表达人JNK基因重组腺病毒的构建和鉴定

目的 制备表达人c-jun氨基末端激酶(JNK)复制缺陷型重组腺病毒.方法 将重组穿梭载体pAdTrack-CMV-WT-JNK线性化后,与pAdEasy-1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体.将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定.结果 JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定JNK基因片段并测序鉴定.动物实验证实构建的Ad-WT-JNK能有效在肝组织表达.结论 该研究成功构建了JNK重组腺病毒载体及相应重组腺病毒颗粒,为进一步研究JNK的作用及应用JNK进行相关疾病的基因治疗奠定了基础.     

表达人单核细胞趋化蛋白-1基因重组腺病毒的制备

目的制备表达人单核细胞趋化蛋白-1(MCP-1)复制缺陷型重组腺病毒。方法RT-PCR法获得人肝组织中MCP-1cDNA片段,与T载体连接后亚克隆至腺病毒穿梭载体的巨细胞病毒启动子下游,构建重组穿梭载体pAdTrack-CMV-MCP-1,将其线性化后与pAdEasy-1共转化大肠杆菌B J5183,进行同源重组得到重组腺病毒载体。将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,PCR扩增鉴定病毒颗粒。结果MCP-1重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5 d后可以观察到绿色荧光蛋白(GFP)明显表达,搜集的病毒经过PCR扩增得到特定MCP-1基因片段。结论MCP-1重组腺病毒载体及相应重组腺病毒颗粒的成功构建和制备,为进一步研究MCP-1的作用及应用MCP-1进行基因治疗奠定了基础。     

HCV NS3解旋酶基因原核表达载体pGEX-4T-1的构建

[目的]构建丙型肝炎病毒(HCV)非结构蛋白NS3解旋酶基因原核表达载体,为进一步研究和解析NS3的解旋酶基因对病毒复制的机制准备条件.[方法]将含有NS3基因的pMD-24/HCV NS3质粒转化感受态菌DH-5α并扩增;提取pMD-24/HCV NS3质粒;从pMD-24/HCV NS3质粒中扩增出NS3解旋酶基因;并将其插入到克隆载体pMD-18T中,再与表达载体pGEX-4T-1重组,以得到重组的原核表达载体pGEX-4T-1/NS3解旋酶.[结果]从pMD-24/HCV NS3质粒中扩增出的NS3解旋酶基因片断大小正确,经测序证明其碱基序列为编码目的基因的正确序列:电泳结果证明已将此片段克隆到pGEX-4T-1内.[结论]成功地构建了HCV NS3解旋酶基因的原核表达载体DGEX-4T-1/NS3解旋酶.     

DN—JNK基因重组腺病毒的构建和鉴定

目的制备表达人c-jun氨基末端激酶（JNK）复制缺陷型重组腺病毒（Ad—DN—JNK）,并通过动物实验进行功能鉴定。方法将重组穿梭载体pAdTrack—CMV—DN—JNK线性化后,与pAdEasy—1共转化大肠杆菌BJ5138,进行同源重组得到重组腺病毒载体。将重组腺病毒载体转染入包装细胞HEK293内制备复制缺陷型重组腺病毒,并经PCR及DNA测序鉴定。Western blot检测Ad—DN-JNK在SD大鼠肝组织中JNK1蛋白表达情况,及胰岛素受体底物1丝氨酸307（IRS-1^Ser307）磷酸化水平。结果JNK重组腺病毒载体能有效转染HEK293细胞并在细胞内成功包装,5d后观察到绿色荧光蛋白（GFP）明显表达,搜集的病毒经过PCR扩增得到了特定JNK基因片段并测序鉴定。所制备的Ad—DN—JNK滴度为2．5×10^10pfu／ml,动物实验证实构建的Ad—DN-JNK能有效在肝组织表达。结论该研究成功构建了DN—JNK重组腺病毒载体及相应重组腺病毒颗粒,动物实验证明Ad—DN—JNK能有效介导DN—JNK基因蛋白表达并下调IRS-1^Ser307磷酸化水平。为进一步研究JNK的作用及应用DN—JNK进行相关疾病的基因治疗奠定基础。     

