制HBV抗原表达的作用 ;在 2 .2 .15细胞中野生型HBV辅助下 ,仍能包装并分泌完整的HBV样颗粒     

RNA干扰对乙型肝炎病毒复制的体外影响

目的:通过一种能够在细胞内表达短发卡RNA的逆转录病毒载体来研究RNA干扰对乙型肝炎病毒复制的影响.方法:首先针对HBV基因组Pol基因RT区寻找RNAi的靶位,并设计相对应的寡核苷酸链.然后再将一对碱基配对寡核苷酸链退火后连接入载体形成重组的质粒,并进行鉴定.在Huh-7细胞中,将通过鉴定的干扰质粒与HBV复制型质粒pHBV3.8共转染,分别应用ELISA方法来检测HBV相关的抗原,应用Northern印迹检测HBV RNA,以及应用实时荧光定量PCR和Southern印迹检测HBV核心颗粒DNA.结果:研究通过计算机方法协助寻找了3条RNAi靶位,并且构建了相应的基于逆转录病毒载体的RNA干扰质粒154i、312i和734i.结果发现312i对pHBV3.8表达有明显的抑制,HBsAg和HBeAg分别为阴性对照组的39%和41%,差异均有显著性(P=0.001,P=0.000).定量荧光PCR结果显示312i组核心颗粒DNA水平显著低于阴性对照组(21.3%±1.1%vs 100.0%±10.6%,P=0.0046).Southern blot和Northern blot结果均显示,312i组病毒复制及mRNA转录水平最低(10.5%,12.0%).结论:在HBV基因组RT区找到了一个可用于RNAi的靶位,并且构建了相对应的干扰质粒,此质粒可成功地抑制HBV复制型质粒在细胞中的复制和表达.     

RNA干扰抑制乙型肝炎病毒复制的实验研究

目的 以乙型肝炎病毒(HBV)核心区为靶位，构建表达小干扰RNA(siRNA)的质粒载体pSilencer3．1-Hlhygro，体外观察siRNA抗HBV的效果。方法 以HepG2 2．2．15细胞为靶细胞，利用脂质体Metafectene与表达siRNA的质粒载体pSilencer3．1-Hlhygro共转染，用定量聚合酶链反应检测细胞上清液中DNA，用逆转录聚合酶链反应检测HBV C-mRNA。结果 成功构建了表达siRNA的转录质粒载体，两条siRNA均可抑制HBV的复制，而且与siRNA浓度成正相关。结论 靶向HBV核心区的siRNA能抑制HBV的复制。     

靶向HBV C区RNAi对HBV复制和HBeAg表达的抑制作用

目的观察靶向HBVC基因区的RNA干扰（RNAi）对HBV复制的抑制作用。方法选择HBV基因组C区的Nt2021-2049作为靶序列，合成相应的正、反义寡核苷酸，退火后形成双链，克隆人shRNA表达质粒，将得到的质粒与HBV质粒共转染HepG2细胞，观察HepG2细胞中HBV的受抑情况。结果病毒复制及HBeAg的表达明显受到抑制。结论所选靶区通过RNAi可有效抑制HBV复制。     

HepG2.2.15细胞分泌前S1、病毒抗原和乙型肝炎病毒DNA的动态变化

HepG 2．2．15细胞是将含有HBV基因组（2个HBV二聚体以尾对尾方向串联）的重组载体质粒转染HepG2细胞，经G418筛选，得到的克隆能稳定分泌HBsAg、HBeAg、HBcAg及Dane颗粒，并可检测到细胞内DNA和RNA等各种中间复制体，还能分泌前s1和前S2。此外，在HepG2．2．15细胞株中，培养细胞的上清液和细胞内均发现HBV ccc DNA，但至今鲜见有关HepG2．2．15细胞分泌前S1动态变化的相关报道。     

HER-2基因靶向RNA干扰重组表达载体的构建及序列分析

目的:利用RNA干扰(RNAi)技术,以HER-2为靶基因,设计构建重组表达载体,并进行测序鉴定.方法:设计具有短发夹结构的两条DNA序列,经退火成互补双链,再克隆至载体pGPU6/ GFP/Neo中构建重组表达载体,转化DH5α菌株,提取质粒行酶切鉴定后,并进行序列测定.再转染人大肠癌HT29细胞,进行荧光摄像和G418抗性筛选.结果:将合成的DNA序列退火后克隆到载体上,经酶切和测序鉴定确实为所需序列.结论:HER-2靶向RNA干扰重组表达载体的构建成功,并可以进行稳定筛选.     

