Trop2病毒样颗粒的制备及纯化研究

目的获得Trop2为靶点的病毒样颗粒(VLPs),为进一步体内诱导实验和疫苗研究提供依据。方法利用分子克隆技术构建Trop2真核表达载体pCAGGs/Trop2和杆状病毒表达载体pFastbac1/Trop2,以pCAGGs/Trop2表达载体转化Hela细胞,用免疫细胞化学方法确定Trop2蛋白表达并锚定于胞膜上;以pFastbac1/Trop2转化大肠杆菌E.coliDH10bac菌株获得重组杆粒Trop2Bacmid,转染Tn5昆虫细胞获得重组病毒Trop2rBV,与本室保存的Gag rBV共感染昆虫细胞得到重组VLPs,透析浓缩后经蔗糖密度梯度离心纯化、蛋白免疫印迹(WB)鉴定,电镜观察病毒形态。结果构建成功的重组杆粒Trop2Bacmid转染Tn5昆虫细胞获得重组病毒Trop2rBV,与Gag rBV共感染昆虫细胞获得重组Trop2VLPs。结论成功制备了Trop2VLPs,为其后续诱导体液和细胞免疫应答研究奠定了基础。     

HPV58型衣壳蛋白L1和L2基因的克隆表达及病毒样颗粒的装配

目的大量表达和纯化HPV58衣壳蛋白,形成病毒相似颗粒。方法制备携带HPV58L1和L2基因的重组杆状病毒,转染昆虫细胞,在真核细胞中大量表达HPV58L1和L2蛋白并进行纯化。电镜观察病毒相似颗粒的形成情况。免疫小鼠制备特异性抗血清。结果 HPV58L1和L2蛋白在真核细胞中被高效表达,电镜下见表达纯化的病毒衣壳蛋白L1与L2共同组装形成病毒相似颗粒(VLPs),用其免疫小鼠能产生针对HPV58L1蛋白的高滴度的特异性抗体。结论在真核细胞中大量表达HPV58L1和L2蛋白,纯化后能形成病毒相似颗粒,并具备很强的免疫原性,有望用其制作宫颈癌预防性疫苗。     

AEG-1肺归巢域与Flagellin融合基因的杆状病毒制备及鉴定

目的通过重叠PCR方法获得AEG-1C-Flic融合基因,采用Bac-To-Bac杆状病毒表达系统获得重组AEG-1C-Flic杆状病毒,为后续AEG-1C-Flic病毒样颗粒疫苗的制备奠定基础。方法从鼠伤寒沙门菌的基因组中PCR扩增细菌鞭毛蛋白Flagellin,采用重叠PCR的方法将AEG1肺归巢域段基因与Flagellin融合,并添加猴免疫缺陷病毒(simian immunodeficiency virus,SIV)包膜蛋白gp41信号肽与跨膜区,构建AEG-1C-Flic pFastBacTM重组表达载体,转座大肠杆菌E.coliDH10Bac感受态细胞后获得重组杆粒,重组杆粒转染昆虫细胞Tn5,获得AEG-1CFlic重组杆状病毒,并采用Western Blot方法对该病毒进行鉴定。结果构建的AEG-1C-Flic杆状病毒表达载体经双酶切及测序正确,通过转化E.coli DH10Bac感受态细胞获得重组Bacmid,经PCR鉴定大小正确,Western Blot结果显示Bacmid转染Tn5细胞后成功获得重组杆状病毒rBv AEG-1C-Flic。结论成功构建的AEG-1C-Flic杆状病毒可用于新型AEG1-病毒样颗粒疫苗的制备。     

