诺如病毒衣壳蛋白重组人3型腺病毒的构建

目的 制备表达诺如病毒衣壳蛋白的重组人3型腺病毒.方法 将诺如病毒衣壳蛋白基因(Noro-orf2)克隆到腺病毒穿梭载体pBSE3CMV-egfp上,与线性化人3型腺病毒骨架质粒pBRAdv3共电转化感受态大肠杆菌BJ5183,使其在细菌内发生同源重组,带Noro-orf2基因的表达框置换腺病毒E3区,PCR及酶切筛选得到重组腺病毒质粒,将重组腺病毒质粒转染Hep-2细胞进行包装,获得感染性的重组腺病毒粒子,免疫组化分析重组腺病毒中诺如病毒衣壳蛋白的表达.结果 同源重组后经酶切和PCR鉴定证明插入Noro-orf2基因的重组腺病毒质粒pBRAdv3E3dNor成功构建,并经转染包装得到高滴度的重组腺病毒Adv3E3dNor,免疫组化证明诺如病毒衣壳蛋白得到表达.结论 成功构建表达诺如病毒衣壳蛋白的重组3型腺病毒Adv3E3dNor,为研制人3型腺病毒-诺如病毒双价疫苗奠定了基础.     

人TH基因重组腺病毒的构建及生物学活性研究

将人酪氨酸羟化酶cDNA表达盒克隆于质粒型腺病毒载体p△Elsp1A，得到重组质粒pAd－TH。随后用脂质体法将重组质粒pAd－TH和拯救型腺病毒质粒pBHG11一起共转染293细胞，通过体内同源重组生成重组腺病毒AdCMVth，THcDNA重组进入腺病毒E1区并受CMV启动子控制。采用形态学、病毒核酸酶切和PCR／RT－PCR等方法进行鉴定正确。重组腺病毒滴度达到1010pfu／ml。初步结果表明，该重组腺病毒感染MN9D细胞后可使细胞内多巴胺水平增加1倍，显示出明显的TH生物学活性。提示TH重组腺病毒AdCMVth可作为高效的基因转移载体用于帕金森氏病基因治疗。     

人神经营养素-3基因重组腺病毒载体的构建、表达和生物活性检测

目的构建具有生物活性的人神经营养素-3（NT-3）基因重组腺病毒表达载体.方法从人脑组织mRNA中扩增NT-3基因全长cDNA,定向克隆于穿梭质粒pShuttle中,获得一个带有CMV启动子的质粒.再与腺病毒骨架DNA（Adeno-X viral DNA）体外连接,形成重组腺病毒质粒（pAd-NT-3）.用pAd-NT-3转染人胚肾293细胞后包装成有感染能力的重组腺病毒颗粒（Adeno-NT-3）. 结果NT-3基因RT-PCR扩增产物约801 bp.Adeno-NT-3 PCR鉴定为正确重组子.RT-PCR、免疫细胞化学、ELISA及Western blot检测显示,Adeno-NT-3能在其转染的293细胞表达和分泌NT-3.这种NT-3能使体外培养的神经干细胞更多地分化为神经元样细胞. 结论应用体外连接法成功构建了人NT-3基因重组腺病毒表达载体,其表达产物具有促进神经干细胞分化为神经元样细胞的活性作用.     

Bax和Fas在人皮肤血管瘤不同时期的表达及意义

目的　探讨Bax和Fax在血管瘤发生、发展及退化过程中的作用。方法　采用免疫组织化学方法 (S P法 )检测4 9例人皮肤血管瘤增生期、退化期及正常组织中Bax和Fax的表达水平。结果　发现Bax和Fas在血管瘤退化期的表达高于增生期 ,差异有显著性 (P <0 0 1)。结论　Bax和Fas与血管瘤的自然退化密切相关。     