长片段RNA抑制HepG2.2.15细胞中HBV基因的表达和复制

目的 评估长的反义RNA干扰片段在培养细胞株中对HBV复制的抑制效应.方法将HBV基因组S区的全部核苷酸序列插入至pTARGETTM载体中,并将重组载体转染入HepG2.2.15细胞中.用酶联免疫吸附法检测HBsAg与HBeAg水平,用荧光定量PCR法检测HBVDNA水平.对数据采用多个独立样本Kruskal-Wallis检验与两两比较的Mann-Whitney U检验.结果 经过处理后,HepG2.2.15细胞上清液中HBsAg表达量(A值)在HBS2组(携带长片段反义RNA)为0.621±0.027,在HBS4组(携带正义RNA)为3.399±0.018,对照组为2.232±0.187;HBeAg表达量(A值)在HBS2组、HBS4组和对照组分别为0.749±0.019、1.548±0.025和1.570±0.044; HBV DNA水平(×104拷贝/ml)在HBS2组、HBS4组、对照组分别为1.597±0.082、3.381±0.297和3.610±0.063.与对照组相比,HBS2组HBsAg、HBeAg和HBV DNA表达量均降低,统计量Z值均为-2.309,P值均＜0.05; HBS4组HBsAg表达量增高(Z=-2.309,P＜0.05),而HBeAg和HBV DNA表达量无明显差异,统计量Z值分别为-0.866、-1.155,P值均＞0.05.结论 长片段反义RNA能抑制HBV基因的表达和病毒复制.

Abstract：

Objective To evaluate the inhibitory effects of long antisense RNA on HBV replication in HepG2.2.15 cells. Methods The coding region of HBV S gene was cloned into pTARGET vector in sense and antisense orientations and the recombinant plasmids were transfected into HepG2.2.15 cells which were divided into HBS2 (antisense RNA) group, HBS4 (sense RNA) group and control group. HBsAg and HBeAg in the culture supernant were detected by ELISA. The HBV DNA in the supernant was quantified by real-time PCR. Results After treatment, the levels of HBsAg in HepG2.2.15 cell supernatants of three groups were 0.621 ± 0.027, 3.399 ± 0.018 and 2.232 ± 0.187 respectively; the levels of HBeAg were 0.749 ± 0.019,1.548 ± 0.025 and 1.570 ± 0.044 respectively and the levels of HBV DNA were 1.597 ± 0.082, 3.381 ± 0.297 and 3.610 ± 0.063 respectively. The expressions of HBsAg and HBeAg and the HBV DNA level in HBS2 group were remarkably reduced as compared to the control (Z = -2.309, P ＜ 0.05); whereas the sense plasmid transfection (HBS4) did not affect HBeAg (Z= -0.866) and HBV DNA (Z = -1.155) levels in the culture supernant but slightly increased the HBsAg level (Z = -2.309). Conclusion Antisense RNA might be a useful tool to repress HBV replication.     

1．2倍基因组长度C基因型乙型肝炎病毒重组体构建及其在HepG2细胞中表达和复制

目的构建基于pBlueBac4．5质粒的1．2倍基因组长度C基因型乙型肝炎病毒（HBV）重组体，并研究其在HepG2细胞中的表达和复制。方法以重组质粒pWT上的1．2倍基因组长度C基因型HBVDNA序列和pBB4．5HBV1．3（D基因型）上的pBlueBac4．5载体序列为模板，构建重组质粒pBB4．5HBV1．2（C基因型）。用FuGENEHD瞬时转染法，将pBB4．5HBV1．2导人HepG2细胞。用化学发光免疫分析法、Southern印迹杂交法、荧光定量PCR法，分别检测转染后不同时间点HBsAg和HBeAg、HBV复制中间体及HBVDNA水平。此外，对转染时重组质粒用量进行优化。结果酶切和序列分析证实，pBB4．5HBV1．2重组质粒构建成功。初步转染实验证实，在转染细胞培养上清中可检测到HBsAg和HBeAg。优化后转染条件为：使用60mm细胞培养皿，8～11tLgpBB4．5HBV1．2，质粒与转染试剂5：9（μg：μl）。在此条件下，5d实验周期内可检测到HBsAg和HBeAg持续表达（峰值一般出现在转染后第3天）、HBVDNA持续复制（10^6～10^8拷贝／ml）及HBVDNA复制中间体形成。结论在HepG2细胞中，建立了以杆状病毒转移载体pBlueBac4．5为基础的1．2倍基因组长度C基因型HBV体外培养体系，有望为研究HBV耐药、筛选新抗病毒药物等提供新技术平台。     

