Blocking effect of hammerhead ribozyme RCP on HBV gene expression in HepG2215 cell line

Objective To investigate the cleavage activities of ribozyme RCP in blocking HBV gene expression and replication in eukaryotic cells. Methods: The recombinant plasmid PCR/pSVL which can transiently express ribozyme RCP in eukaryotic cells was constructed and introduced into HepG2215 cell line with the technique of lipofectamine-mediated gene transfer. The vector pSVL transfection group served as the control. HB-sAg and HBsAg from culture medium of the cells were tested with solid-phase radioimmunoassay. Results:The cpm obtained from the medium samples showed that the inhibition rate of ribozyme RCP for HBsAg andHBeAg was 26. 7% and 24. 8% respectively in this transient expression system. Conclusion:Hammerhead ribozyme RCP can inhibit to some extent the expression of HBV gene.

献血者HBVs基因变异的生物信息学分析

目的分析HBV DNA+/HBsAg-献血者的传染性指标、S区基因序列突变情况及生物信息学特征变化。方法通过PCR方法筛查出1份HBV DNA+/HBsAg-的标本,应用酶联免疫和化学发光方法检测该标本乙肝病毒5项指标,对该标本HBVs区基因片段测序并分析变异情况,应用分析软件对所测序列进行生物信息学分析。结果该标本HBV DNA+、HBsAg-、HBsAb+、HBeAg+、HBeAb-、HBcAb+;HBVs区基因序列内发生氨基酸单位点突变(P151L),空间结构发生改变。结论HBVs区基因序列空间结构改变,可能是导致检测结果出现血清学HBsAg-/HBsAb+/HBV DNA+的原因。

Construction of eukaryotic expression vector of HBV x gene

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THE EXPRESSIONS OF HBV X GENE AND ets-2, IGF-Ⅰ, c-myc AND N-ras ONCOGENES IN HUMAN HEPATOCELLULAR CARCINOMA AND TUMOR-ADJACENT TISSUES

The expressions of HBV X gene and ets-2, IGF-I, c-myc and N-ras were studied in 7 pairs of human primary hepatocellular carcinoma (PHC) and tumor-adjacent tissues, using RNA hybridization and im-munoblot methods. The results showed that specific 17 and 28 kD HBV X gene products (HBxAg) were existed in a portion of PHC and tumor-adjacent tissues. The 17 kD HBxAg was detected in the sera of 3 patients who also had 17 kD HBxAg in their liver tissues. Multiple expressions of oncogenes such as ets-2, c-myc and N-ras were observed in PHC and tumor-adjacent tissues that had HBxAg expressed, indicating HBxAg might function as a transactivator in the course of intracellular proto-oncogene activation. It is also observed that in some tumor-adjacnet tissues the expressions of ets-2, c-myc and N-ras were higher than those in corresponding PHC. The relationship of HBxAg to the expression of est-2, IGF-Ⅱ, c-myc and their possible roles in the carcinogenesis of PHC are discussed.

Construction of retrovirus vector with HBV Pol gene and its expression in vitro

Objective: To construct retroviral vector with HBV Pol gene and study its expression in vitro. Methods:The recombinant plasmid HBV2/pBR322 containing 2 copies of HBV full-length gene was provided as the amplification template. The PCR product was cloned into pMD 18-T and subcloned into pMSCVneo and pLNCX2 to construct the recombinant retroviral vectors with HBV Pol gene named by HBV P/pMSCVneo and HBV P/pLNCX2. HBV Pol gene was detected by RTPCR after transfecting the recombinant plasmids into SMMC7721 cells with liposome and G418 selection. Results: The retroviral vector with HBV Pol gene was successfully constructed and the expression of HBV Pol gene in vitro was detected by RTPCR. Conclusion: The retroviral vector with HBV Pol gene can be obtained, which will provide a new insight on the function of HBV Pol gene.

