利用RNA干扰技术干扰HBeAg表达的研究

构建针对乙肝病毒HBeAg前c/c区(HBV prec/c)的短发夹环RNA(shRNA)质粒(psiHBV),观察其在体内外对HBV复制的抑制作用。构建RNA干扰真核表达载体psiHBV,与1.5倍HBV真核表达质粒pHBV1.5共转染HeLa细胞,用微粒子化学发光分析仪(MEIA)分别检测细胞上清和细胞裂解液中HBeAg表达水平;用半定量PCR检测prec/cmRNA的转录情况。随后用水动力学方法,向小鼠尾静脉注射pHBV1.5建立小鼠急性乙型肝炎病毒感染模型。采用此感染模型,将pHBV1.5与在体外实验筛选到有明显抑制作用的shRNA表达载体(psiHBV4)共注射,注射后第6天用同样方法检测其干扰效果。结果显示,成功构建了针对HBV前c/c区的shRNA表达载体psiHBV4、psiHBV5、psiHBV6及无关shRNA表达载体psiC,筛选到在体外对HBeAg有明显的抑制作用的psiHBV4载体;注射pHBV1.5的动物血清高表达HBsAg和HBeAg。而共注射干扰性psiHBV4明显抑制了HBeAg的表达,与单纯感染组相比有显著差异,RT-PCR显示肝内HBV C mRNA水平亦明显降低。上述结果表明,siRNA能特异抑制HBV的复制和表达,对乙型肝炎的治疗有潜在应用前景。     

RNA干扰体内抑制HBeAg表达

目的 在机体水平观察小干扰RNA(siRNA)对HBeAg表达的影响.方法 通过尾静脉注射1.5倍的HBV真核表达质粒pHBV1.5建立小鼠急性乙型肝炎病毒感染模型,再将其与针对乙型肝炎病毒核心区的siRNA表达载体(psiHBV4)共注射,于注射后第6天取血测HBeAg的表达,并用RT-PCR检测肝组织HBV prec/c mRNA转录子的水平.结果 注射后第6天血清中,HBsAg、HBeAS在造模组中高表达.psiHBV4干扰组中HBeAg的表达受到了明显的抑制,与对照组相比有显著差异(P<0.05),RT-PCR显示肝内HBV prec/c mRNA水平亦明显降低,而无关干扰对照组则无相应的干扰作用.结论 RNAi能特异,高效的抑制小鼠体内HBeAS的表达,为乙型肝炎的治疗带来了新的希望.     

靶向HBVs和e抗原基因的shRNA表达载体共转染对HBV抗原表达的抑制作用

目的:探索靶向乙型肝炎病毒HBsAg基因的shR-NA表达载体Pgs1、Pgs2、Pgs3及靶向HBeAg基因shRNA表达载体psiHBV4、psiHBV5、psiHBV6共转染,对体外培养HepG2.2.15细胞中的HBV抗原表达的抑制作用。方法:以质粒PTZ为阴性对照,将自行构建的靶向HBVs和e抗原基因的shRNA表达载体按不同组合共转染HepG-2.2.15细胞;继续培养24 h后用MEIA分别检测细胞裂解液和培养上清中HBsAg和HBeAg的表达水平。结果:psiHBV4+PgS2、psiH-BV4+PgS3在联合转染时,在细胞上清和裂解液中对HBsAg和HBeAg表达都有显著抑制作用(P0.05),psiHBV5+PgS1、psiHBV6+PgS3对裂解液HBeAg表达抑制作用不显著(P0.05)。结论:靶向HBs和HBe基因的两种载体psiH-BV4+PgS2、psiHBV4+PgS3共转染比单个载体转染更能显著减少HBV抗原的表达。     