编码与大鼠ERα-甘露糖苷酶同源的人6A8 cDNA在真核细胞的表达

目的观察６Ａ８ｃＤＮＡ（１３５８ｂｐ）在真核细胞的表达。方法Ｎｏｒｔｈｅｒｎｂｌｏｔ，构建６Ａ８ｃＤＮＡ表达载体ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ，转染ＣＯＳ－７细胞，及基因免疫小鼠。结果６Ａ８ｃＤＮＡ有４．３ｋｂ和３．５ｋｂ两种ｍＲＮＡ转录形式。４．３ｋｂｍＲＮＡ以外周血白细胞最丰富，其次为淋巴结、脾脏和骨髓，胸腺组织表达较少，胎肝则缺如。３．５ｋｂ者也以外周血白细胞最丰富，其次为肝脏、淋巴结和骨髓，胸腺和脾脏表达量最少。ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ在ＣＯＳ－７细胞表达了分子量４５０００左右的蛋白质，与单抗６Ａ８有反应。ｐＲｃ／ＣＭＶ－６Ａ８ｃＤＮＡ基因免疫小鼠，６／１０只小鼠血清与人活化Ｂ细胞株３Ｄ５细胞膜有反应，５／５只对照小鼠血清呈阴性反应。结论６Ａ８ｃＤＮＡ（１３５８ｂｐ）在真核细胞获得表达，但此ｃＤＮＡ非为全长。     

重组人肝细胞生长因子-NK4腺病毒的制备及初步鉴定

目的制备表达人肝细胞生长因子（HGF）-NK4蛋白的复制缺陷型重组腺病毒。方法酶切pcDNA3-hNK4质粒获得NK4基因编码区序列并克隆至穿梭载体构建重组pAdTrack—CMV—NK4载体，线性化后与pAdEasy-1共转化BJ5183，通过同源重组得到重组pAd—NK4病毒载体。将重组腺病毒载体转染HEK293包装细胞制备重组腺病毒，用病毒悬液感染人肝癌细胞株HepG2。RT—PCR法检测感染肿瘤细胞中NK4mRNA表达。结果酶切鉴定得到阳性pAd—NK4重组腺病毒载体，该载体能有效转染HEK293细胞并在细胞内成功包装。在转染2d后能观察到绿色荧光蛋白（GFP）表达。制备的Ad—NK4在体外能有效感染HepG2细胞并获得NK4基因高水平表达。结论成功构建了NK4基因的重组腺病毒载体并制备重组腺病毒颗粒，为进一步研究NK4基因的功能及应用NK4进行基因治疗提供实验依据。     

人骨形态发生蛋白2基因重组腺病毒的构建与鉴定

BACKGROUND:Bone morphogenetic protein 2 plays a key role in inducing osteogenesis. It involves in a series of bioprocess, including cell proliferation, determining the differentiation direction of germ line and cell death.  OBJECTIVE: To construct and identify the recombinant adenovirus vectors encoding human bone morphogenetic protein 2 gene by using AdMax system. METHODS:First, human bone morphogenetic protein 2 gene sequencing was amplified by PCR from human cDNA template and then cloned. Second, the recombinant shuttle plasmid was constructed and transformed into Escherichia coli competent cells DH5α. After the positive colonies were identified by colonies PCR and sequencing, the expression vectors were amplified and extracted. Next, the adenovirus expression vectors with target gene and the helper packaging plasmid carrying a majority of adenovirus genes were co-transfeced into 293 cells for virus packaging and amplification. Finally, target genes were detected by PCR, and target protein was detected by Western blot method, as well as infectious titer of the recombinant adenovirus was detected by end point dilution method. RESULTS AND CONCLUSION:Gene fragment of a length of 1 223 bp human bone morphogenetic protein 2 was obtained by PCR. The expression vectors constructed by homologous recombination techniques were identified by PCR cloning and sequencing; the results were correct. After virus packaging and amplification in 293 cells were identified by Western blot and PCR methods, the virus titer of recombinant adenovirus was 5×1013 pfu/L. These results suggest that the recombinant adenovirus vectors carrying human bone morphogenetic protein 2 gene have been constructed successfully.     

靶向腺病毒载体介导的EGFP基因在甲胎蛋白阳性肝癌细胞的特异表达

目的：建立一种在甲胎蛋白(AFP)阳性的肝癌细胞中靶向表达目的基因的重组腺病毒载体。方法： 基于腺病毒载体Adeno-XTM Expression system,以300 bp AFP特异启动子替换穿梭质粒Pshuttle中CMV启动子，将增强型绿色荧光蛋白(EGFP)基因作为报告基因亚克隆至Pshuttle，HEK293细胞包装腺病毒，收集病毒后分别转染人正常肝脏LO2细胞，人肝癌HepG2细胞及HeLa细胞；通过Northern杂交检测EGFP基因在3种细胞中的转录水平，荧光显微镜下观察3种细胞中绿色荧光蛋白的表达。结果：Northern杂交显示，HepG2细胞中有大量EGFP基因的转录，而正常肝细胞LO2和HeLa细胞中仅能检测到微量基因的转录；荧光显微镜检测发现HepG2细胞内有强绿色荧光表达，而在LO2以及HeLa细胞内见极弱绿色荧光。 结论： 在AFP特异启动子作用下，腺病毒携带的目的基因在AFP阳性的肝癌细胞中得到显著转录和表达，而在非AFP阳性细胞仅微量转录，蛋白表达极弱。该腺病毒载体可作为AFP阳性的肝癌基因靶向治疗的适宜载体。     

