摘要
研究背景载脂蛋白B mRNA编辑酶催化多肽3G(APOBEC3G)是最近发现的胞嘧啶脱氨酶家族成员,由384个氨基酸组成,分子量为46kD,共含有两个胞嘧啶脱氨酶结构域,可以催化单链DNA胞嘧啶脱氨基生成尿嘧啶。APOBEC3G是重要的天然免疫因子,参与机体的抗病毒防御,APOBEC3G在病毒包装过程中以出芽的方式包装入病毒颗粒,随感染病毒颗粒进入新感染细胞发挥作用。APOBEC3G通过催化HIV和HBV等逆转录病毒基因组发生dC脱氨基生成dU或直接抑制病毒逆转录酶活性而发挥抗病毒作用。APOBEC3G可以在肺脏、肝脏、脾脏、睾丸、卵巢等组织以及外周血粒细胞和淋巴细胞中广泛表达,但是对于A3G转录水平调节的研究甚少,目前的研究主要集中在肝细胞和淋巴细胞中的表达调控。研究表明佛波酯可以通过PKCα/βⅠ/MEK/ER途径诱导AOPBEC3G的表达;IFNα则可以通过不依赖STAT1途径诱导AOPBEC3G的表达;IFNγ、IL2、IL15和IL7等多种细胞因子亦可诱导A3G转录。HCV病毒也可以影响APOBEC3G的表达,其NS5A蛋白可以激活APOBEC3G的转录从而上调APOBEC3G蛋白表达。核转录因子Sp1和Sp3可以与APOBEC3G启动子区域的GC-box结合,参与APOBEC3G基本转录的调控。但是对于其他核转录因子对APOBEC3G转录的调控以及HBV能否影响AOPBEC3G的转录则未见报道。
研究目的:
1、研究上游激活转录因子1(upstream stimulatory transcription factor1,USF1)基因对APOBEC3G的转录调控作用。
2、研究HBV对APOBEC3G转录调控的影响。
研究方法:
1、通过在线软件对AOPBEC3G启动子全长分析,寻找潜在的转录因子结合位点。
2、pcDNA3.1(+)-USF1载体构建:抽提HepG2细胞总RNA,逆转录成cDNA,PCR扩增USF1基因后连接到pMD18-T载体,以M13F和M13R为引物PCR鉴定有目的片段插入证明pMD-USF1载体构建成功,测序并与UCSC数据库序列比对正确无误后抽提质粒,用BamHⅠ和XhoⅠ双酶切连接到真核表达载体pcDNA3.1(+)上构建pcDNA3.1(+)-USF1重组体,大量抽提质粒,转染Huh7细胞48小时后提取细胞总蛋白通过western blot鉴定其表达情况。
3、过表达USF1基因对A3G的影响:分别以pcDNA3.1(+)和pcDNA3.1(+)-UF1质粒瞬时转染Huh7细胞,转染后48小时抽提细胞总RNA,用Real-time RT-PCR检测APOBEC3G mRNA的变化;分别以pcDNA3.1(+)和pcDNA3.1(+)-SUF1质粒转染Huh7细胞,加入G418筛选稳定转染细胞系,抽提细胞总RNA,用Real-time RT-PCR检测APOBEC3G mRNA的变化。
4、降低USF1基因表达对APOBEC3G的影响:合成USF1小干扰RNA片段瞬时转染Huh7细胞,转染后48小时提取细胞总蛋白通过western blot检测对USF1基因的干扰效果。提取细胞总RNA,用Real-time RT-PCR检测APOBEC3G mRNA的变化。
5、利用双报告基因系统检测USF1基因对APOBEC3G启动子的影响:pcDNA3.1(+)和pcDNA3.1(+)-UF1质粒分别与pGL3-A3G载体共转染Huh7细胞和HepG2细胞,在转染过程中为平衡转染效率,加入phRL-SV40质粒作为内参同时为了保证转染的DNA分子数和质量数一致,加用鱼精DNA补足质量。转染后48小时裂解细胞检测双萤光素酶的活性,观察在不同细胞中USF1基因对APOBEC3G启动子的影响,保持转染DNA总量以及pGL-A3G的量不变的情况下,采用不同剂量的pcDNA3.1(+)-USF1质粒与pGL-A3G质粒共转染Huh7细胞,观察USF1基因对APOBEC3G启动子影响的剂量效应。
6、确定USF1基因在APOBEC3G启动子上的作用位点:对APOBEC3G启动子全长进行分析,根据APOBEC3G启动子上E-box序列所在位置,设计引物PCR扩增一系列5'截短的APOBEC3G启动子序列扩增位点分别位于转录起始位点上游-1130、-795、-232、-159、-84和-54处,酶切后连接到pGL3载体上,测序确认与与GenBank登录序列一致然后抽提质粒分别与pcDNA3.1(+)和pcDNA3.1(+)-SUF1质粒共转染Huh7细胞,检测双萤光素酶的活性,确定在APOBEC3G启动子上可能与USF1蛋白结合的E-box位点。