APOBEC3F剪接亚型与全长APOBEC3F、APOBEC3G对乙肝病毒作用的比较

目的比较APOBEC3F剪接亚型3F79与完整APOBEC3F以及APOBEC3G对HepG2.2.15细胞中HBV DNA复制及HBsAg、HBeAg抗原分泌的影响。方法用PCR合成法扩增人APOBEC3F剪接亚型3F79,构建真核表达载体pEGFPC1-3F79和原核表达质粒pET28a-3F79,将pEGFPC1-3F79与含有全长APOBEC3F和APOBEC3G的质粒Pflag-APOBEC3F、PC-APOBEC3G-HA转染入HepG2.2.15中,检测转染后细胞上清中HBV DNA以及HBsAg和HBeAg的水平。结果构建的重组载体经酶切和PCR鉴定,表明3F79基因正确地插入。将pEGFPC1-3F79与Pflag-APOBEC3F、PC-APOBEC3G-HA转染HepG2.2.15细胞后,pEGFPC1-3F79对细胞中HBV的复制及HBsAg和HBeAg的分泌无明显的抑制作用(P0.05),而转染含有完整APOBEC3F和APOBEC3G质粒的HepG2.2.15细胞与对照组相比,HBsAg、HBeAg、HBV DNA含量明显下降,差异有统计学意义(P0.05)。结论 3F79不能像完整APOBEC3F和APOBEC3G一样抑制HepG2.2.15细胞中HBV DNA复制以及抗原的分泌。     

Construction and expression of eukaryotic plasmids containing lamivudine-resistant or wild-type strains of Hepatitis B Virus genotype C

AIM： To construct eukaryotic expression plasmids of full-length Hepatitis B Virus （HBV） genotype C genome, which contain lamivudine-resistant mutants （YIDD, YVDD） or wild-type strain （YMDD）, and to observe the expression of HBV DNA and antigens [hepatitis B surface antigen （HBsAg） and hepatitis B e antigen （HBeAg）] of the recombinant plasmids in HepG2 cells. METHODS： Three HBV full-length genomes were amplified from the plasmids pMD18T-HBV/YIDD, pMD18T-HBV/YVDD and pMD18T-HBV/YMDD, using PCR. Three recombinant plasmids were generated by inserting each of the PCR products into the eukaryotic expression vector pcDNA3.1 （＋）, between the EcoRI and HindⅢ sites. After being characterized by restriction endonuclease digestion, and DNA sequence analysis, the recombinant plasmids were transfected into HepG2 cells. At 48 and 72 h post-transfection, the levels of intracellular viral DNA replication were detected by real-time PCR, and the expression of HBsAg and HBeAg in the cell culture supernatant was determined by ELISA.
RESULTS： Restriction endonuclease digestion and DNA sequence analysis confirmed that the three recombinant plasmids were correctly constructed. After transfecting the plasmids into HepG2 cells, high levels of intracellular viral DNA replication were observed, and HBsAg and HBeAg were secreted into the cell culture supernatant.
CONCLUSION： Eukaryotic expression plasmids pcDNA3.1 （＋）-HBV/YIDD, pcDNA3.1 （＋）-HBV/YVDD or pcDNA3.1 （＋）-HBV/YMDD, which contained HBV genotype C full-length genome, were successfully constructed. After transfection into HepG2 cells, the recombinant plasmids efficiently expressed HBV DNA, HBsAg and HBeAg. Our results provide an experimental basis for the further study of HBV lamivudine-resistant mutants.     

Inhibition of hepatitis B virus gene expression and replication by artificial microRNA

AIM： To investigate the inhibitory effects of hepatitis B virus （HBV） replication and expression by transfecting artificial microRNA （amiRNA） into HepG2.2.15 cells. METHODS： Three amiRNA-HBV plasmids were constructed and transfected into HepG2.2.15 cells. HBV antigen secretion was detected in the cells with transient and stable transfection by time-resolved fluoroimmunoassays （TRFIA）. HBV DNA replication was examined by ？ uorescence quantitative PCR, and the level of HBV S mRNA was measured by semi- quantitative RT-PCR. RESULTS： The efficiency of transient transfection of the vectors into 2.2.15 cells was 55%-60%. All the vectors had significant inhibition effects on HBsAg and HBeAg at 72 h and 96 h after transfection （P 〈 0.01 for all）. The secretion of HBsAg and HBeAg into the supernatant was inhibited by 49.8% ± 4.7% and 39.9% ± 6.7%, respectively, at 72 h in amiRNA- HBV-S608 plasmid transfection group. The copy of HBV DNA within culture supernatant was also significantly decreased at 72 h and 96 h after transfection （P 〈0.01 for all）. In the cells with stable transfection, the secretion of HBsAg and HBeAg into the supernatant was significantly inhibited in all three transfection groups （P 〈 0.01 for all, vs negative control）. The copies of HBV DNA were inhibited by 33.4% ± 3.0%, 60.8% ± 2.3% and 70.1% ± 3.3%, respectively. CONCLUSION： In HepG2.2.15 cells, HBV replication and expression could be inhibited by artif icial microRNA targeting the HBV S coding region. Vector-based artificial microRNA could be a promising therapeutic approach for chronic HBV infection.     