Alveolar macrophages and T cells from sarcoid, but not normal lung, are permissive to adenovirus infection and allow analysis of NF-kappa b-dependent signaling pathways

Adenovirus (Adv)-mediated gene transfer requires efficient infection of target cells. The objective of this study was to establish whether alveolar macrophages (AM) and T cells (AT) from sarcoid patients were permissive to infection with Adv vectors and if this property could be used to investigate cytokine gene regulation. Sarcoid and normal bronchoalveolar lavage (BAL) specimens infected with Adv vectors expressing either beta-galactosidase or a green fluorescent protein were analyzed for transgene expression by fluorescence-activated cell sorter (FACS) and direct immunofluorescence, respectively. Expression of surface antigens previously associated with Adv infection, the coxsackie/adenovirus receptor (CAR), alpha v beta 3, and alpha v beta 5 integrins, was also assessed using FACS analysis. Sarcoid AM and AT were found to efficiently express Adv transgenes, unlike AM from normal volunteers, peripheral blood monocytes, and peripheral blood T cells. Cells permissive to Adv infection expressed the CAR and alpha v beta 5 integrin (also alpha v beta 3 integrin for AM). The data indicate that the upregulation of Adv receptors and the ability to infect sarcoid AM and AT are related to the inflammatory environment within the lung. Having demonstrated efficient Adv-mediated transgene delivery to sarcoid AM and AT, a construct encoding porcine I kappa B alpha was then used to investigate the requirement for nuclear factor (NF)-kappa B in the regulation of cytokine gene expression in pulmonary sarcoidosis. Overexpression of I kappa B alpha in sarcoid BAL specimens indicated that tumor necrosis factor-alpha and interleukin (IL)-6 production by AM and interferon (IFN)-gamma production by AT is NF-kappa B dependent, whereas IL-4 production by AT is NF-kappa B independent. This is the first occasion that the requirement for NF-kappa B in IFN-gamma gene expression within primary human T cells has been demonstrated. The results of this study have implications for the future investigation of molecular pathways in inflammatory lung disease.     

表达Cre酶复制缺陷型重组腺病毒载体的构建及鉴定

目的构建Cre-loxP条件性基因敲除系统中Cre酶的重组腺病毒表达载体,作为体细胞条件性基因敲除的基础,并为传统ES细胞条件性基因敲除提供新的选择。方法用pAd-easy系统在大肠杆菌内经同源重组的方法构建Cre酶的复制缺陷型重组腺病毒载体;W estern b lot方法鉴定Cre蛋白的表达。结果在大肠杆菌内构建出重组腺病毒质粒,在包装细胞系内包装出重组病毒颗粒;测定病毒滴度为106pfu/L;重组病毒在细胞内表达Cre蛋白。结论表达Cre酶的重组腺病毒载体构建成功,阳性重组质粒的鉴定方法得以改进,为进一步的体细胞Cre-loxP条件性基因敲除提供了基础。     

Budding and secretion of HIV Gag-Env virus-like particles from recombinant human adenovirus infected cells

We have characterized the assembly, budding and extra-cellular release of human immunodeficiency virus (HIV) Gag-Env virus-like particles (VLPs) from human embryonic kidney cells (293 cells expressing the E1a protein of adenovirus) infected with recombinant replication-defective human adenovirus type 5. Recombinant human adenovirus vectors expressing the chimeric Gag-Env protein were constructed by inserting the gag-env fusion gene into the E1a region of the human adenovirus type 5. Biochemical and immunological analyses of VLPs recovered from the culture supernatant revealed that these particles contain the HIV-2 Gag protein and segments of Env protein from the HIV-1 gp120. This chimeric Gag-Env protein interacted with HIV-1 positive patient sera and with HIV-2 Gag monoclonal antibody. Immunoelectron microscopy of the 293 cells infected with the recombinant adenoviruses showed that the HIV Gag-Env antigen is present in the VLPs. Thin-section electron microscopy (EM) revealed that the Gag-Env VLPs bud through the cytoplasmic membrane, as well as through membranes of intracellular vacuoles. The thin-section EM showed that the Gag-Env VLPs have a spherical morphology with an electron-dense ring. The size of VLPs range from 110 to 140 nm in diameter, which is slightly larger than that of the Gag particles without Env protein fusion. Mice immunized with recombinant adenoviruses generated antibodies that specifically reacted with Gag-Env chimeric proteins. Our results support the idea that the replication-defective adenovirus could be used to induce immune responses that might be useful in a vaccine against HIV/AIDS.     