RNA干扰体外抑制HBeAg的表达

目的：构建乙型肝炎病毒前C／C基因小发夹RNA（shRNA）表达载体，探索shRNA表达载体对HBeAg表达的抑制作用。方法：用基因重组技术构建shRNA表达载体，经酶切、测序鉴定后，与HBeAg表达载体pcDNA3．1-preC／C按7：1的比例，共转染HeLa细胞，采用半定量PCR和微粒子酶免疫试验（MEIA）分析shRNA表达载体对HBeAg表达的抑制情况。结果：经限制性内切酶、测序证实成功构建shRNA表达载体；筛选到psiHBV2载体对HBeAg表达有一定抑制作用，对mRNA的抑制率为75％，对蛋白质的抑制率为53％，其余两序列无明显抑制作用。结论：RNAi技术可以有效抑制载体pcDNA3．1-preC／C表达HBeAg。     

针对S基因的siRNA抑制HepG2 2.2.15细胞中HBV基因的表达

目的 构建针对乙型肝炎病毒（HBV）S基因的siRNA（short interfering RNA）表达载体pSUPER-S1和pSUPER-S2,观察其对HepG2 2.2.15细胞中的HBV基因表达的影响.方法 设计并合成针对HBV S区基因的siRNA寡核苷酸,经退火形成双链后克隆入pSUPER载体,构建成功的siRNA表达载体与pTK-Hyg质粒共转染稳定表达HBV的HepG2 2.2.15细胞,经200μg/ml潮霉素抗性筛选,4周后获得稳定细胞克隆,对所得细胞培养上清中的HBsAg和HBeAg进行定量检测,免疫荧光染色检测细胞内抗原的表达,同时用RT-PCR检测靶基因mRNA的抑制效果.结果 成功构建了针对HBV S基因的siRNA表达载体pSUPER-S1和pSUPER-S2,两种siRNA均能明显抑制HepG2 2.2.15细胞的HBsAg和HBeAg分泌,抑制率分别为83%和78%,免疫荧光染色显示siRNA能抑制细胞内抗原的合成,RT-PCR结果证实HBV的mRNA表达降低,而无关序列的siRNA和对照则无此作用.结论 载体产生的针对HBV S基因的siRNA能稳定、高效、特异地抑制HBV基因的表达.     

preS2反义RNA肝癌特异性表达载体的构建及相关特性研究

目的 构建针对preS2基因的肝癌特异性反义RNA表达载体，并对其特异性和有效性进行研究。方法 PCR扩增preS2(3203—340)基因，克隆至含甲胎蛋白启动子的EB病毒表达载体pEBAF，构建反义RNA表达载体pEBAF-as—preS2。脂质体转染肝癌细胞和ECV304细胞，3d后Northern blot检测preS2 mRNA的表达，ELISA检测HBV抗原，荧光定量PCR检测HBV DNA。结果序列分析表明，pEBAF-as-preS2构建成功，Northern blot证实反义RNA仅在AFP阳性的肝癌细胞中表达，pEBAF-as-preS2转染3d后，可显著抑制HepG2．2．15细胞HBV复制和抗原表达，对HBsAg、HBeAg表达的抑制率分别为33．4％和41．5％；对HBV复制抑制率为86％。结论 反义RNA表达载体pEBAF-as-preS2仅在肝癌细胞中特异表达、并可有效抑制HBV，有良好的开发应用前景。     

shRNA表达载体的构建及对HBV复制和表达的抑制

目的:构建shRNA的表达载体,检测针对乙型肝炎病毒(HBV)的shRNA的稳定表达质粒对HBV复制和表达的影响。方法:扩增U6启动子构建shRNA表达载体pU6。针对HBV基因组序列设计并化学合成双链核苷酸克隆到pU6载体得到质粒pU6B/HBVi,脂质体介导转染带有HBV基因组的2.2.15细胞,定量PCR和ELISA法检测pU6B/HBVi对HBV复制和表达的影响。结果:成功构建了shRNA的真核表达载体;针对HBV的pU6B/HBVi质粒对HBV的复制和表达有明显的抑制作用。结论:稳定表达shRNA的pU6B/HBVi质粒可以抑制HBV在2.2.15细胞中的复制和表达。     