7、观察USF1对定点突变E-box后的APOBEC3G启动子影响:采用QuickChange定点突变试剂盒对APOBEC3G启动子上-91/-86处的E-box进行定点突变,使其由CAGCTG序列变为CAATTG,测序确认突变成功后抽提质粒酶切后重新与pG13载体连接,构建成pGL3-A3Gmut重组载体,然后分别与pcDNA3.1(+)和pcDNA3.1(+)-SUF1质粒分别共转染,同时将未突变的pGL3-A3G质粒分别与pcDNA3.1(+)和pcDNA3.1(+)-SUF1质粒共转染作为阳性对照。检测双萤光素酶活性,观察USF1基因是否能激活突变后的pGL-A3G载体萤光素酶表达。
8、研究HBV对APOBEC3G启动子影响:将pUC19-1.24HBV载体与pGL3-A3G共转染Huh7细胞,48小时后裂解细胞检测双萤光素酶的活性,观察HBV是否影响APOBEC3G启动子活性。
9、HBV病毒蛋白对AOPBEC3G启动子的影响:将编码HBV病毒大S抗原、核心抗原、e抗原的真核表达载体与pGL3-A3G载体共转染Huh7细胞,检测双萤光素酶活性,观察上述三种蛋白是否影响APOBEC3G启动子活性。
10、HBV P基因RT区段重组腺病毒表达研究:采用PCR方法从pUC19-1.24HBV质粒中扩增RT/RNaseH区段,将该片段与pCMV-Tag2载体连接构建pCMV-tag-RT载体,随后以pCMV-tag-RT载体为模板扩增含有Flag标签的HBVRT区段基因酶切后与腺病毒穿梭载体pShuttle-CMV连接,将其在Bi5183细菌中与腺病毒骨架载体重组,获得的重组质粒转染到Ad293细胞中包装,待细胞裂解后收集含病毒的上清并感染Huh7细胞,48h后抽提细胞总RNA通过RT-PCR鉴定目的基因转录。
11、利用杆状病毒系统表达重组HBV聚合酶RT区段研究:采用PCR方法从pUC19-1.24HBV质粒中扩增RT/RNaseH区段,构建含有HBVP基因RT区段的杆状病毒穿梭载体pFastBacHT-RT,转化大肠埃希菌DH10Bac与杆状病毒骨架质粒进行重组,提取重组Bacmid DNA质粒,转染昆虫细胞Sf9获得含有HBV P基因RT区段的重组杆状病毒。重组的杆状病毒再感染Sf9细胞48小时后收集细胞,利用针对载体自身携带的His标签的抗体通过Western blot检测表达情况。
12、研究HBV P基因RT区段对APOBEC3G启动子的影响:将pCMV-tag-RT与pGL3-A3G载体共转染Huh7细胞,同时以pCMV-tag与pGL3-A3G共转染做为阴性对照。转染后48小时收集细胞,检测双萤光素酶活性。
13.用SPSS13.0统计学软件包进行统计学处理,两组均数比较用独立样本t检验,多组比较采用完全随机设计方差分析(One-wayANOVA),多重比较采用LSD检验,P＜0.05具有统计学意义。
结果
1、通过在线对APOBEC3G启动子进行分析结果显示有大量潜在的的转录因子结合位点,我们选择USF1基因做为研究对象来进行验证。
2、pcDNA3.1(+)-USF1载体构建:经过测序后与UCSC上的标准序列比对证明pMD-USF1序列是正确的。酶切以及PCR鉴定pcDNA3.1(+)-USF1载体构建成功,转染Huh7细胞后western blot可见在34KD处有明显特异条带,证明我们所构建的pcDNA3.1(+)-USF1载体可以表达USF1蛋白。
3、过表达USF1基因对APOBEC3G mRNA的影响:在瞬时转染pcDNA3.1(+)-USF1基因的Huh7细胞中APOBEC3G的mRNA表达水平是转染了pcDNA3.1(+)质粒的对照组的两倍左右。在稳定表达pcDNA3.1(+).USF1的Huh7细胞系中APOBEC3G的mRNA水平是pcDNA3.1(+)对照组的1.7倍。
4、降低USF1基因表达对APOBEC3G的影响:通过转染将USF1基因的小干扰RNA转染到Huh7细胞中,western blot检测USF1的表达显示转染了USF1小干扰RNA片段转染Huh7细胞后USF1与对照组相比的表达降低,而做为内参的GAPDH则基本一致。Real-time RT-PCR检测结果显示APOBEC3G的表达比对照组降低约30%。