靶向S区和C区基因的M1GS RNA核酶共同抑制HBV的表达

目的:研究靶向HBVS区和C区基因的M1GSRNA核酶共同作用对HBV基因表达的影响.方法:选择HBVayw亚型S区基因294nt和C区基因2333nt为切割位点,以含有编码M1RNA的DNA序列的质粒pTK117为模板,通过PCR扩增得到M1GSRNA核酶的DNA模板,并将其克隆至真核表达载体pEGFP-C1得到重组质粒pEGFP-GSS和pEGFP-GSC.将2个重组质粒共转染HepG2.2.15细胞,转染后ELISA法测细胞培养液中的HBsAg和HBeAg,RT-PCR检测HBVmRNA.结果:成功构建了分别靶向HBVS区基因和C区基因的真核表达载体.共转染HepG2.2.15细胞后,HBsAg和HBeAg的表达分别被抑制了33.2%和39.1%,HBVCmRNA和SmRNA分别被抑制了32.5%和29.7%,而HepG2.2.15细胞的增殖无明显变化.结论:靶向HBVS区和C区基因的M1GSRNA核酶共同作用可特异性抑制HBVS区和C区基因的表达.     

Inhibition of hepatitis B virus replication in 2.2.15 cells by expressed shRNA

Summary.  Hepatitis B virus (HBV) infection is a worldwide health problem. To determine whether RNA interference (RNAi) could inhibit ongoing HBV replication in 2.2.15 cells, we constructed shRNA-producing vector pU6P based on the mouse U6 RNA promoter and cloned 12 targeted sequences against HBV into the vector, resulting in a series of pU6-siHBV vectors. The recombinant vectors were transfected into 2.2.15 cells, HBsAg and HBeAg in cultured media were assayed using enzyme-linked immunosorbent assay at various days after transfection. The amount of HBV DNA in the culture medium was quantitated by real-time polymerase chain reaction. HBsAg and HBeAg expression were inhibited by 72.8 ± 5.4% ( P  = 0.00003) and 55.8 ± 6.2% ( P  = 0.000026), respectively, 4 days after transfection with pU6-siHBV5. The greatest inhibition of HBV DNA was decreased by approximately 1.9-fold ( P  = 0.013) on day 6 post transfection with pU6-siHBV11 compared with that of empty vector. No change was found for HBV protein expression and DNA replication on pU6-siGFP (negative control) transfected cells. Our data demonstrate that the transfection of HBV-targeted shRNA-producing vector in 2.2.15 cells could inhibit the HBV protein expression and HBV DNA replication specifically. RNAi may be considered as a potential antiviral approach for human HBV infection.     

三螺旋形成寡核苷酸抑制乙型肝炎病毒复制及抗原合成的实验研究

目的 探讨三螺旋形成寡核苷酸抗乙型肝炎病毒（ＨＢＶ）作用。方法 针对ＨＢＶ核心启动子ＳＰ１位点,合成２１ｍｅｒ硫代磷酸三螺旋形成寡核苷酸及２１ｍｅｒ无关对照寡核苷酸。采用ＬＥＩＳＡ,斑点杂交法分别检测了经寡核苷酸处理的ＨｅｐＧ２．２．１５细胞及空白对照组细胞培养上清ＨＢｓＡｇ,ＨＢｅＡｇ及ＨＢＶ ＤＮＡ水平。结果 ＴＦＯ２１组２．２．１５细胞ＨＢｓＡｇ及ＨＢＶ ＤＮＡ分泌量明显低于空白对照组。ＴＦ     

Inhibition of hepatitis B virus expression and replication by RNA interference in HepG2.2.15

INTRODUCTION Hepatitis B is a severe infectious disease threatening peoples’ health all over the world. There is still no efficient therapy to control HBV persistent replication, which may lead to the development of liver cirrhosis and hepatocellualar ca…