HBsAg腺病毒疫苗载体的构建及其免疫学研究

目的 构建含HBsAg基因的重组复制缺陷型腺病毒疫苗载体,观察小鼠抗HBsAg免疫反应. 方法以pEcob-6为模板,PCR扩增目的 基因HBsAg,腺病毒穿梭载体PAd-track-cmv与HBsAg经T4连接酶连接,重组为PAd-track-cmv-HBs穿梭质粒;腺病毒骨架载体质粒PAd-Easy-1再与PAd-track-cmv-HBs在大肠杆菌BJ-5183中同源重组PAd-Easy-1-HBs质粒,PAd-Easy-1-HBs脂质体介导转染293细胞,包装HBsAg重组腺病毒.30只小鼠随机分成3组,分别肌肉注射PAd-Easy-1(载体组)、HBsAg重组腺病毒(实验组)、PBS(正常对照组),免疫2次.2月后取小鼠血清检测抗HBsAg抗体反应. 结果 PAd-track-cmv-HBs穿梭质粒经上海基康生物技术公司进行DNA测序,结果与预期结果相同,含有完整的HBsAg目的 基因.PAd-Easy-1-HBs用Pac-1酶切,Pac-1能将载体切成两个片段,大片段约为30 kb,小片段约为3 kh.HBsAg重组腺病毒组抗HBsAg的OD值明显升高,与正常对照组比较,差异有统计学意义(P＜0.05).与PAd-Easy-1载体对照组比较,差异有统计学意义(P＜0.05). 结论 HBsAg腺病毒疫苗载体构建成功,且能够诱导小鼠产生抗HBsAg.     

表达肺孢子菌p55多表位串联基因腺病毒重组载体的构建

目的构建携带肺孢子菌p55多表位串联基因(p55TAG)的腺病毒重组载体,鉴定其在真核细胞HEK293中的表达情况。方法将人工合成的p55TAG目的片段与腺病毒载体(p HBAd-EF1-MCS-GFP)连接后转化到DH5α大肠杆菌感受态中,经Amp抗性筛选得到重组载体p HBAd-p55TAG,将重组载体与腺病毒骨架质粒p BHGlox(delta)E1,3Cre共转染HEK293细胞,通过绿色荧光蛋白(GFP)验证转染结果,收集包装成功的病毒进行PCR扩增,扩增产物测序验证,用Western blot检测病毒在感染细胞后的目的蛋白表达情况。结果构建的p HBAd-p55TAG腺病毒重组载体转染HEK293细胞后检测到绿色荧光蛋白表达,包装好的病毒PCR扩增产物测序结果与原始序列一致,Western blot检测结果显示在60 k Da处有成功表达目的条带。结论本研究成功构建了表达肺孢子菌p55多表位串联基因的腺病毒重组载体,并验证其能在真核细胞中有效表达。构建的载体为进一步研究抗肺孢子菌感染疫苗奠定了基础。     

重组人Bcl-2腺病毒载体的构建与鉴定

目的　构建人bcl-2重组腺病毒载体。方法　通过酶切分析获得正向插入目的基因的真核表达载体pcDNA/bcl -2 ,然后将bcl -2定向克隆到腺病毒穿梭质粒pAdCMV中 ,通过体内同源重组 ,重组质粒与质粒pJM17共转染 2 93细胞 ,获得复制缺陷型重组腺病毒AdCMV/bcl -2 ,进行扩增、纯化。通过PCR检测病毒DNA ,空斑实验检测病毒滴度。结果　腺病毒穿梭质粒与pJM17体内同源重组后获得含目的基因的重组腺病毒 ,病毒滴度为 2 0× 10 1 0 pfu/ml。结论　基因重组腺病毒载体能成功获得高滴度含目的基因的腺病毒     

