乙型肝炎病毒核心蛋白突变体干扰HBV包装的实验研究

目的：构建乙型肝炎病毒（HBV）核心蛋白突变体基因的真核表达载体，转染HepG2细胞，观察其表达及干扰HBV颗粒包装的显性负调节作用。方法：采用PCR从质粒pHBVadrl-A1中扩增HBVadr1-A1中扩增HBV C基因和S基因，分别克隆到pGEM-T载体上，构建成pGEM-T-C和pGEM-T-S，进而构建成pGEM-T-CS载体，用HindⅢ切出克隆基因片段与pcDNA3.1连接，经PCR鉴定后构建成真核表达载体pcDNA3.1^ -CS,DNA测序显示基因融合正确，表达载体转染HepG2细胞，经G418筛选得到高拷贝转化子，逆转录－聚合酶链反应（RT－PCR）检测重组蛋白体外表达，用HBV阳性血清感染HepG2细胞，72h后提提细胞内HBV DNA，斑点杂交法分析各组DNA，结果：核心蛋白突变体在HepG2细胞内得到表达，且表达了该重组蛋白的HepG2细胞内HBV DNA量不同程度地低于对照组，表明重组蛋白具有抗HBV包装的DN突变体作用，结论：HBV核心蛋白与表面蛋白融合基因的真核表达载体pcDNA3.1-CS能够体外表达核心蛋白突变体，该突变体具有干扰HBV颗粒包装的显性负调节作用。     

HDV核酶对乙型肝炎病毒抑制作用的研究

目的研究HDV核酶在细胞内对乙型肝炎病毒(HBV)复制及其抗原表达的抑制作用。方法1)以HBV前基因组mRNA为靶基因,体外筛选出HDV核酶有效作用位点,构建HDV核酶并进行体外测活;2)分别选用tRNA-Val、U6和hCMV 3种真核启动子,重组构建HDV核酶真核表达载体ptVHRz、pSURz和pcDHRz,分别用3种载体转染HepG2.2.15细胞;3)用点杂交、ELISA和实时荧光定量PCR方法分别检测核酶在细胞内的表达及对HBV的抑制作用。结果在HBV C基因区筛选到一位点,所构建的HDV核酶在体外条件下对该位点能产生有效切割。3种核酶表达载体在细胞内均能高效表达,在转染48 h后,ptVHRz和pcDHRz对HBeAg的表达产生了明显的抑制作用,而对HBsAg没有抑制作用。三者对HBV的复制均未产生明显影响。结论HDV核酶在细胞内对HBV抗原的表达能产生特异性抑制作用,但未能有效抑制HBV的复制,对其原因需进一步深入研究。     

1.3倍乙型肝炎病毒全基因真核表达载体的构建及在HepG2细胞中的表达

目的 构建1.3倍全基因乙型肝炎病毒(HBV)真核细胞表达载体,观察其在HepG2细胞中的表达。方法 从pGEM—HBV载体上将约4.1kb的1.3倍HBV全基因克隆至真核表达载体pCDNA3.1的Hind Ⅲ位点,将构建的pHBV经Lipofectamine2000导入肝癌细胞系HepG2细胞中。分别在转染后24、48、72h测定HepG2上清液乙型肝炎表面抗原(HBsAg)、乙型肝炎e抗原(HBeAg)的表达,细胞内HBsAg、乙型肝炎核心抗原(HBcAg)免疫组织化学染色,Southern、northern杂交鉴定HBV的复制与表达。结果成功构建了1.3倍HBV全基因真核表达载体,转染后24、48、72h细胞上清液中HBsAg分别为5.36±0.25,13.42±1.24,7.52±0.43;HBeAg分别为9.16±0.32,22.75±1.49,15.96±1.03。免疫荧光结果显示转染后24 h细胞内HBsAg表达最强,主要呈胞浆内弥漫性分布,HBcAg为核浆型分布,以浆型为主。Southern、northern杂交均证实细胞内存在各种病毒复制中间体和特异的病毒复制转录物。结论 该表达载体可介导高水平病毒复制,其转染细胞可望成为一种新型的HBV 体外感染模型。     