5、USF1基因对APOBEC3G启动子的影响:双萤光素酶报告基因检测结果表明在Huh7和HepG2细胞中萤光素酶的活性与转染了空质粒的对照组相比都可以升高约5倍((t＝-40.476,P＝0.000;t＝-42.960,P＝0.000)。说明USF1基因在不同的肝细胞中均可以激活APOBEC3G启动子的转录。不同量的USF1基因转染结果表明,萤光素的活性随着USF1的表达增强而增强,提示USF1基因表达增强APOBEC3G启动子活性也相应的增强(F＝204.750,P＝0.000)。
6、确定USF1基因在APOBEC3G启动子上的作用位点:对APOBEC3G启动子全长进行分析显示,APOBEC3G启动子上共有四处E-box位点,分别位于-1439/-1434、-757/-752、-288/-283和.91/-86,PCR扩增后通过酶切和测序鉴定证实一系列的5'端截短的APOBEC3G启动子报告基因构建成功,转染Huh7细胞后检测萤光素酶活性结果表明,随着截短的片段越短,启动子的活性越弱。转染了USF1基因组与对照组相比较,当截短到-159位点的时候USF1基因仍然可以激活APOBEC3G的表达(t＝-96.058,P＝0.000),当截短至-84位点时转染pcDNA3.1(+)-USF1和pcDNA3.1(+)质粒的两组萤光素酶活性基本相同(t＝-1.482,P＝0.213)。
7、观察USF1对定点突变E-box后的APOBEC3G启动子影响:对pGL3-A3Gmut测序证实-91/-86位点CAGCTG成功突变为CAATTG,随后的转染结果表明在Huh7细胞中,USF1基因对pGL3-A3Gmut载体无激活作用,从而证实USF1通过对-91/-86位点CAGCTG起作用而激活APOBEC3G的转录(F＝1833.717,P＜0.001)。
8、研究HBV对APOBEC3G启动子影响:pUC19-1.24HBV与pGL3-A3G共转染后检测萤光素酶活性显示,对照组的萤光素酶活性是转染了HBV组的4倍左右(t＝-13.335,P＝0.000)。HBV明显抑制了APOBEC3G启动子的活性。
9、确认可能与AOPBEC3G启动子作用的病毒蛋白:转染了大S抗原、核心抗原和E抗原的各组萤光素酶的活性并没有明显升高(F＝2.878,P＝0.103),说明大S抗原,核心抗原和E抗原并不会影响APOBEC3G启动子的活性。
10、利用腺病毒系统表达重组HBV聚合酶RT区段研究:成功扩增HBV RT区并克隆到pCMV-Tag2载体,成功将带有Flag标签的HBV RT区克隆到穿梭载体,并与腺病毒骨架质粒重组成功,转染ad293细胞成功包装成病毒颗粒,病毒颗粒感染Huh7细胞后RT-PCR能在Huh7细胞中检测到HBV RT区段mRNA转录。
11、利用杆状病毒系统表达重组HBV聚合酶RT区段研究:成功扩增HBV RT区并克隆到pFastBacHT载体,在DH10Bac细菌中与杆状病毒骨架质粒重组成功,转染Sf9细胞后成功包装出杆状病毒颗粒,随后再感染Sf9细胞,利用抗His抗体通过western blot检测可以见到特异的条带,证实HBV RT区段可以表达。
12、研究HBV P基因RT区段对APOBEC3G启动子的影响:HBV P基因RT区段与pGL3-A3G共转染后检测萤光素酶活性显示HBV P基因RT区段组与对照组相比较萤光素酶的活性无明显差别(t=-0.404,P=0.707),说明HBV P基因的RT区段并不影响APOBEC启动子的活性。
结论
1、在肝细胞中USF1基因参与APOBEC3G基因的基础转录调节,USF1可能通过与APOBEC3G转录起始位点上游-91/-86相作用而激活APOBEC3G的转录。
2、HBV可以抑制APOBEC3G启动子的活性,但是大S抗原、核心抗原、e抗原和HBV聚合酶RT区段对APOBEC3G无抑制作用。
Background