Inhibition of HBV Replication by Delivering the Dual-gene Expression Vector pHsa-miR16-siRNA in HepG2.2.15 Cells

This study aimed to construct the dual-gene expression vector p Hsa-miR16-siRNA which can express human miR-16 and HBV X siRNA, and examine its regulatory effect on HBV gene expression in the HepG2.2.15 cell line. The expression vectors siR-1583 and pHsa-miR16-siRNA were designed and constructed. Hep G2.2.15 cells were transfected with the empty vector, siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively. ELISA was performed to measure the expression of HBsAg and HBeAg in the culture supernatant 48 and72 h post transfection. Fluorescence quantitative PCR was used to measure the HBV mRNA degradation efficiency and HBV DNA copy number. The results showed that the expression of HBV genes was significantly inhibited in Hep G2.2.15 cells transfected with siR-1583, pmiR-16 and pHsa-miR16-siRNA, respectively, when compared with that in cells transfected with the empty vectors, with the inhibitory effect of pHsa-miR16-siRNA being the most significant. ELISA showed that the inhibitory rates of HBs Ag and HBeAg in pHsa-miR16-siRNA transfected cells were correspondingly 87.3% and 85.0% at 48 h, and 88.6% and 86.5% at 72 h post transfection（P〈0.01 vs. control group）. RT-PCR showed that the level of HBV mRNA decreased by 80.2%（t=–99.22, P〈0.01）, the genomic HBV DNA by 92.8%（t=–73.06, P〈0.01）, and the supernatant of HBV DNA copy number by 89.8%（t=–47.13, P〈0.01） in pHsa-miR16-siRNA transfected group. It was suggested that the dual-gene expression vector pHsa-miR16-siRNA can inhibit the replication of HBV more efficiently than a single-gene expression vector.

Study of Hepatitis B Virus Genotypes and Mutation in 1762 &1764 Nucleotides of X Gene in Chronic HBV Patients from Golestan Province—Iran

Introduction: More than 350 million people are chronic carriers of HBV and many of them develop progressive diseases, including cirrhosis and hepatocellular carcinoma. Many of those infected develop persistent disease and a proportion goes on to develop liver failure and cancer. Researchers showed that double mutations of the x gene at position 1762 and 1764, have been found in chronic hepatitis B. These mutations were proposed to be associated with fulminant hepatitis B increasing risk of hepatocellular carcinoma. This project aimed to investigate mutation in the x gene region of HBV infected patients in Golestan province, Iran. Method: 100 patients were entered in this study. Hepatitis B viral DNA was extracted from plasma and PCR was performed using specific primers. Direct sequencing and alignment of x gene were applied using reference sequence from Gene Bank database (Okamoto, 1988;Accession number AB033559). Results: Among the chronic HBV patients 51% were male. The results showed that 49% of patients had A1762T, G1764A mutations changing AGG to stop codon TGA. 27% and 24% of cases were showed mutation only in A1762T and G1764A positions respectively. Conclusion: This study was shown presence of X gene mutation in HBV infected people in Golestan province, Iran. The rate of mutation in two positions 1762 and 1764 of HBV genotype D X gene was higher than the average rate of the world (34%).

Effects of Variant rs346473 in ARHGAP24 Gene on Disease Progression of HBV Infection in Han Chinese Population

Host genetic,environmental and viral factors are classified as three categories that determine clinical outcomes of hepatitis B virus（HBV） infection.The objective of this study was to detect the associations between polymorphisms rs346473 and rs346482 in Rho GTPase-activating protein 24（ARHGAP24） gene and disease progression of HBV infection in Han Chinese population.These two SNPs were found by our DNA pooling using Affymetrix Genome-Wide Human Mapping SNP6.0 Array in HBV carriers,and verified by using TaqMan 7900HT Sequence Detection System with 758 progressed HBV carriers versus 300 asymptomatic HBV carriers（AsC） in a discovery phase and 971 progressed HBV carriers versus 328 AsC in a replication phase.Multivariable logistic regression revealed that individuals with genotype TT at variant rs346473 displayed remarkable correlations with disease progression of HBV infection both in the discovery phase（OR,2.693;95% CI,1.928-3.760;P=6.2×10-9;additive model） and the replication phase（OR,1.490;95% CI,1.104-2.012;P=9.0×10-3;additive model）.These two SNPs were in strong linkage disequilibrium with D＇=0.99 and r2=0.951,and haplotype TT disclosed an increased susceptibility to HBV progression（OR,1.980;95% CI,1.538-2.545;P=8.1×10-8）.These findings suggest that polymorphism rs346473 in the ARHGAP24 gene might be a part of the genetic variants underlying the susceptibility of HBV carriers to disease progression.