乙型肝炎病毒HBeAg与AK026018基因表达产物的结合能力

目的验证乙型肝炎病毒HBeAg与AK026018基因表达蛋白在细胞内及细胞外的结合作用.方法应用逆转录聚合酶链反应（RT-PCR）技术,从HepG2细胞中扩增AK026018基因序列,构建真核表达载体,与HBeAg真核表达载体共转染营养缺陷酵母细胞,观察生长情况.同时进一步应用网织红细胞裂解体外翻译及免疫共沉淀系统,验证AK026018蛋白与HBeAg间的确切结合作用.结果经限制性内切酶分析和DNA序列测定构建的AK026018重组表达载体正确.共转染后的酵母细胞在营养缺陷培养基中生长良好.体外免疫共沉淀实验可见特异放射自显影带.结论HBeAg与AK026018基因编码蛋白在细胞内及细胞外均能相互结合,推测该分子在HBV的致病过程中起着重要作用.     

β2-微球蛋白对乙型肝炎病毒核心启动子转录活性的调节作用

目的:研究β2-m对核心启动子表达的调节作用.方法:根据HBV核心启动子及β2-微球蛋白的序列设计引物,用聚合酶链反应(PCR)的方法分别扩增HBV核心启动子和β2-微球蛋白基因,分别构建HBV核心启动子的报告载体及β2-微球蛋白的真核表达载体,脂质体法瞬时转染HepG2细胞.结果:成功构建HBV核心启动子的报告载体及β2-微球蛋白(β2m)的真核表达载体.脂质体法瞬时转染HepG2细胞48 h后,核心启动子在β2-微球蛋白(β2_m)的影响下,其启动子活性降底2倍.结论:β2-微球蛋白(β2-m)明显HBV核心启动子的表达.     

乙型肝炎病毒核心蛋白c/el表位处TC标签的基因标记对S和e抗原表达的影响

目的 研究乙型肝炎病毒核心蛋白c/el表位处TC标签的基因标记对S和e抗原表达的影响,探索TC标签标记HBV病毒体的可行性.方法 以含有1.3倍HBV基因组的载体为模板,在HBV C基因中插入编码TC标签的碱基序列,得到重组的突变型HBV载体,其编码的核心蛋白的c/el表位处嵌入了TC标签.将野生型和突变型HBV载体分别瞬时转染HepG2细胞,Westernblot检测野生型和各种TC嵌合型核心蛋白的表达,酶联免疫吸附法分析病毒S和e抗原的表达.多组间比较用单因素方差分析.结果 Western blot显示各种嵌合型核心蛋白在细胞内成功表达,并且同野生型蛋白的表达水平无显著差别,酶联免疫吸附法检测表明各组细胞S和e抗原表达水平没有明显差别.结论 HBV核心蛋白c/el表位处TC标签的基因标记不会明显影响突变HBV载体表达病毒蛋白.     

包膜蛋白和核心蛋白共突变体对乙型肝炎病毒在 HepG2细胞中表达的抑制作用

目的探讨包膜蛋白和核心蛋白共突变体对乙型肝炎病毒(HBV)在HepG2细胞中表达的抑制作用。方法构建包膜蛋白和核心蛋白共突变的HBV载体pHBV-mSIS／△C。瞬时转染HepG2细胞,以adwR9转染HepG2细胞为对照;用ELISA方法检测上清液中和细胞内HBV S抗原;重组载体与adwR9共转染HepG2细胞,以pcDNA3与adwR9共转染为对照组。用荧光定量PCR检测胞内和上清液中病毒量。结果突变载体转染细胞胞内和上清液中S蛋白表达量及分泌量与adwR9无明显差别;突变载体与adwR9共转染组上清液和胞内病毒量较pcDNA3与adwR9共转染组低。结论包膜蛋白和核心蛋白共突变对HBV S蛋白的表达量和分泌量没有影响,但干扰病毒复制和包装,具有抗HBV作用。     