Apolipoprotein B mRNA-editing enzyme catalytic polypeptide-like 3G (APOBEC3G),the new member of cytosine deaminase family,is composed of 384 amino acid,with a molecular weight 46kD.APOBEC3G comprises two domains of cytosine deaminase,which can deaminate deoxycytidine(dC) residues to deoxyuridine(dU) in the growing minus-strand of viral DNA.APOBEC3G exert antiviral defense as an important factor of innate immunity.APOBEC3G is packed into virions by budding during the assembly of virions,and then enter the new infected cells with the viron to exert its antiviral effect either by catalyzing the deamination of dC to dU in the genome of retrovirus such as HIV and HBV or directly inhibiting the activity of reverse transcriptase independently of deaminase.
APOBEC3G can be expressed in lung,liver,spleen,testiculus,ovary and granulocytes and lymphocytes of peripheral blood extensively,but the researches on the modulation of APOBEC3G transcription are limited.Current researches have focused on the regulation of APOBEC3G expression in hepatocytes and lymphocytes. Researches show that the phorbol ester can induce the expression of AOPBEC3G by the pathway of PKCα/βI/MEK/ER while the inducible expression of AOPBEC3G by IFNαmay be independent of STAT1.A variety of cytokines such as IFNγ、IL2、IL15、IL7 can also induce the transcription of AOPBEC3G..HCV can upregulate the expression of AOPBEC3G.by the stimulation of NS5A on the transcription of AOPBEC3G.Nuclear factor Sp1 and Sp3 can bind with the GC-box in the promoter of AOPBEC3G and participate the modulation of basic transcription of AOPBEC3G.. However the regulation of other nuclar factor and HBV on the transcription of AOPBEC3G.have not been reported at the present.
Objective
1.To investigate the modulation of USF1 gene on the transcription of APOBEC3G..
2.To investigate the modulation of HBV on the the transcription of APOBEC3G.
Methods
1.In order to search for one or more potential binding sites of transcription factors,the on-line bio-software was used to analyze the full-length of the APOBEC3G promoter.