IL-2和IL-15诱导扩增的脐带血NK细胞生物学特性研究

本研究探讨脐血CD3-CD56+NK细胞及其在IL-2、IL-15诱导扩增后的免疫表型、细胞毒活性等生物学特性的改变。分离脐血单个核细胞,在无血清培养条件下,分别加入IL-2或(和)IL-15,培养14 d,流式细胞术检测CD3-CD56+NK细胞亚群水平以及NK表面CD16、CD62L、NKG2D、NKG2A、NCR44、NCR46、颗粒酶B、穿孔蛋白的表达;用WST-1法检测NK细胞对K562细胞毒活性。结果表明,扩增14 d后IL-2、IL-15、IL-2/IL-15 3组NK细胞分别扩增了10.78±2.51、10.42±3.72、10.54±6.24倍,3组之间无显著差异;扩增后的NK细胞表达CD16的比例显著减低,IL-2和IL-15组之间差异显著;细胞因子扩增后CD62L表达无改变;IL-2有下调NKG2A、NCR46表达的作用,IL-15的作用则相反;两种细胞因子均有上调NKG2D、穿孔蛋白、NCR44表达的作用,且细胞因子之间也存在差异;IL-15有上调NK细胞颗粒酶B表达的作用;经细胞因子扩增的NK细胞毒活性显著增高,但细胞因子之间作用无显著差异。结论:在无血清培养条件下,IL-2和IL-15均能有效地扩增脐血中NK细胞。虽然2种细胞因子扩增后的CD3-CD56+NK细胞与功能相关的免疫表型呈现出不同的改变特征,但细胞毒活性均显著增加,且2种细胞因子之间作用无显著差别,协同作用不明显;NK细胞毒活性是各种活性分子共同作用的结果。     

人乳头瘤病毒58型L1/E7“假病毒”疫苗的制备及免疫效果评价

目的构建人乳头瘤病毒58型L1/E7"假病毒"疫苗,并研究其免疫学特性。方法采用sf9昆虫-杆状病毒表达系统表达HPV58L1蛋白,体外解离重组病毒颗粒,向已经解离的衣壳中加入mE7-pcDNA3.1+质粒,CaCl2室温透析促使病毒样颗粒(VLP)重新聚合,用"假病毒"颗粒滴鼻免疫小鼠,检测小鼠血清IgG,IgA中和抗体以及脾淋巴细胞IFN-γ的分泌水平和细胞毒性T淋巴细胞(CTL)反应。结果 SDS-PAGE,Western blot和红细胞凝集实验证实sf9昆虫-杆状病毒表达系统有效表达有功能活性的HPV58L1蛋白,HPV58L1蛋白能够有效的折叠、解聚以及再折叠为VLP。小鼠免疫保护实验发现,用包含有mE7-pcDNA3.1~+质粒的HPV58L1"假病毒"免疫小鼠,抗HPV58L1特异性IgG,IgA明显升高(P0.001),"假病毒"免疫组产生的IFN-γ明显高于VLP免疫组(P0.001);"假病毒"免疫过的小鼠脾淋巴细胞可以特异性杀伤Tc-1细胞,引起CTL反应。结论成功制备了人乳头瘤病毒58型L1/E7"假病毒"疫苗,能诱发免疫动物较强的体液免疫反应,激发保护性抗体的产生。     

用人乳头瘤病毒6bL1病毒样颗粒检测尖锐湿疣血清抗体

目的　采用人乳头瘤病毒 (HPV) 6bL1病毒样颗粒 (VLPs) ,检测HPV6感染的尖锐湿疣(CA)病人血清抗体 ,以评价HPV感染的血清学诊断方法。方法　采用重组杆状病毒———昆虫细胞系统制备HPV6bL1VLPs ,经氯化铯梯度离心纯化 ;以酶联免疫吸附试验 (ELISA)分别检测CA、宫颈癌和健康对照组的 136份标本的血清抗体。结果　CA组、健康对照组和宫颈癌组抗HPV6bL1VLPs的血清抗体阳性率分别为 75 % [吸光度 (A)均值 0 111± 0 0 94],2 9% (A均值 0 0 12± 0 0 2 4) ,14 3% (A均值0 0 2 9± 0 0 2 2 ) ,3组间比较差异均有显著性 (P <0 0 1)。结论　HPV6bL1VLPs作ELISA抗原 ,可在CA病人体内检测到HPV6型特异性抗体 ,提示该方法可用于HPV6感染的血清学诊断和流行病学调查。     