携带人生长抑素2型受体的腺病毒载体的构建和表达

目的： 构建表达SSTR2的重组腺病毒载体，用于胰腺癌的基因治疗。 方法: 构建重组腺病毒穿梭质粒pDC316-SSTR2，通过脂质体与腺病毒骨架质粒pBHGlox(delta)E1,3Cre共转染293细胞，经位点特异性重组获得重组腺病毒Ad-SSTR2。通过PCR鉴定Ad-SSTR2。经293细胞扩增，纯化制备高滴度病毒感染液。以病毒感染液感染人胰腺癌细胞capan-2，应用免疫印迹检测SSTR2蛋白的表达。 结果: 成功构建腺病毒穿梭质粒pDC316-SSTR2，与pBHGlox(delta)E1,3Cre共转染293细胞获得了重组腺病毒Ad-SSTR2，以Ad-SSTR2基因组DNA为模板同时扩增出了人SSTR2 cDNA基因片段和腺病毒骨架基因片段。Ad-SSTR2感染capan-2 48 h后，免疫印迹检测到SSTR2蛋白的表达。 结论: 表达人SSTR2的重组腺病毒载体构建成功，并在胰腺癌细胞中得到正确表达，为SSTR2基因治疗胰腺癌奠定了基础。     

TR基因重组腺病毒载体的构建和鉴定

目的 :构建含人硫氧还蛋白还原酶 (TR)基因的重组腺病毒载体 ,探讨TR的抗氧化功能与神经退行性疾病的相关性。方法 :从重组质粒pGEM TR上用内切酶切下编码 5 0 0个氨基酸的全长TRcDNA片段 ,并连接穿梭质粒pShuttle,再双酶切pShuttle TR。将带有CMV启动子的目的片段 ,插入E1、E3缺失的Adeno X病毒DNA中 ,以Adeno TRDNA通过脂质体转染HEK2 93细胞 ,获得重组腺病毒Adeno TR进行PCR鉴定及病毒滴度测定。用重组腺病毒感染CV1细胞 ,通过免疫荧光染色与Westernblot分别检测重组腺病毒感染的细胞上和裂解液中TR蛋白的表达。结果 :重组腺病毒Adeno TR的病毒滴度为 4 .4× 10 11pfu/L。PCR、荧光显微镜证实 ,以及Westernbolt分析 ,在相对分子质量 (Mr)约 5 5 0 0 0处均出现特异性条带。结论 :成功地构建了重组腺病毒载体 ,并能介导外源基因TR表达 ,为进一步研究TR的功能及其与疾病的相关性奠定了基础。     

7型腺病毒疫苗株载体的构建及β—半乳糖苷酶基因 …

目的 构建Ｅ３区缺失的７型腺病毒疫苗株（Ａｄ７ｖ）载体并表达β－半乳糖苷酶基因。方法 从人二倍体细胞的Ｗ１３８培养的Ａｄ７ｖ中分离病毒ＤＮＡ，利用Ａｄ７ｖＤＮＡ天然的酶切位点，经过多步亚克隆，克隆的同时将Ｅ３区７８．８－８７ｍｕ片段缺失，并将多克隆酶切位点带入Ａｄ７ｖ载体。为了验证载体的功能，将带有巨细胞病毒（ＣＭＶ）早期启动子β－半乳糖苷酶基因插入缺失的Ｅ３区。将这一重组质粒和ＥｃｏＲＩ酶切Ａｄ     