蛋白转导结构域介导乙型肝炎病毒聚合酶末端蛋白重链可变区抗体体外抑制病毒的复制

目的 蛋白转导结构域(TAT)介导HBV聚合酶末端蛋白(TP)重链可变区(VH)抗体,研究特异性TAT-VH抗体体外对HBV复制的影响.方法 将TAT-VH基因克隆入原核表达载体pET28a(+),在大肠埃希菌BL21(DE3)LysS内诱导融合蛋白表达并进行纯化.纯化的TAT-VH加入培养的HepG2.2.15细胞,间接免疫荧光法检测其导入HepG2.2.15细胞的效率,四甲基偶氮唑盐(MTT)法检测其对细胞生长代谢的影响,将TAT-VH加入培养的HepG2.2.15细胞,定量PCR法检测HBV DNA水平.数据行单因素方差分析和t检验.结果 成功制备了TAT-VH融合蛋白,间接免疫荧光及MTT证实TAT-VH可以跨膜导入HepG2.2.15细胞,且对细胞生长无影响;加入5 000 nmol/L TAT-VH的HepG2.2.15细胞培养上清液内HBV DNA为(1.211±0.132)lg拷贝/mL,对照组为(5.325±0.041)lg拷贝/mL(t=72.91,P＜0.05);细胞内分别为(3.521±0.411)和(8.532±0.132)Ig拷贝/mL(t=28.41,P＜0.05).结论 HBV聚合酶TP区特异性TAT-VH抗体在体外可抑制HBV复制,为应用细胞内抗体治疗HBV感染提供了良好的实验基础.     

rAAV载体介导的HDV核酶对HBV基因表达的抑制作用

目的探讨重组腺相关病毒介导的HDV核酶在细胞内抑制乙型肝炎病毒基因表达的作用。方法将针对HBV基因序列C区的反式HDVR z与U6启动子和绿色荧光蛋白基因EGFP及其启动子CMV共同插入腺相关病毒载体pSNAV中并取代其原有CMV启动子区域,构建为pSNAV-CR z重组载体。并将pSNAV-CR z、pSNAV及pLEGFP质粒转染HepG2.2.15培养细胞,荧光显微镜下观察绿色荧光,对转染后培养细胞内蛋白进行HBeAg和HBsAg的ELISA分析。结果所构建的重组载体经双酶切及PCR验证与实验设计一致。重组载体转染培养细胞后,荧光显微计数表明,pSNAV-CR z的转染效率为30%。HBeAg和HBsAg的ELISA检测显示,pSNAV-CR z重组载体对HepG2.2.15细胞中HBV病毒的HBeAg和HBsAg表达抑制率分别为63.5%和50.07%。结论细胞水平试验证明,重组腺相关病毒载体介导的反式HDVR z在体外培养细胞水平对HBV的复制起到一定的抑制作用。     

HBV核心和前S1融合蛋白在大肠杆菌中的表达及真核表达载体的构建

目的:表达可用于乙型肝炎治疗性疫苗研究的乙型肝炎病毒(HBV)核心抗原(HBcAg)与前S1抗原(PreS1)氨基酸(AA)3～55肽段融合蛋白。方法:以原核表达载体pET-11d表达HBV融合蛋白,采用免疫印迹技术和酶联免疫吸附试验(ELISA)分析表达产物的抗原性。构建以CMV为启动子的表达HBV核心和前S1融合蛋白的真核表达载体。结果:HBV融合蛋白在大肠杆菌中有效稳定表达,表达产物具有核心抗原和前S1特异杭原性。成功构建了pCI-CS1真核表达载体。结论:本方法有效表达了HBV融合蛋白,表达产物具有特异抗原性。融合蛋白的表达及真核表达载体的构建为进一步进行HBV CS1融合蛋白和基因疫苗研究奠定了基础。     

Mutations within a furin consensus sequence block proteolytic release of ectodysplasin-A and cause X-linked hypohidrotic ectodermal dysplasia