肾上腺髓质素基因腺病毒表达载体的构建及其对骨髓间充质干细胞增殖的影响

目的:构建表达大鼠AM基因的重组腺病毒载体,探讨其对MSCs增殖的影响.方法:设计AM基因上下游引物,以大鼠肾脏组织总RNA为模板,经RT-PCR扩增获得AM基因全部序列.片段回收后经酶切,定向插入腺病毒穿梭质粒,获得重组质粒pshuttle-CMV-AM.经双酶切、PCR及插入片段测序鉴定后,将正确重组体转化包含腺病毒骨架质粒pAdEasy-1的BJ5183菌.同源重组后用选择性培养基筛选阳性克隆,提取质粒,将重组腺病毒质粒分别进行酶切、线性化、纯化,用脂质体转染293细胞.制备病毒上清并测定其滴度,并感染大鼠骨髓间充质干细胞,用MTT方法评估其对MSCs增殖的影响.结果:完成构建含有AM基因的重组腺病毒,RT-PCR及Western blot检测证实AM在MSCs中表达,并能够促进其增殖(缺氧组与缺氧 AM组相比,P＜0.01).结论:构建重组腺病毒载体,并在MSCs表达AM蛋白.     

转录因子Bach2的构建及表达

目的构建高效表达人Bach2基因的慢病毒重组质粒,并探索其在免疫疾病中的机制。方法取健康人外周血分离单个核细胞并提取总RNA逆转录为c DNA,扩增Bach2基因CDS序列,插入p LVX-IRES-Zs Green1慢病毒质粒。磷酸钙共转染法包装慢病毒,感染人HEK293T细胞。空白质粒感染HEK293T细胞作为对照。免疫印迹分析空白质粒和过表达质粒HEK293T细胞中Bach2基因表达。结果 PCR结果显示,Bach2成功插进p LVX-IRES-Zs Green1质粒中,real-time PCR以及Western blot证实,Bach2基因成功在HEK293T细胞中高效表达。结论成功构建高效表达Bach2基因的慢病毒穿梭质粒,并成功在HEK293T细胞表达。     

Construction and immunogenicity of recombinant adenovirus expressing the capsid protein of porcine circovirus 2 (PCV2) in mice

Porcine circovirus 2 (PCV2) has been implicated as the etiological agent of some diseases, mainly postweaning multisystemic wasting syndrome (PMWS) and porcine dermatitis and nephropathy syndrome (PDNS). The capsid (Cap) protein encoded by the PCV2 ORF2 gene may be an excellent candidate for vaccination. In this study, the Cap protein gene was amplified by PCR, and cloned into the transfer vector pShuttle-CMV. After co-transformation of PmeI-linearized recombinant plasmid pShuttle-CMV-ORF2 and the bone vector pAdEasy-1 into Escherichia coli bacteria strain BJ5183, recombinant plasmid containing Cap protein gene (pAd-ORF2) was obtained and identified with PCR. Upon transfection of PacI-linearized plasmid pAd-ORF2 in 293 cell line, a recombinant adenovirus was obtained and named as rAd-Cap with viral titer of 10(13.0) TCID(50)/ml. The expression of the Cap protein in the 293 cells infected with rAd-Cap was confirmed with specific antibody to PCV2 by Western blotting and IPMA. Mice were inoculated with 10(8), 10(10) and 10(12) TCID(50)/mouse of rAd-Cap and boosted 2 weeks later, and they could generate antibody against PCV2 detected by indirect ELISA, Western blot and neutralizing activity assay. It indicated that the rAd-Cap was able to express the capsid of PCV2 and could elicit immune responses against the PCV2 in mice. The recombinant adenovirus might be an attractive candidate vaccine for preventing the disease associated with PCV2 infection.     

Vaccination with an adenoviral vector encoding hepatitis C virus (HCV) NS3 protein protects against infection with HCV-recombinant vaccinia virus

Cellular immune response plays an important role in the clearance of hepatitis C virus (HCV). Thus, development of efficient ways to induce anti-viral cellular immune responses is an important step toward prevention and/or treatment of HCV infection. With this aim, we have constructed a replication-deficient recombinant adenovirus expressing HCV NS3 protein (RAdNS3). The efficacy of RAdNS3 was tested in vivo by measuring the protection against infection with a recombinant vaccinia virus expressing HCV-polyprotein (vHCV1-3011). Immunisation with 10(9)pfu of RAdNS3 induced anti-NS3 humoral, T helper and T cytotoxic responses. We identified eight epitopes recognised by IFN-gamma producing cells, five of them exhibiting lytic activity. Moreover, we show that RAdNS3 immunised mice were protected against challenge with vHCV1-3011 and that this protection was mediated by CD8(+) cells. In conclusion, our results suggest that adenoviral vectors encoding NS3 might be useful for the induction of prophylactic and/or therapeutic anti-HCV immunity.     