HBV-HCC中HBx与免疫微环境的交互作用

乙型肝炎病毒X蛋白(HBx)是乙型肝炎病毒X基因(HBX)将自身DNA整合至人基因组,进而合成的多功能蛋白。HBX基因的表达受肝细胞免疫、微环境和机体免疫的监视和调控,其表达的蛋白也可通过激活肝星状细胞、参与机体免疫调节、调控炎性细胞因子和诱导细胞外基质重塑等参与肝细胞癌(HCC)抑制性免疫微环境的形成。HBx与免疫微环境的相互作用是影响乙肝病毒相关肝细胞癌(HBV-HCC)发生、发展的主要因素之一。深入研究HBx与免疫微环境相互作用机制,探索促进HBV-HCC抑制性免疫微环境形成的机制,有助于开发新型抗HCC药物,改善患者预后。本文就HBx与HBV-HCC免疫微环境的研究进展进行综述。

HBV前C区基因变异与中医证候的相关性研究

研究慢性乙型肝炎患者HBV前C区基因变异的变化规律与中医证候的相关机制。基因芯片检测慢性乙型肝炎不同证候的HBV前C区基因变异。不同位点变异率(%)与证候相关,肝肾阴虚1764(38%),1896(68%),1899(38%);瘀血阻络1762(93%),1764(90%),1899(50%)。不同证候变异株信号强度差异显著,1762、1764;瘀血阻络>肝肾阴虚>肝郁脾虚;1862:肝肾阴虚>肝郁脾虚;>1896:肝肾阴虚>瘀血阻络>湿热中阻>肝郁脾虚;1899:瘀血阻络>肝肾阴虚>湿热中阻>肝郁脾虚。慢性乙型肝炎HBV前C区基因变异与中医证候相关,为分子证候辨证提供了一定实验依据和研究基础。

HBeAg阳性的慢性乙型肝炎患者应用阿德福韦治疗过程中HBVP区变异情况分析

目的研究HBeAg阳性的慢性乙型肝炎(CHB)患者应用阿德福韦治疗过程中P区变异情况。方法选取4例服用阿德福韦患者的系列血清,包括用药前的基线血清(Baseline,简称BL)、用药后半年(28周)、一年(52周)、二年(92周)的血清,将4份系列血清用一片段PCR法进行HBV全基因组克隆、测序,并将测序结果进行基因型及血清型分型、P区变异分析。结果4例系列血清的基因型及血清型分型:1号为C基因型/drq+血清型;2号为B、C混合基因型/adw2、adrq+混合血清型;3号、4号为B基因型/adw2血清型。在P基因的反转录酶区,有几处非常有意义的有义突变,其中有2例病人在P-ORF与S-ORF重叠区,即反转录酶区存在1处完全相同的热点变异,很可能会影响反转录酶的活性,而使HBV对ADV产生耐药逃逸。结论HBeAg阳性的HB患者应用阿德福韦治疗过程中,P区有几处非常有意义的氨基酸变异,很可能与HBV对阿德福韦的耐药逃逸有关。

母儿传播的HBV初步基因分型及变异株检出

以4名HBsAg、HBeAg 均阳性的女性携带者及其子宫内感染HBV 的4 例胎儿为对象,研究母儿间病毒的基因分型与S区变异株。以双脱氧链末端终止法检测母儿所携HBVS区451～660 位核苷酸序列。母儿间同源性98% ～100% ,检出530、546、581位点变异致使126 位、131 位、143 位氨基酸替代。根据当前的基因分型方法, 2对母儿均为B型, 2对母儿母亲为B型, 胎儿为A、B混合型。HBV 子宫内传播存在S基因变异株。