2.Construction of pcDNA3.1(+)-USF1 vector:total RNA from HepG2 cells was isolated,reverse transcribed into cDNA.USF1 gene was amplified and then ligated with the pMD18-T vectors.The pMD-USF1 vector construction was identified by polymerase chain reaction by using the M13F and M13R primers pairs and further sequenced.Sequencing results alignment with UCSC database confirmed the target gene.The plasmid was extracted,and double digested by BamHⅠand XhoⅠ, subsequently ligated with the eukaryotic expression vector pcDNA3.1(+) to yield pcDNA3.1(+)-USF1 recombinant vector.After transfection into Huh7 cells for 48 hours,the total proteins were isolated,and the expression levels of the USF1 gene was identified by western blotting
3.Effects of USF1 gene overxpression on the APOBEC3G:total RNA was extracted from transient transfected Huh7 cell lines for 48 hours with pcDNA3.1(+) and pcDNA3.1(+)-UF1 plasmids.The Real-time RT-PCR was performed to determine the mRNA of APOBEC3G.pcDNA3.1(+) and pcDNA3.1(+)-USF1 plasmids transfected in Huh7 cells,respectively.Selection of the stable cell lines was obtained by G418.Total RNA was isolated from transfected cells,and the mRNA of APOBEC3G was detected by Real-time RT-PCR.
4.Effects of decreased USF1 gene expression on APOBEC3G:The short interfering RNAs(siRNAs) were synthesized,and transiently transfected into Huh7 cells.After transfection for 48 hours,the total proteins were extracted,and the USF1 protein expression was determined by western blotting to examine the interfering capacity of USF1 gene.Meanwhile,the total RNA was isolated,and the mRNA of APOBEC3G was detected by Real-time RT-PCR.
5.The effect of USF1 on APOBEC3G promoter was determined by Dual-Luciferase(?)Reporter Assay System:pcDNA3.1(+) and pcDNA3.1(+)-UF1 were co-transfected with pGL3-A3G vector into Huh7 cells and HepG2 cells, respectively.In order to balance the transfection efficiency,phRL-SV40 plasmid as endogenous control,was added into the transfection.To be consistent with the number of molecules and mass of DNA transfected,salmon DNA was also added in the transfection as the supplement for quality control.Transfected cells were lyzed at 48-hour posttransfection,and dual luciferases enzymatic activity was detected to observe the effect of USF1 on APOBEC3G promoter in different cell lines.Dosage effect was investigated by transfecting different doses of pcDNA3.1(+)-USF1 plasmid and pGL-A3G into Huh7 cell under fixed total quality of DNA and pGL-A3G in transfection.
6.Identification of the binding siteof the USF1 gene on APOBEC3G promoter: we analyzed the full-length of APOBEC3G promoter,obtained the position of E-box, and designed primers to amplify a series of 5'deletion fragemnts of APOBEC3G promoter from upstream transcription initiation site,including -1130、-795、-232、-159、-84 and -54.The sequences described previously were digested and inserted into the luciferase reporter vector,pGL3-basic,The target fragemnts were confirmed by comparing with the sequence in GenBank.And then,plasmids were extracted and co-transfeted with pcDNA3.1(+) or pcDNA3.1(+)-SUF1 plasmid into Huh7 cells, respectively.The activities of dual luciferases were determined to study the potential bingding-site of the USF1 protein on APOBEC3G promoter.
7.The effects of USF1 E-box on APOBEC3G promoter by site-directed mutagenesis:the two nucleotide sites 91/-86 of E-box on APOBEC3G promoter were mutated by using the QuickChange site-directed mutagenesis kit,changing CAGCTG to CAATTG.After sequencing,the plasmid was extracted,digested and inserted into pGL3 -basicvector again to constructe recombinant pGL3-A3Gmut vector.The pGL3-A3Gmut vector was cotransfected with either pcDNA3.1(+) or pcDNA3.1(+)-USF1 plasmid.Meanwhile,wild type pGL3-A3G plasmid was also cotransfected with pcDNA3.1(+) or pcDNA3.1(+)-USF1 plasmid as positive control. Dual luciferases enzymatic activity was monitored to detect if USF1 could activate the luciferase expression of pGL3-A3Gmut vector after mutation.
8.Impact of integral HBV particle on APOBEC3 G promoter:pUC 19-1.24HBV and pGL3-A3G vectors were co-transfected into Huh7 for 48 hours.,The transfected cells were lysed to determine the activities of dual luciferases.Therefore,we could observe if integral HBV particle has an effect on the activity of APOBEC3G promoter or not.