轮状病毒组特异性抗原VP6在重组腺病毒中的表达及其免疫学效果的研究

目的 构建可表达A组轮状病毒组特异性抗原VP6的非复制型重组腺病毒载体，并对其生物学和免疫学性质进行研究。方法 将人轮状病毒VP6基因插入腺病毒载体pShuttle-CMV中，通过细胞内同源重组方法获得重组腺病毒DNA，将其转染293细胞获得重组腺病毒。用聚合酶链反应（PCR）及Southern blot方法，检测目的基因在重组腺病毒中的整合，用Western blot检测VP6的表达。通过灌胃和滴鼻两种途径对小鼠进行免疫，并对免疫后小鼠体液和粘膜免疫进行分析。结果 得到了重组腺病毒rvAd-VP6,VP6基因整合于腺病毒基因且中，在293细胞中可稳定表达。2次免疫后灌胃和滴鼻两组小鼠均产生明显免疫应答，血清IgG抗体滴度分别为1：1000和1：10000－1：100000。除了血清IgG外，小鼠还产生了较强的针对轮状病毒的血清IgA，滴度为1：10－1：100。滴鼻组在肺灌洗液和肠匀浆液中均可检测到分泌型IgA(sIgA)，灌胃组仅在肠道检测到sIgA。滴鼻组的免疫学效果明显优于灌胃组。结论 人轮状病毒VP6基因重组腺病毒载体的成功构建及所取得的良好免疫学效果，为我国具有自主知识产权的新型轮状病毒基因工程疫苗的研制奠定了基础。     

Characterization of host immunity to cytomegalovirus pp150 (UL32)

The basic phosphoprotein 150 (pp150), the product of UL32 (unique long domain 32) gene of human cytomegalovirus (CMV), is an abundant component of the viral tegument and a target of human leukocyte antigen (HLA)-restricted cytotoxic T cells (CTLs) after infection. Identification of minimal cytotoxic epitopes (MCEs) from this CMV protein is of importance for peptide-based vaccines and immunotherapeutic approaches. Several pp150-specific CTL clones were derived from peripheral blood mononuclear cells of healthy CMV-positive donors with autologous fibroblasts infected either with CMV AD169 or with a recombinant vaccinia virus expressing full-length pp150 protein. HLA A*0301- and HLA A*6801-restricted CD8+ pp150 T-cell clones derived from different donors were found to efficiently kill autologous CMV-infected fibroblasts. Fine mapping of each MCE first used a T-cell epitope prediction algorithm. Overlapping peptides within the recognized regions were screened. The analysis identified pp150(792-802) and pp150(945-955) as MCEs for the HLA A*6801 and the HLA A*0301 pp150 clones, respectively. In vitro stimulation by recombinant modified vaccinia Ankara virus expressing full-length pp150 elicited high frequencies of CMV-CTL and interferon gamma production specific for the MCE identified in all subjects. The consistent presence of pp150 T cells in CMV-exposed individuals supports a role for this antigen in shaping the antiviral CTL response and indicates that pp150 could be a pivotal constituent of prophylactic and therapeutic CMV vaccines.     

Mesenchymal stem cells and adipogenesis in hemangioma involution

Hemangioma is a benign tumor of infancy whose hallmark is rapid growth during the first year of life followed by slow regression during early childhood. The proliferating phase is characterized by abundant immature endothelial cells, the involuting phase by prominent endothelial-lined vascular channels and endothelial apoptosis, and the involuted phase by few remaining capillary-like vessels surrounded by loose fibrofatty tissue. Nothing is known about the mechanisms that contribute to the adipogenesis during this spontaneous regression. We postulated that mesenchymal stem cells (MSCs) reside in the tumor and preferentially differentiate into adipocytes. To test this hypothesis, we isolated MSCs from 14 proliferating and five involuting hemangiomas by taking advantage of the well known selective adhesion of MSCs to bacteriologic dishes. These hemangioma-derived MSCs (Hem-MSCs) are similar to MSCs obtained from human bone marrow, expressing the cell surface markers SH2 (CD105), SH3, SH4, CD90, CD29, smooth muscle alpha-actin, and CD133 but not the hematopoietic markers CD45 and CD14 or the hematopoietic/endothelial markers CD34, CD31, and kinase insert domain receptor (KDR). Hem-MSCs exhibited multilineage differentiation with robust adipogenic potential that correlated with the proliferating phase. The numbers of adipogenic Hem-MSCs were higher in proliferating-phase than in involuting-phase tumors and higher than in normal infantile skin. Furthermore, Hem-MSCs exhibited a random pattern of X-chromosomal inactivation, indicating that these cells are not clonally derived. In summary, we have identified MSCs as a novel cellular constituent in infantile hemangioma. These MSCs may contribute to the adipogenesis during hemangioma involution.     