截短的丙型肝炎病毒核心蛋白基因和绿色荧光蛋白基因在Sf9细胞中的融合表达

目的构建含截短的HCV核心蛋白基因和绿色荧光蛋白基因的重组杆状病毒表达载体,探讨其在Sf9细胞中的表达和抗原性。方法用PCR法扩增截短的HCV核心蛋白基因片段(Ct)和增强型绿色荧光蛋白(EGFP)基因,将其插入转座子载体pFastBac1,构建成重组表达载体pFastCt-EGFP,转化大肠埃希菌DH10Bac,获得重组杆状病毒穿梭质粒BacmidCt-EGFP,转染昆虫Sf 9细胞。结果SDS-PAGE和western blot鉴定表明成功表达了分子量约40 000的融合蛋白。以ELISA法检测发现该融合蛋白能与28份抗HCV抗体阳性血清中的15份发生反应,阳性率为53.6%。结论该融合蛋白在昆虫Sf 9细胞中成功表达并具有一定的抗原性。     

人CD13基因克隆及其基因转染细胞株的构建

目的 克隆人CD13全长cDNA,构建稳定表达人CD13 分子的基因转染细胞株.方法 提取人外周血单个核细胞总RNA,通过RT-PCR 克隆人CD13基因,将人CD13基因重组入逆转录病毒表达载体pEGZ-Term.将重组逆转录病毒载体pEGZ-Term/CD13 和辅助病毒载体用脂质体法共转染包装细胞293T,收集含有完整重组逆转录病毒颗粒的293T细胞培养上清感染L929细胞,筛选获得Zeocin抗性的基因转染细胞.结果 成功克隆人CD13基因和构建逆转录病毒载体pEGZ-Term/ CD13,并成功获得稳定表达人CD13的基因转染细胞株L929/CD13.结论 成功克隆了人CD13基因及其逆转录表达载体,成功制备了稳定表达人CD13的基因转染细胞株,为研究CD13生物学功能奠定了基础.     

系统性红斑狼疮患者CD40-CD40L的检测及临床意义

目的探讨CD40-CD40L在系统性红斑狼疮(SLE)患者淋巴细胞上的表达及意义.方法采用流式细胞术检测SLE患者淋巴细胞CD40及CD40L的表达,并与对照组比较.结果SLE患者淋巴细胞及CD4+T淋巴细胞CD40L的表达显著高于对照组(P＜0.05),而CD40在淋巴细胞及CD3-淋巴细胞上的表达,两组无显著性差异(P＞0.05).结论CD40-CD40L共刺激分子途径在SLE发病机制中起重要作用,以CD40L表达上调为重要特点.     

CD40-CD40L在移植免疫中的研究进展

CD40-CD40L交联在体液免疫和细胞免疫中是一条重要的细胞信号传导途径,参与了体液免疫和细胞免疫的调节。近年在骨髓移植和器官移植中应用anti-CD40/CD40L单克隆抗体或基因敲除等手段来干预CD40-CD40L的异常表达,发现阻断CD40-CD40L共刺激信号可诱导免疫耐受和抑制移植物抗宿主反应等,因此阻断CD40-CD40L共刺激途径将成为临床移植领域中防治排斥反应的新途径。本文就CD40/CD40L及其交联作用在移植免疫中的研究进展作一综述。     

Papillomavirus virus-like particles as anticancer vaccines

Papillomavirus virus-like particles (VLPs) are empty, non-replicative, non-infectious particles that retain conformationally correct epitopes for the generation of antibody responses to the viral capsid proteins. Chimeric human papillomavirus (HPV) virus-like particles incorporating non-structural virus proteins offer an exciting approach for combined prophylactic and therapeutic vaccines against HPV-induced lesions. Both HPV VLPs and chimeric VLPs can induce potent humoral and cellular immune responses when injected into mice, leading to the generation of virus-neutralizing antibodies, priming of CD8+ T-cells and activation of cytotoxic T-cell effector functions. This review summarizes recent advances in the production of chimeric VLPs, the immune response elicited by VLPs and chimeric VLPs, and their ability to generate strong protective and therapeutic antitumor immune responses.     