9.The effects of HBV viral proteins on APOBEC3G promoter:the recombinant eukaryotic expression vectors of HBV large S antigen,core antigen or E antigen were co-transfected with pGL3-A3G vector into Huh7 cells.After 48 hours,the activities of dual luciferases were detected to study the potential effects of these viral antigens on the activity of APOBEC3G promoter.
10.Expression of HBV P gene(RT/RnaseH domain) by Using the adenoviral expressing system:HBV/P(RT/RNAaseH) gene fragment was amplified from pU19-1.24HBV and inserted into pCMV-Tag2 vector,which was named as pCMV-tag-RT vector.Subsequently,,pCMV-tag-RT was amplified as the template, and HBV RT portion containing Flag tag was generated and double digested.The target gene was inserted into adenovirus shuttle vector,pShuttle-CMV,then transformed into the bacteria Bj5183,in order to recombinate with the adenovirus skeleton vector.The recombinant plasmid was transfected into Ad293 cells for packaging virus particles.After the tranfected cells were lyzed,the supernatant containing virus was collected to infect Huh7 cells.At 48 hours postinfection,the transcription of HBV/P gene(RT/RnaseH domain) was detected by reverse-transcript PCR.
11.Expression of recombinant HBV polymerase RT domain by the baculovirus system:RT/RNaseH gene fragment was amplified by PCR from pUC19-1.24HBV plasmid,and inserted into the baculovirus shuttle vector pFastBacHT-RT.In order to generate recombinant Bacmid DNA,the recombinant vectors were transformed into DH10Bac.The recombinant plasmid was extracted and transfected into Sf9 cells,to generate recombinant baculovirus containing HBV P gene RT domain.The supernatant from recombinant baculoviurs was used to re-infect Sf9 cells for 48 hours, The infected cells were lyzed,and the expression was detected by western blotting using anti His antibody which recognizes His tag in the vectors.
12.Effects of HBV P gene RT domain on APOBEC3G promoter:pCMV-tag-RT and pGL3-A3G vectors were co-transfected into Huh7 cells.Meanwhile,pCMV-tag and pGL3-A3G vectors were also co-transfected into Huh7 cells for 48 hours as negative control,.The transfected cells were harvested,and the activities of luciferase were detected.
13.Statistic analysis:data were analyzed by the SPSS 13.0 software with one-way ANOVA,student's t test or LSD test,when P＜0.05 was regarded as significant.
Result
1.A number of potential transcription factor binding sites in APOBEC3G promoter including USF1 gene were identified.Therefore,we selected USF1 gene to study its function.
2.Construction of pcDNA3.1(+)-USF1 vector:sequencing and alignment with UCSC database confirmed the sequence of target gene.Double digestion and PCR identified the pcDNA3.1(+)-USF1 vector was constructed successfully.A 34 KD protein displayed as a strip were observed obviously by western blotting in transfected Huh7 cells,which demonstrated the pcDNA3.1(+) -USF1 vector we constructed could express USF1 protein.
3.Effects of USF1 gene overxpression on the APOBEC3G:the expressions of APOBEC3G mRNA in Huh7 cells with transiently transfected pcDNA3.1(+)-USF1 plasmid and stably expressed pcDNA3.1(+)-USF1 were appropriately twice and 1.7 times in pcDNA3.1(+) plasmid as control,respectively.
4.Effects of decreased USF1 gene expression on APOBEC3G:the expression of USF1 in Huh7 cells which transiently transfected with siRNA was determined by western blotting was lower than control,but the expressions of GAPDH,which was regarded as endogenous control were similar in all groups.The expression of APOBEC3G was lower about 30%than the control group when detected by Real-time RT-PCR.