shRNA双表达载体pCSH1的构建及抗肿瘤活性研究

目的构建一种可同时表达靶向两种基因shRNA的瞬时表达载体,以提高RNA干扰效率和降低载体的非特异毒性。方法载体采用pUC19的质粒基本序列,RNA聚合酶III识别的H1启动子,并利用同尾酶的特性设计两个多克隆位点,用DNA重组技术构建了载体pCSH1;以pCSH1为基础,构建靶向N-Ras或c-Myc的单干扰载体,以及靶向N-Ras和c-Myc的双干扰载体,并验证其干扰效率;并用克隆形成法验证了载体的抗肿瘤活性;此外还将靶向N-Ras两个shRNA转录单位串联到一个载体中,检测该连接方式的优势。结果成功设计并构建了可同时表达双基因shRNA的瞬时表达载体pCSH1。对该载体的验证结果表明,靶向N-Ras的单干扰shRNA表达载体pCSH1-shNR或靶向c-Myc的单干扰shRNA表达载体pCSH1-shMyc对靶基因mRNA和蛋白具有明显的干扰作用;双干扰载体pCSH1-shNM也能同时抑制N-Ras和c-Myc的mRNA和蛋白表达水平,且对细胞克隆形成的抑制率最高。细胞生长曲线法检测两个N-Ras-shRNA表达单位串联的载体pCSH1-2shNR对细胞生长的抑制作用,结果显示在质粒质量相同条件下,双表达载体pCSH1-2shNR对细胞的生长抑制作用显著强于单N-Ras-shRNA表达载体pCSH1-shNR,而相同质量的对照质粒pCSH1-2shMock、pCSH1-shMock对细胞生长的抑制没有明显差异。结论利用同尾酶原理构建了一个可同时表达双shRNA的瞬时表达载体pCSH1,该载体可实现双基因同时干扰;pCSH1还具有分子量小、shRNA表达单位可以串联和串联后RNA干扰活性增强的优点。该载体通过同时抑制两个靶基因或增加对单个靶基因的shRNA表达单位可以取得更好的抗肿瘤活性。     

shRNA抑制人肺癌细胞A549 SURVIVIN基因表达

目的 构建存活素(SURVIVIN)基因的短发夹RNA(shRNA)表达质粒,为肺癌RNA干扰(RNAi)途径的基因治疗打下基础.方法 设计并合成SURVIVIN短发夹结构模板序列,与穿梭载体重组质粒后转染人肺癌细胞A549,通过RT-PCR法筛选出抑制效果最强的质粒;MTT和Western blot法检测该质粒对细胞增殖和SURVIVOIN表达的影响.结果 构建和筛选出抑制作用最强的质粒;该质粒能明显抑制A549细胞的增殖和SURVIVIN的表达.结论 在RNAi的介导下,SURVIVIN的表达被下调,为肺癌的基因治疗提供实验依据.     

Slug基因RNAi慢病毒载体的构建与鉴定

目的构建Slug基因RNA干扰(RNA interference,RNAi)慢病毒表达载体。方法针对人Slug基因的序列,设计出RNA干扰的靶序列,合成靶序列Oligo DNA,退火形成双链DNA,通过Age I和EcoR I酶切后的pGCSIL-GFP载体连接产生shRNA慢病毒载体,质粒转化感受态细菌,筛选阳性克隆并用插入鉴定引物进行PCR鉴定阳性克隆并测序,同时应用Real-time PCR和Western blot方法检测HCT116结肠癌细胞中Slug基因mRNA和蛋白的表达情况。结果 PCR鉴定与DNA测序证实合成的含Slug shRNA慢病毒载体寡核苷酸链插入正确。Western blot证实Slug RNAi慢病毒载体能够抑制Slug的表达。结论成功构建Slug基因RNAi慢病毒表达载体,为后续感染结肠癌细胞,为探索在结直肠癌发生和发展中的作用奠定基础。     