X-linked hypohidrotic ectodermal dysplasia (XLHED) is a heritable disorder of the ED-1 gene disrupting the morphogenesis of ectodermal structures. The ED-1 gene product, ectodysplasin-A (EDA), is a tumor necrosis factor (TNF) family member and is synthesized as a membrane-anchored precursor protein with the TNF core motif located in the C-terminal domain. The stalk region of EDA contains the sequence -Arg-Val-Arg-Arg156-Asn-Lys-Arg159-, representing overlapping consensus cleavage sites (Arg-X-Lys/Arg-Arg( downward arrow)) for the proprotein convertase furin. Missense mutations in four of the five basic residues within this sequence account for approximately 20% of all known XLHED cases, with mutations occurring most frequently at Arg156, which is shared by the two consensus furin sites. These analyses suggest that cleavage at the furin site(s) in the stalk region is required for the EDA-mediated cell-to-cell signaling that regulates the morphogenesis of ectodermal appendages. Here we show that the 50-kDa EDA parent molecule is cleaved at -Arg156Asn-Lys-Arg(159 downward arrow)- to release the soluble C-terminal fragment containing the TNF core domain. This cleavage appears to be catalyzed by furin, as release of the TNF domain was blocked either by expression of the furin inhibitor alpha1-PDX or by expression of EDA in furin-deficient LoVo cells. These results demonstrate that mutation of a functional furin cleavage site in a developmental signaling molecule is a basis for human disease (XLHED) and raise the possibility that furin cleavage may regulate the ability of EDA to act as a juxtacrine or paracrine factor.     

Cleavage of the HPV16 Minor Capsid Protein L2 during Virion Morphogenesis Ablates the Requirement for Cellular Furin during De Novo Infection

Infections by high-risk human papillomaviruses (HPV) are the causative agents for the development of cervical cancer. As with other non-enveloped viruses, HPVs are taken up by the cell through endocytosis following primary attachment to the host cell. Through studies using recombinant pseudovirus particles (PsV), many host cellular proteins have been implicated in the process. The proprotein convertase furin has been demonstrated to cleave the minor capsid protein, L2, post-attachment to host cells and is required for infectious entry by HPV16 PsV. In contrast, using biochemical inhibition by a furin inhibitor and furin-negative cells, we show that tissue-derived HPV16 native virus (NV) initiates infection independent of cellular furin. We show that HPV16 L2 is cleaved during virion morphogenesis in differentiated tissue. In addition, HPV45 is also not dependent on cellular furin, but two other alpha papillomaviruses, HPV18 and HPV31, are dependent on the activity of cellular furin for infection.     

The cystatin-related epididymal spermatogenic protein inhibits the serine protease prohormone convertase 2

The cystatin-related epididymal spermatogenic (CRES) protein is related to the family 2 cystatins of the cystatin superfamily of cysteine protease inhibitors. However, CRES lacks sequences important for cysteine protease inhibitory activity and is specifically expressed in reproductive and neuroendocrine tissues. Thus, CRES is distinct from cystatins and may perform unique tissue-specific functions. The purpose of the present study was to determine whether CRES functions as a protease inhibitor in in vitro assays. In contrast to mouse recombinant cystatin C, recombinant CRES did not inhibit the cysteine proteases papain and cathepsin B, suggesting that it probably does not function as a typical cystatin. CRES, however, inhibited the serine protease prohormone convertase 2 (PC2), a protease involved in prohormone processing in the neuroendocrine system, whereas cystatin C showed no inhibition. CRES did not inhibit subtilisin, trypsin, or the convertase family members, PC1 and furin, indicating that it selectively inhibits PC2. Kinetic analysis showed that CRES is a competitive inhibitor of PC2 with a K(i) of 25 nM. The removal of N-terminal sequences from CRES decreased its affinity for PC2, suggesting that the N terminus may be important for CRES to function as an inhibitor. These studies suggest that CRES is a cross-class inhibitor that may regulate proprotein processing within the reproductive and neuroendocrine systems.     