应用S基因转染的Sp2/0细胞作为靶细胞研究HBV基因疫苗的细胞免疫应答

构建编码ＨＢｓＡｇ 蛋白的重组真核表达质粒ｐＣＲ３－１Ｓ作为ＨＢＶ 基因疫苗， 免疫接种Ｂａ１ｂ／ｃ 小鼠。以重组质粒ｐＣＲ３－１Ｓ转染的Ｓｐ２／０ 细胞作为靶细胞， 采用５１Ｃｒ ４ｈ 释放法， 体外检测免疫小鼠的淋巴细胞杀伤功能。结果显示， 与空载体对照组相比较， ＨＢＶ基因疫苗可诱导Ｂａｌｂ／ｃ 小鼠产生ＨＢＶ 特异性细胞毒性Ｔ 细胞应答（ Ｐ＞ ０－０５） ， 提示以Ｓｐ２／０ 基因转染的细胞作为靶细胞， 检测免疫Ｂａｌｂ／ｃ 小鼠淋巴细胞的杀伤功能是可靠的。

PCR扩增HBV x基因特征序列检测乙肝病毒DNA

提出了通过扩增特征序列检测乙肝病毒DNA的新方法.根据乙肝病毒DNA保守区的特点,利用Primer Premier5.0设计出乙肝病毒x基因的特异性引物,并在以血清DNA为模板的聚合酶链反应体系中进行扩增,由此检测待测血清中是否存在乙肝病毒DNA.实验结果表明,该方法较传统的酶联免疫法灵敏,可以检测出酶联免疫法所检测不到的乙肝病毒,具有与免疫印迹一样的准确度;通过与以乙肝病毒s基因为靶序列的PCR反应体系比较,发现乙肝病毒x基因比s基因在序列上更为保守,更适合做乙肝病毒DNA检测的特征序列.

以HBc颗粒为呈现载体的HBV多表位复合基因真核表达载体的构建

目的 构建以HBVHBc颗粒为呈现载体的HBV多表位复合基因真核表达质粒 ,以探讨HBV基因免疫的新途径。方法 PCR扩增HBc第 1～ 72aa及 93～ 183aa编码序列并定向克隆至pcDNA3.1的NheⅠ /XhoⅠ位点 ,构建真核表达载体pHBcdM。合成HBV多表位基因并插入HBc原脊区基因位点 ,构建HBc Mep融合基因真核表达质粒。经PCR、酶切及序列测定等方法筛选阳性质粒。结果 双酶切及测序结果证实阳性质粒正确克隆了约 885bp、编码HBc与HBV多表位的复合基因。结论 成功构建了以HBc颗粒为呈现载体的HBV多表位复合基因真核表达载体pHBc Mep ,为研究pHBc Mep作为DNA疫苗的免疫活性奠定基础。

IFN-γ基因多态性与HBV感染不同结局之间的关系

目的:探讨IFN-γ基因多态性与HBV感染不同临床转归之间的相关性。方法:应用聚合酶链反应-序列特异性引物法(PCR-SSP)检测60例自限性HBV感染者(SR组)和118例慢性持续性HBV感染者[包括无症状HBV携带者(AsC组)36例和进展性肝病者82例(慢性乙型肝炎50例、乙肝后肝硬化32例)]的IFN-γ基因+874T→A、+2109A→G两个位点的多态性,比较各组间基因型和等位基因频率。结果:①+874T→A位点多态性中,慢性持续性感染者总AA基因型频率比SR组显著升高(P<0.05),差异有显著性。等位基因A的频率,进展性肝病组均显著高于SR组(P<0.05)。②+2109A→G位点多态性中,进展性肝病组总GG基因型显著高于SR组(P<0.05),而肝硬化和慢乙肝组之间,SR和AsC组之间差别无显著性。结论:IFN-γ基因+874T→A、+2109A→G两个位点的多态性,与HBV感染不同结局之间有关,携带+874T→AAA基因型和A等位基因的HBV感染者容易发展为慢性,A等位基因更趋向于进展性肝病,而携带+2109A→GGG基因型患者则容易进展为慢性乙型肝炎甚至肝硬化。