针对S基因的siRNA或shRNA表达框对HepG2.2.15细胞中HBV基因表达和病毒复制的抑制作用比较

目的 化学合成针对乙型肝炎病毒（HBV）S基因的siRNA，同时制备针对相同区段的shRNA表达框，并比较二者对HepG2．2．15细胞中HBV基因表达和病毒复制的抑制作用。方法 化学合成针对HBVS基因的带有FITC标记的siRNA，设计合成带有FTrc标记的引物，PCR扩增含有RNA聚合酶Ⅲ启动子H1序列和shRNA编码序列的表达框，并对扩增产物进行纯化，将等摩尔数siRNA和shRNA表达框分别用脂质体转染稳定表达HBV的HepG2．2．15细胞，转染1d后流式细胞仪检测转染效率，转染3d后RT-PCR检测靶基因mRNA的水平，用SDS-PAGE、Westem blot及间接免疫荧光染色检测HBsAg的表达。收集转染前和转染后1、3、5和7d的细胞培养上清，检测HBsAg水平，同时用荧光定量PCR方法检测HBV　DNA的含量。结果成功制备了针对HBVS基因的shRNA表达框，将其与等摩尔数siRNA分别转染HepG2．2．15细胞后，RT-PCR证实细胞中HBV　mRNA水平降低，SDS-PAGE、Westem blot及间接免疫荧光染色检测到HBsAg的表达受到抑制，细胞培养上清中HBsAg和HBV DNA含量下降。与siRNA相比，shRNA表达框对HBV的抑制作用虽然起效较慢，但持续时间更长。结论shRNA表达框和siRNA均可明显抑制HBV的转录和表达，与siRNA相比，shRNA表达框能够更持久地抑制靶基因的表达。     

乙型肝炎病毒外膜蛋白和核壳蛋白双表达质粒的构建和表达

目的 构建同时表达乙型肝炎病毒外膜蛋白和核心蛋白的真核表达质粒并研究其表达效果.方法 利用DNA重组技术,构建由两个CMV启动子分别表达乙型肝炎病毒外膜蛋白和核心蛋白的双表达真核质粒VR-SC,体外真核表达后ELISA检测HBsAg和HBeAg.结果 在乙肝病毒外膜蛋白表达质粒上插入多克隆位点,并插入乙肝病毒核心蛋白的完整表达调控单元,体外真核表达后成功检测到HBsAg和HBeAg.结论 成功构建双表达质粒vR-sc,其表达效果良好,为进一步改造成HBV DNA疫苗打下了基础.     

B7-DC-silenced dendritic cells induce stronger anti-HBV immunity in transgenic mice

Hepatitis B virus (HBV) is a noncytopathic DNA virus and is the pathogen of acute and chronic hepatitis. Interferon and nucleotide analogues such as lamivudine and adefovir are the current treatment strategies of HBV infection; however, it is still a serious disease. Therefore, the development of new therapeutic options against HBV is needed. In the present study, we have investigated whether the vectors carrying short hairpin RNA (shRNA) targeting the murine B7-DC gene could silence the expression of B7-DC and analyzed the function of gene-modified dendritic cells (DCs) by mixed lymphocyte reaction. The results demonstrated that two shRNA vectors efficiently suppressed the expression of B7-DC. The MLR assay showed that shRNA-B7-DC-transfected DCs induced markedly higher allogeneic lymphocyte proliferation than transfected DCs with the vector plasmid pAS and untreated DCs at all dilutions. The most efficient shRNA plasmid vector against B7-DC was then used to silence the expression of B7-DC on DCs, the gene-modified DCs were pulsed with HBV-specific peptides, and HBV transgenic mice were immunized. After three rounds of immunization, the splenocytes were stimulated in vitro and tested for cytotoxicitic T lymphocyte activity, while the sera were used to detect the level of HBsAg and HBV DNA. The data demonstrated that blockade of B7-DC on DCs augmented the cytolytic activity induced by immunization with peptide-pulsed DCs and significantly reduced the concentration of serum HBsAg and HBV DNA, suggesting that silencing of B7-DC is of potential value in DC-based therapy of HBV infection.     

乙型肝炎病毒核心蛋白小干扰RNA载体的构建与验证

目的 构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,并验证其干扰效果.方法 设计针对HBV基因组（ayw亚型）HBc基因的小干扰RNA序列（位于2147 bp ～2165 bp序列）,目的片段与载体成功连接后转入大肠埃希菌DH5α,酶切及测序验证其正确性.最后以脂质体转入HepG2.2.15细胞中,检测其干扰效果.结果 成功构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,并命名为pshRNA-HBc..酶切及测序验证正确,pshRNA-HBc对HepG2.2.15细胞中HBc表达具有特异性干扰效果.结论 构建针对乙型肝炎病毒核心蛋白的小干扰RNA真核表达载体,为后续研究HBc的生物学功能奠定良好基础.     