Chimeric Trop2 virus-like particles: a potential immunotherapeutic approach against pancreatic cancer

Trop2 is a recently discovered cell surface glycoprotein overexpressed in pancreatic cancer which could potentially be used as an immunotherapeutic target. Enveloped virus-like particles (VLPs) are highly immunogenic and versatile immune stimulatory agents which can be modified to incorporate exogenous proteins on their membrane envelope to use as cancer vaccines. In this study, we investigated the effects of murine Trop2 (mTrop2) VLP immunization in a pancreatic cancer syngeneic murine model. VLPs incorporating mTrop2 were used to immunize C57BL/6 tumor-bearing mice. Immunization with mTrop2 VLPs led to a significant reduction in tumor growth accompanied by a broad activation and tumor infiltration of CD4(+) and CD8(+) T cells as well as natural killer and natural killer T cells. VLP immunization generated mTrop2-specific cytotoxic T lymphocytes and antibodies with no measurable induction of autoimmunity. Importantly, VLP immunization decreased the population of regulatory T cells and myeloid-derived suppressor cells inside the tumor tissue resulting in decreased levels of immunosuppressive cytokines like interleukin-10 and transforming growth factor-β while promoting the activation of immature macrophages and dendritic cells. Furthermore, combination of VLP immunization with gemcitabine treatment showed an improved effect significantly increasing the survival of tumor bearing mice. Our results demonstrate that mTrop2 VLP immunization can activate broad antitumor immune responses and affect key players in the tumor microenvironment overcoming a major barrier, which has limited the efficacy of cancer vaccines. This study presents a novel immunotherapeutic approach which could potentially be used as an alternative treatment option in combination therapies to treat pancreatic cancer patients.     

Distinct regulatory roles of lymphocyte costimulatory pathways on T helper type-2 mediated autoimmune disease

We assessed the role of CD40-CD40L, cytotoxic T lymphocyte (CTL)A4/CD28- B7s, and CD2-CD48/CD58 lymphocyte costimulatory pathways in the development of mercury chloride (HgCl2)-induced autoimmune disease in mice, which is believed to be mediated by T helper (Th) subset Th2. Inhibition of CD40-CD40-L and CTLA4/CD28-B7s interactions by anti-CD40- L antibody and soluble CTLA4-immunoglobulin (Ig) fusion protein, respectively, abrogated the autoimmune disease without affecting interleukin 4 (IL-4) production, showing the importance of physical contact between T and B lymphocytes in the Th2-mediated process. In contrast, two anti-CD2 antibodies that have been shown to induce immunosuppression of Th1-mediated events exacerbated the autoantibody response and augmented IgG1, IgE, and IL-4 production, transforming a mild mesangial glomerulopathy into a severe systemic immune complex disease. These observations demonstrate that manipulation of lymphocyte accessory counterreceptor interactions may affect the course of Th2- associated autoimmune disease and suggest that signals resulting from CD2 engagement play an essential role in the regulation of the Th1-Th2 effector equilibrium.     

Transient blocking of both B7.1 (CD80) and B7.2 (CD86) in addition to CD40-CD40L interaction fully abrogates the immune response following systemic injection of adenovirus vector

Blockade of the CD40-CD40L and CD80/CD86-CD28 costimulatory pathways represents a strategy to inhibit the immune response against Ad vectors designed for gene therapy applications. Since most previous studies have used a CTLA4-Ig fusion molecule binding to both CD80 and CD86, the respective roles of these B7 molecules remained undefined. We have studied the effect of blocking monoclonal Abs (mAbs) directed against the costimulatory molecules CD40L, CD80 and CD86, alone or in different combinations, on the humoral and cellular immune responses against Ad. Groups of mice were transiently treated with each combination of blocking mAbs upon systemic injection of a first Ad vector. Combinations of anti-CD80 + anti-CD86 or anti-CD40L + anti-CD86 mAbs resulted in strong inhibition of the immune response against Ad. Using either of these mAb pairs, a second vector could be administered 1 month after the first injection but with lower efficiency than in naive animals. Thus, CD86 stands as the pivotal B7 molecule involved in the development of the immune response against Ad. However, only the blockade of both CD80 and CD86 in addition to CD40L fully inhibited the humoral and cellular responses against the Ad vector, such that readministration after 1 month was as efficient as in naive animals. At the time of readministration, treated animals had regained their ability to mount a normal immune response to the second Ad vector, showing that tolerance was not induced.     