Interactions between helper T-cell epitopes of hepatitis C virus

The premise of this work is that within a given hepatitis C virus (HCV) protein there exists an array of Th1 and Th2 epitopes, each of which can provide synergistic (positive or negative) effects upon other epitopes by intramolecular, cytokine-mediated immunoregulation of helper T-cell responses. To address this question, we constructed minigene plasmids pHCVTh1, pHCVTh1X3 and pHCVThR, and HCV NS3 full-length plasmid pHCVNS3. 293T cells were transfected with these plasmids and cell lysates from the transfected cells were used to stimulate PBMC from a patient with chronic HCV infection. IL-2 and IFN-gamma in the supernatant of the cultured PBMC were tested and proliferation of the PBMC was measured. The results demonstrate that interactions exist among helper T-cell epitopes; the synergistic effects of suppressive Th2 epitopes upon Th1 epitopes will inhibit the responses induced by Th1 epitopes, which may contribute to chronic infection by HCV; synergistic effects among Th1 epitopes induce higher levels of IFN-gamma, which may suggest a new strategy for HCV vaccine development. Further, stimulation of an HCV NS3 specific clone with cell lysates from 293T cells transfected with different constructs shows that the HCV NS3 clone could respond to all suggesting that the epitope-specific suppression may be due to an imbalance of Type 1 and Type 2 cytokines or regulatory T-cells.     

Construction and immunogenicity of recombinant adenovirus expressing the major outer membrane protein (MOMP) of Chlamydophila psittaci in chicks

Avian chlamydiosis is caused by Chlamydophila psittaci. The major outer membrane protein (MOMP) encoded by the outer membrane protein 1 (omp1) gene is an excellent candidate for genetic engineering of a vaccine against avian chlamydiosis. In this study, the MOMP gene was amplified by PCR and cloned into the transfer vector pShuttle-CMV. The recombinant plasmid was obtained by recombination between the plasmid pShuttle-CMV-MOMP and skeleton vector pAdEasy-1 in Escherichia coli strain BJ5183. The titer of recombinant adenovirus containing the MOMP gene (rAd-MOMP) of C. psittaci was 3.4x10(10)TCID(50)/ml in human embryonic kidney 293 (HEK293) monolayer cells. The expression of the MOMP in HEK293 cells infected with rAd-MOMP was confirmed by an indirect immunofluorescence assay. Specific pathogen free (SPF) chicks were inoculated with 10(6), 10(8), and 10(10)TCID(50) of rAd-MOMP/chick. Inoculated chicks generated antibodies against MOMP of C. psittaci, which were detected by an indirect hemagglutination test (IHA). The vaccinated chicks were challenged with a virulent Chinese field isolate. Nine out of 10 chicks in the vaccinated group were protected, while birds in the wild-type adenovirus control group and the PBS control group all showed clinical signs after challenge. The results indicate that the recombinant adenovirus containing the MOMP gene of C. psittaci might be a candidate vaccine against avian chlamydiosis.     

丙型肝炎病毒E2蛋白DNA疫苗诱导小鼠产生中和抗体的研究

目的探讨丙型肝炎病毒(HCV)包膜E2蛋白DNA疫苗诱导小鼠产生中和抗体的可行性。方法构建截除疏水性羧基末端的HCV包膜蛋白表达质粒pCI-1b661以及同时截除疏水性羧基末端和高变区1(HVR1)的表达质粒pCI-1b661Δ,转染293T细胞,以Western blot和ELISA检测细胞内和培养上清中的HCVE2蛋白,将两种表达质粒及空载体分别肌注免疫BALB/c小鼠,以ELISA检测小鼠血清中的HVR1抗体,以HCV假病毒颗粒(HCVpp)分析小鼠血清的中和活性。结果 2种表达质粒均能表达分泌性截短型E2蛋白。pCI-1b661免疫的8只小鼠血清中均可检测到HVR1抗体,而pCI-1b661Δ免疫血清中未检测到HVR1抗体。pCI-1b661和pCI-1b661Δ免疫血清对HCVpp的中和率分别为(78.5±13.8)%和(38.7±6.5)%,差异有显著性(P<0.01)。pCI-1b661免疫组小鼠血清的中和率与HVR1抗体水平呈正相关(r=0.967,P<0.01)。结论表达截短型E2蛋白的DNA疫苗能诱导产生HCV中和抗体,其主要成员为HVR1抗体。     