5.The effect on APOBEC3G promoter by USFI:Dual-Luciferase(?)Reporter Assay results suggestedthat the luciferases enzymatic activities in Huh7 cells and HepG2 cells were about five times higher than the control that transfected blank plasmid(t＝-40.476,P＝0.000;t＝-42.960,P＝0.000).It suggested that USF1 could activate the APOBEC3G promoter in different hepatocytes.Simultaneously,the luciferases enzymatic activity increased accompany with the expression of USF1 enhanced,suggesting that USF1 gene expression upregulated may enhance the activity of APOBEC3G promoter(F＝204.750,P＝0.000).
6.Identification of the site on APOBEC3G promoter binding to USF1 gene:we analyzed the full-length of APOBEC3G promoter,and found that there were four E-box sites in APOBEC3G promoter,the detail position including -1439/-1434,-757/-752,-288/-283 and -91/-86.A series of 5'deletion fragemnts of APOBEC3G promoter report gene were constructed successfully which were confirmed by PCR,double digestion and sequencing.The luciferases enzymatic activity detected in Huh7 cells after transfection suggested that the shorter in 5'deletion fragment,the weaker in activity of promoter.Compared to the control, the USF1 gene still could activate the expression of APOBEC3G when the APOBEC3G promoter was deleted to -159 position(t＝-96.058,P＝0.000).However, when the APOBEC3G promoter was deleted to -84 position,the luciferase enzymatic activity was similar to the control(t＝--1.482,P＝0.213).
7.The effects of USF1 E-box on APOBEC3G promoter by site-directed mutagenesis:pGL3-A3Gmut vector was confirmed by sequencing that in position -91/-86,changing CAGCTG to CAATTG successfully.The following results of transfection into Huh7 cellssuggested USF1 gene had no activation on pGL3-A3Gmut vector,therefore,it demonstrated that USF1 gene could activate the transcription of APOBEC3G through CAGCTG in position -91/-86(F＝1833.717, P＜0.001).
8.Impact of integral HBV particle on APOBEC3G promoter:the luciferase enzymatic activity in the control was four times compared to transfected group,when pUC 19-1.24HBV and pGL3-A3 G cotransfection(t＝-13.335,P＝0.000).it suggested that HBV inhibit the activity of APOBEC3G promoter.
9.The effects of HBV viral proteins on APOBEC3G promoter:there were no significant difference in the luciferase enzymatic activity between the groups transfected with large S antigen,core antigen and E antigen(F＝2.878,P＝0.103). therefore,we thought that the activity of APOBEC3G promoter were not affected by different HBV viral antigens.
10.Expression of HBV P gene(RT/RnaseH domain) by Using the adenoviral expressing system:HBV RT fragment was amplified and inserted into pCMV-Tag2 vector successfully.Subsequently,HBV RT fragment containing Flag tag was cloned into shuttle vector,and recombined with adenovirus skeleton plasmid successfully. The recombinant vector transfected into AD293 cells could package virus particle, when the transcription of HBV RT mRNA could be detected in infected Huh7 by RT-PCR.
11.Expression of recombinant HBV polymerase RT domain by the baculovirus system::HBV RT fragment was amplified and inserted into pFastBacHT vector successfully.Subsequently,the vector recombined with baculorvirus skeleton plasmid in DH10Bac successfully,and transfected into Sf90 cells,which could package baculovirus particle.When the packaging viral particle re-infected Sf90 cells,western blotting dectection through anti-His antibody could be observed specific strip,which confirmed HBV RT domain could be expressed inbaculovirus system.
12..Effects of HBV P gene RT domain on APOBEC3G promoter::the luciferase enzymatic activity after HBV P gene RT section and pGL3-A3G cotransfection was no difference compared to the control(t＝-0.404,P＝0.707),which suggested that HBV P gene RT section could not affect the activity of APOBEC3G promoter.
Conclusion:
1.USF1 gene can regulate basal transcription regulation of the human APOBEC3G gene in hepatocyte,maybe the USF1 gene can bind at position -91/-86 of the APOBEC3G promoter to transactivate APOBEC3G transcription.
2.HBV virus can inhibit the expression of APOBEC3G,but the large S antigen,core antigen,e antigen and HBV polymerase RT domain have no effect on the expression of APOBEC3G.
引文
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