RNA interference against hepatitis B virus with endoribonuclease-prepared siRNA despite of the target sequence variations

RNA interference (RNAi) has proven to be very powerful in inhibiting hepatitis B virus (HBV) replication by cell culture and mouse model studies. We have previously reported that endoribonuclease-prepared short interfering RNAs (esiRNAs) were able to inhibit HBV replication more efficiently than synthesized siRNAs. Here we tested the hypothesis that esiRNAs are able to inhibit gene expression with limited mutations within the target region. Target sequences with different similarities to esiHBVP (esiRNA targeting the DNA polymerase and S antigen of Hepatitis B virus) were amplified and cloned into the 3' untranslated region of HBsAg, respectively. When the obtained expression vectors were co-transfected with esiHBVP into CHO cells, HBsAg expression was suppressed with same efficiency regardless of the target sequence similarities. In HepG2 cells, esiHP9 based on one of the amplified sequence that sharing 87% similarity to the target region suppressed HBsAg expression effectively and dose dependently. In vivo experiment showed that a single dose of 5 microg esiHP9 was able to reduce HBsAg and HBeAg level in the mouse sera by 88 and 77% despite of its 87% similarity to the target sequence, which was as good as esiHBVP that is 100% similar to the target sequence. All the data suggest that esiRNA can tolerate limited target sequence variations without losing its inhibitory capacity. It would be very helpful to suppress virus replication by RNAi despite of their high mutation rate.     

Co-expression of hepatitis B virus antigens by a non-defective adenovirus vaccine vector

Summary Adenovirus type 7 vaccine strain was engineered to express foreign antigens from both the E 3 early promoter in the E 3 region and the major late promoter inserted between the E 4 region and the right inverted terminal repeat. This multiple expression vector was used to express hepatitis B core antigen (HBcAg), hepatits B e antigen (HBeAg), and hepatitis B surface antigen (HBsAg). The gene inserted in the E 3 region was derived from the core gene of the hepatitis B virus genome. When the precore region was present, an immunoreactive group of proteins with molecular weights ranging from 15,000 to 19,000 was secreted into the media. Velocity sedimentation centrifugation of media and lysates from cells infected with recombinants containing the core gene with the precore region resulted in peaks of HBeAg at the top of the gradient where authentic HBeAg should be found. In addition to the core gene in the E 3 region, the surface antigen gene of hepatitis B virus was inserted behind the major late promoter in the E 4 region resulting in an adeno-hepatitis recombinant virus capable of expressing both the core gene and the HBsAg cells. Cells infected with the adeno-hepatitis recombinants could also be stained with peroxidase-conjugates after reacting to antibody against HBcAg. Inoculation of dogs with the recombinant viruses which contained the core gene, with and without the precore sequence, resulted in a significant antibody response to HBcAg/HBeAg. The dogs also produced a significant antibody response to HBsAg as well as neutralizing antibody to adenovirus.     

Response of pre-core mutant chronic hepatitis B infection to lamivudine

The proportion of chronic liver disease associated with the pre-core mutant of hepatitis B virus (HBV) infection is increasing, particularly in Mediterranean Europe and in Asia. The pre-core mutant HBV is unable to produce hepatitis B e antigen (HBeAg), so that patients with this variant do not present with HBV characterised by HBeAg in the serum. Pre-core mutant chronic hepatitis B infection usually proceeds to serious liver disease. Wild-type HBV infection may be mild and respond relatively well to interferon (IFN) alpha therapy, but IFN alpha is not an effective therapeutic option in pre-core mutant hepatitis B infection and new therapeutic options are needed. Clinical data show that lamivudine is an effective treatment for patients with pre-core mutant hepatitis B. There is profound suppression of HBV replication and improvement in indicators of liver disease in most patients. In conclusion, lamivudine is suitable for treatment of a wide range of patients with chronic hepatitis B, including those with pre-core mutant HBV infection.