Induction of multispecific Th-1 type immune response against HCV in mice by protein immunization using CpG and Montanide ISA 720 as adjuvants

Recent studies demonstrate that Th1-type immune responses against a broad spectrum of hepatitis C virus (HCV) gene products are crucial to the resolution of acute HCV infection. We investigated new vaccine approaches to augment the strength of HCV-specific Th1-type immune responses. ELISPOT assay revealed that single or multiple protein immunization using both CpG ODN and Montanide ISA 720 as adjuvants induced much stronger IFN-gamma-producing Th1 responses against core, NS3 and NS5b targets than did the formulation without these adjuvants. Protein vaccination using CpG ODN and Montanide ISA 720 as adjuvants also greatly enhanced humoral responses to HCV core, E1/E2 and NS3. When specific IgG isotypes were assayed, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants produced higher titers of IgG2a dominant antibodies than did protein immunization alone, indicating a more Th1-biased pathway. This increase in IgG2a is consistent with the induction of Th1 cells secreting IFN-gamma demonstrated by ELISPOT assay. In conclusion, protein immunization using CpG ODN and Montanide ISA 720 as adjuvants greatly enhanced cellular (Th1 type) as well as humoral immune responses against HCV in Balb/c mice. The use of adjuvants appears critical to the induction of Th1 immune responses during HCV vaccination with recombinant proteins.     

Enhanced cellular immunity to hepatitis C virus nonstructural proteins by codelivery of granulocyte macrophage-colony stimulating factor gene in intramuscular DNA immunization

Hepatitis C virus (HCV) nonstructural (NS) proteins appeared to be important targets for HCV vaccine development, since NS-specific T-helper-cell responses are associated with clearance from acute HCV infection. In this report, we have constructed a plasmid, pTV-NS345, that encodes the HCV NS3, NS4 and NS5 proteins (NS345) and a bicistronic plasmid, PTV-NS345/GMCSF, in which the HCV NS345 polyprotein and GMCSF are translated independently. Intramuscular inoculation with pTV-NS345 plasmid DNA into the Buffalo rats generated both antibody and T-cell proliferative responses to each NS protein. The expression of GMCSF, together with HCV NS345 proteins, appeared to significantly increase T-cell proliferative responses. In particular, the inoculation of a bicistronic plasmid generated higher T-cell proliferative responses to each NS protein than did the coinjection of two separate plasmids, pTV-NS345 and pTV-GMCSF. These results demonstrate that the codelivery of GMCSF augmented HCV NS345-specific cellular immunity and that the intensity of the immunity was differed depending on how GMCSF gene is codelivered.     

Enhancement of CD4 and CD8 immunity by anti-CD137 (4-1BB) monoclonal antibodies during hepatitis C vaccination with recombinant adenovirus

The induction of protective or therapeutic cellular immunity against hepatitis C virus (HCV) is a difficult goal. In a previous work we showed that immunization with a recombinant adenovirus encoding HCV-NS3 (RAdNS3) could partially protect mice from challenge with a vaccinia virus encoding HCV antigens. We sought to investigate whether systemic administration of an immunostimulatory monoclonal antibody directed against the lymphocyte surface molecule CD137 could enhance the immunity elicited by RAdNS3. It was found that treatment with anti-CD137 mAb after the administration of a suboptimal dose of RAdNS3 enhanced cytotoxic and T helper cell responses against HCV NS3. Importantly, the ability of RAdNS3 to induce protective immunity against challenge with a recombinant vaccinia virus expressing HCV proteins was markedly augmented. Thus, combination of immunostimulatory anti-CD137 mAb with recombinant adenoviruses expressing HCV proteins might be useful in strategies of immunization against HCV.