Recombinant bovine adenovirus type 3 expressing bovine viral diarrhea virus glycoprotein E2 induces an immune response in cotton rats

Recombinant bovine adenovirus is being developed as a live vector for animal vaccination and for human gene therapy. In this study, two replication-competent bovine adenovirus 3 (BAV-3) recombinants (BAV331 and BAV338) expressing bovine viral diarrhea virus (BVDV) glycoprotein E2 in the early region 3 (E3) of BAV-3 were constructed. Recombinant BAV331 contains chemically synthesized E2 gene (nucleotides modified to remove internal cryptic splice sites) under the control of BAV-3 E3/major late promoter (MLP), while recombinant BAV338 contains original E2 gene under the control of human cytomegalovirus immediate early promoter. Since E2, a class I membrane glycoprotein, does not contain its own signal peptide sequence at the 5' end, the bovine herpesvirus 1 (BHV-1) glycoprotein D signal sequence was fused in frame to the E2 open reading frame (ORF) for proper processing of the E2 glycoprotein in both the recombinant viruses. Recombinant E2 protein expressed by BAV331 and BAV338 recombinant viruses was recognized by E2-specific monoclonal antibodies as a 53-kDa protein, which also formed dimer with an apparent molecular weight of 94 kDa. Insertion of an E2-expression cassette in the E3 region did not effect the replication of recombinant BAV-3s. Intranasal immunization of cotton rats with these recombinant viruses generated E2-specific IgA and IgG responses at the mucosal surfaces and in the serum. In summary, these results show that the pestivirus glycoprotein can be expressed efficiently by BAV-3. In addition, mucosal immunization with replication-competent recombinant bovine adenovirus 3 can induce a specific immune response against the expressed antigen.     

采用绿色荧光蛋白腺病毒载体表达HBsAgCTL优势表位肽MHC单链三聚体

目的 采用携带GFP报告基因腺病毒载体表达HBsAg细胞毒性T细胞(CIL)表位MHC单链三聚体,为增强HBV特异性免疫提供新方法.方法 构建小鼠MHC Ⅰ类分子(H-2Ld)限制的HBsAg优势CTL表位肽与MHCⅠ类分子重链(Ld)和β2M链(β2-microglobulin,β2微球蛋白)的单链三聚体(HBsAg-SCT).并将HBsAg-SCT亚克隆到GFP标记腺病毒载体,以HBsAg和OVA-SCT作为对照,转染293A细胞,包装产生携带HBsAs-SCT,HBsAg和OVA-SCT的重组腺病毒.结果 经双酶切和克隆测序鉴定,HBsAg-SCT能成功克隆到编码GFP标记的腺病毒载体,通过荧光显微镜观察到转染的293A细胞含有绿色荧光素蛋白.通过Western Blot进一步证实表达的重组蛋白HBsAg-SCT能与抗-腿抗体发生反应.结论 编码HBsAg-SCT的荧光蛋白腺病毒载体成功构建,并能在293A细胞中完整包装成重组腺病毒颗粒.     

采用绿色荧光蛋白腺病毒载体表达HBsAgCTL优势表位肽MHC单链三聚体

目的 采用携带GFP报告基因腺病毒载体表达HBsAg细胞毒性T细胞(CIL)表位MHC单链三聚体,为增强HBV特异性免疫提供新方法.方法 构建小鼠MHC Ⅰ类分子(H-2Ld)限制的HBsAg优势CTL表位肽与MHCⅠ类分子重链(Ld)和β2M链(β2-microglobulin,β2微球蛋白)的单链三聚体(HBsAg-SCT).并将HBsAg-SCT亚克隆到GFP标记腺病毒载体,以HBsAg和OVA-SCT作为对照,转染293A细胞,包装产生携带HBsAs-SCT,HBsAg和OVA-SCT的重组腺病毒.结果 经双酶切和克隆测序鉴定,HBsAg-SCT能成功克隆到编码GFP标记的腺病毒载体,通过荧光显微镜观察到转染的293A细胞含有绿色荧光素蛋白.通过Western Blot进一步证实表达的重组蛋白HBsAg-SCT能与抗-腿抗体发生反应.结论 编码HBsAg-SCT的荧光蛋白腺病毒载体成功构建,并能在293A细胞中完整包装成重组腺病毒颗粒.