Cleavage of the papillomavirus minor capsid protein, L2, at a furin consensus site is necessary for infection

Papillomaviruses (PV) comprise a large family of nonenveloped DNA viruses that include the oncogenic PV types that are the causative agents of human cervical cancer. As is true of many animal DNA viruses, PV are taken into the cell by endocytosis and must escape from the endosomal compartment to the cytoplasm to initiate infection. Here we show that this step depends on the site-specific enzymatic cleavage of the PV minor virion protein L2 at a consensus furin recognition site. Cleavage by furin, a cell-encoded proprotein convertase, is known to be required for endosome escape by many bacterial toxins. However, to our knowledge, furin has not been previously implicated in the viral entry process. This step is potentially a target for PV inhibition.     

Post-translational modification of Fibroblast Growth Factor 23

Fibroblast Growth Factor (FGF) 23 has been shown to play important roles in the development of hypophosphatemic rickets/osteomalacia. Complementary DNA predicts that the FGF23 protein is composed of 251 amino acids and N-terminal 24 amino acids seem to be a signal peptide. In vitro experiments indicate that a part of the FGF23 protein is processed between arginine179 and serine180. When full-length, N-terminal and C-terminal processed fragments of FGF23 were injected into mice, only the full-length FGF23 reduced serum phosphate levels indicating that the processing of FGF23 abolished its effect to cause hypophosphatemia. This processing was shown to be prevented by an inhibitor of furin indicating that the cleavage is mediated by subtilisin-like proprotein convertase. In addition to this processing, FGF23 protein seems to have O-linked glycosylation. Further studies are necessary to clarify the importance of O-glycosylation for FGF23 activity.     

α1-Antitrypsin Portland, a bioengineered serpin highly selective for furin: Application as an antipathogenic agent

The important role of furin in the proteolytic activation of many pathogenic molecules has made this endoprotease a target for the development of potent and selective antiproteolytic agents. Here, we demonstrate the utility of the protein-based inhibitor α₁-antitrypsin Portland (α₁-PDX) as an antipathogenic agent that can be used prophylactically to block furin-dependent cell killing by Pseudomonas exotoxin A. Biochemical analysis of the specificity of a bacterially expressed His- and FLAG-tagged α₁-PDX (α₁-PDX/hf) revealed the selectivity of the α₁-PDX/hf reactive site loop for furin (K_i, 600 pM) but not for other proprotein convertase family members or other unrelated endoproteases. Kinetic studies show that α₁-PDX/hf inhibits furin by a slow tight-binding mechanism characteristic of serpin molecules and functions as a suicide substrate inhibitor. Once bound to furin’s active site, α₁-PDX/hf partitions with equal probability to undergo proteolysis by furin at the C-terminal side of the reactive center -Arg³⁵⁵-Ile-Pro-Arg³⁵⁸-^↓ or to form a kinetically trapped SDS-stable complex with the enzyme. This partitioning between the complex-forming and proteolytic pathways contributes to the ability of α₁-PDX/hf to differentially inhibit members of the proprotein convertase family. Finally, we propose a structural model of the α₁-PDX-reactive site loop that explains the high degree of enzyme selectivity of this serpin and which can be used to generate small molecule furin inhibitors.     

Regulation of matrix metalloproteinase MT1-MMP/MMP-2 in cardiac fibroblasts by TGF-beta1 involves furin-convertase