HBV_x基因真核表达载体的构建及对肝癌细胞恶性表型的影响

目的 构建乙型肝炎病毒 x基因真核表达载体并在肝癌细胞中表达 ,以研究其对肝癌细胞恶性表型的影响 .方法 应用 PCR、基因克隆等方法构建乙型肝炎病毒 x基因真核表达载体 pc DNA3.1 -HBX,用基因转染的方法将 pc D-NA3.1 -HBX转染入肝癌细胞 HCC-92 0 4,筛选稳定表达乙型肝炎病毒 X蛋白的肝癌细胞 .MTT比色实验、平板集落形成实验检测肝癌细胞恶性表型 .结果 成功构建了乙型肝炎病毒 x基因真核表达载体 pc DNA3.1 -HBX并获得了稳定表达乙型肝炎病毒 X蛋白的肝癌细胞系 HCC-92 0 4细胞克隆 ,且MTT比色实验、平板克隆形成实验显示稳定表达乙型肝炎病毒 X蛋白的肝癌细胞恶性度增高 .结论 乙型肝炎病毒 X蛋白具有生长因子样作用 ,可刺激肝癌细胞生长 ,在肝癌细胞中稳定表达乙型肝炎病毒 X蛋白可增加肝癌细胞恶性表型 .

慢性乙肝病毒感染者HBV DNA前C区A83基因突变的研究

用错配聚合酶链反应（Ｍ－ＰＣＲ）及限制性片段长度多态性分析（ＲＦＬＰ）方法，对９７例慢性乙肝病毒感染（ＨＢＶ）者血中ＨＢＶＤＮＡ前Ｃ区Ａ８３基因突变进行检测。结果显示：共检出Ａ８３基因突变株感染３７例（３８．１％），其中重症肝炎和重度慢性肝炎患者的检出率均明显高于轻度慢性肝炎和ＨＢＶ携带者；ＡＬＴ明显升高者的检出率明显高于ＡＬＴ正常或升高不明显者；ＨＢｅＡｂ（＋）者明显高于ＨＢｅＡｇ（＋）者。

构建不同筛选特性的HBV X基因真核表达载体

目的:构建不同筛选特性的两个HBV X基因真核表达载体。方法:从质粒pEcob6中PCR扩增HBV X全基因,利用pCEP4和pcDNA3.1(+)两者多克隆位点的特点,选用pGEMR○-T Easy Vector构建中间载体pEasy-X,分别酶切后连接特异性片段构建载体pCEP4-X和pcDNA3.1(+)-X。结果:从质粒pEcob6成功PCR扩增出HBV X全基因并克隆至质粒pEasy-X,酶切、PCR及测序均证实真核表达载体质粒pCEP4-X和pcDNA3.1(+)-X构建成功。结论:具有不同筛选特性的两个HBV X基因真核表达载体业已成功构建。

HBVC区基因克隆和PCR条件优化

目的建立PCR优化条件和方法,探讨HBVC区基因在乙型肝炎PCR检测中的应用价值,为荧光定量PCR检测HBV摸索条件。方法将HBV基因组导入DH5α大肠杆菌,SDS碱裂解法小量提取大肠杆菌阳性构建质粒DNA,设计合适引物对HBVC区基因进行PCR扩增,对PCR条件中的退火温度、Mg2+浓度进行了优化,并比较三种不同Taq DNA聚合酶的灵敏度。结果转化的大肠杆菌质粒DNA最佳退火温度为58℃,最佳Mg2+浓度为1.5mM,同时确定天源公司的Biostar Taq DNA聚合酶灵敏度最高,扩增到10-6。结论确立了HBVC区PCR的优化条件,将为后期进行荧光定量PCR检测HBV感染奠定基础以及为探索更有效的检测方法提供实验依据。