OBJECTIVE: Heart failure is characterized by an imbalance of matrix synthesis/turnover, finally resulting in fibrosis. Cardiac myocytes and fibroblasts play a pivotal role in the remodeling process. Cardiac remodeling involves the expression of TGF-beta1 and matrix metalloproteinases (MMPs) in cardiac fibroblasts (CFBs). Furin, a subtilisin/kexin-like proprotein convertase (PC), activates TGF-beta1 and membrane-bound MT1-MMP, which facilitates pro-gelatinase A (MMP-2) activation. Even though several reports identified TGF-beta1 as a pro-fibrotic cytokine in the heart, it increases MMP-activity and cell migration/invasion in several cell types. The present study was done to investigate the contribution of TGF-beta1 and furin to CFBs MMP-activity and motility. METHODS AND RESULTS: Stimulation of CFBs from adult Sprague-Dawley rats with TGF-beta1 (20 ng/ml) induced furin, but had no effect on the closely related PC5. Inhibition of furin inhibited angiotensin II-induced TGF-beta1 activation, indicating that TGF-beta1 amplifies its activating convertase in CFBs. Pretreatment of CFBs with TGF-beta1 (20 ng/ml, 24 h) increased their migration by about two-fold (p<0.05), which was accompanied by an enhanced expression and activity of MT1-MMP and MMP-2. Brefeldin A (BFA), a Golgi-disturbing agent, inhibited MT1-MMP activation, indicating that it occurs in the trans-Golgi network (TGN), where furin is concentrated and colocalized with MT1-MMP. Inhibition of furin significantly inhibited TGF-beta1-induced MT1-MMP/MMP-2 activation. Furthermore, inhibition of furin attenuated TGF-beta1-enhanced migration on gelatin-coated membranes (p<0.05). This was comparable to the effects of the MMP-inhibitor GM6001, pointing out that MMPs are major mediators of TGF-beta1-enhanced CFB motility. CONCLUSION: We demonstrate that TGF-beta1 induces MMP-activity in CFBs, thereby facilitating CFBs motility. Furthermore, TGF-beta1 amplifies its activating convertase furin, which is also required for MT1-MMP/MMP-2 activation in CFBs. Thus, furin is central for TGF-beta1 and MT1-MMP activation and might be a novel target in cardiac remodeling.     

Processing of a fusion protein by endoprotease in COS-1 cells for secretion of mature peptide by using a chimeric expression vector.

The subtilisin-related proprotein convertase furin is expressed in various mammalian tissues. Expecting that COS-1 cells have a furin-like endoprotease, we constructed a fusion expression vector for production of a recombinant foreign protein having no signal peptide or a protein in truncated form into secreted mature protein. A cDNA fragment encoding N-terminal procalcitonin (pro-CT) of human calcitonin precursor was inserted into the mammalian expression vector pME18S. We used PCR techniques to generate four kinds of cDNAs encoding the C terminus of the pro-CT with Arg residues at P4 (Arg-Xaa-Lys-Arg), P6 (Arg-Xaa-Xaa-Xaa-Lys-Arg), or both (Arg-Xaa-Arg-Xaa-Lys-Arg), in addition to the Lys-Arg motif at the cleavage site, in order to determine the conditions for efficient processing in nonendocrine cells, such as COS-1 cells. The cDNA coding for the Fc fragment of human immunoglobulin G1 was fused in-frame to the cDNA encoding pro-CT at its C terminus. Upon transfection of the chimeric plasmids into COS-1 cells, almost all of the fusion protein with the Arg residues at both P4 and P6 were processed into secreted Fc product, even without cotransfection of furin. These results indicate that COS-1 cells have a furin-like endoprotease and suggest that pro-CT, with the Arg residues at both P4 and P6, can be used as a carrier peptide for expression of a foreign protein having no signal peptide or a protein in truncated form in COS-1 cells.     

The neuroendocrine polypeptide 7B2 is an endogenous inhibitor of prohormone convertase PC2.

The subtilisin-like prohormone convertase PC2 and the polypeptide 7B2 (an intracellularly cleaved protein of unknown function) are both selectively present in the regulated secretory pathway of neurons and endocrine cells. Here we demonstrate that intact recombinant 7B2 is a potent inhibitor of PC2 and prevents proPC2 cleavage in vitro, whereas the 7B2 cleavage product is virtually inactive. The PC2-related proteinase PC1/PC3 is not inhibited by 7B2. Furthermore, the carboxyl-terminal half of the 7B2 protein sequence is distantly related to the so-called potato inhibitor I family (which includes subtilisin inhibitors). Our findings indicate that 7B2 is a physiological inhibitor of PC2 and may provide alternative avenues for the manipulation of peptide hormone levels.