结核分枝杆菌rdESAT-6抗原在耻垢分枝杆菌中的制备及其血清学诊断研究

目的构建结核分枝杆菌(MTB)融合基因2×esat-6原核表达载体,利用耻垢分枝杆菌诱导表达、纯化早期分泌抗原ESAT-6重组二聚体(rdESAT-6),Western blot分析其抗原性,并评估其在结核病患者中的血清学诊断价值。方法以DNA疫苗HG856A2为模板扩增2×esat-6基因,构建pMF41-2×esat-6原核表达载体,电转化耻垢分枝杆菌,诱导表达rdESAT-6蛋白,Western blot分析其抗原性。收集126例确诊的结核病患者和42名健康体检者的血清,用间接酶联免疫吸附试验(ELISA)和结核斑点金免疫渗滤试验(TB-DOT)检测血清结核抗体,评估rdESAT-6在结核血清学诊断中的价值。结果成功构建了pMF41-2×esat-6重组质粒,以包涵体形式表达了rdESAT-6蛋白,纯化的蛋白纯度在95%以上,可与小鼠抗ESAT-6血清发生特异性反应。126例结核病患者血清检测结果表明,rdESAT-6蛋白在MTB阳性组和阴性组患者血清学诊断的敏感性分别为79.75%(63/79)、61.70%(29/47),好于TB-DOT的62.03%(49/79)、44.68%(21/47)(P0.05)。2种方法对42名健康体检者血清诊断特异性均为95.24%(40/42)。结论 MTB的融合蛋白rdESAT-6用于结核病血清学检测具有较好的敏感性和特异性,可作为结核病血清学诊断的优选抗原。     

人偏肺病毒F1、F2亚单位原核表达系统的构建及多克隆抗体制备

目的 构建人偏肺病毒融合蛋白亚单位F1、F2原核表达系统,初步获得抗F1、F2重组蛋白的多克隆抗体,为相关疫苗的研究奠定基础。 方法 聚合酶链反应(PCR)法扩增F1、F2基因片段,经T-A克隆确保其准确性,双酶切后插入原核表达载体pET32a(+)中,转化大肠埃希菌BL21(DE3),按优化后条件大量诱导表达,纯化后经Western Blot检测特异性。取纯化蛋白免疫BALB/c小鼠制备多克隆抗体,ELISA法检测效价,间接免疫荧光法(IFA)检测多克隆抗体是否能与人偏肺病毒发生特异性反应。 结果 F1、F2基因均正确插入pET32a(+)中,并具有正确的读码框架。0.5 mmol/L IPTG 37 ℃诱导培养5 h可获得大量带His标签的重组蛋白,纯化后浓度分别达到200 g/ml和300 g/ml。Western Blot结果显示,重组蛋白可被抗His标签抗体特异识别;免疫小鼠获得多克隆抗体效价分别为抗pET32a-F1最高1 : 640,抗pET32a-F2最高1 : 40 960。IFA显示抗pET32a-F1、抗pET32a-F2多克隆抗体均可与人偏肺病毒作用产生特异性荧光。 结论 成功构建了F1、F2的原核表达质粒, 并在大肠埃希菌中获得高效表达,该蛋白具有良好的抗原性;免疫小鼠获得特异性多克隆抗体,有利于人偏肺病毒的深入研究。     

Fusion of Epstein-Barr virus nuclear antigen-1-derived glycine-alanine repeat to trans-dominant HIV-1 Gag increases inhibitory activities and survival of transduced cells in vivo

Trans-dominant human immunodeficiency virus type 1 (HIV-1) Gag derivatives have been shown to efficiently inhibit late steps of HIV-1 replication in vitro by interfering with Gag precursor assembly, thus ranking among the interesting candidates for gene therapy approaches. However, efficient antiviral activities of corresponding transgenes are likely to be counteracted in particular by cell-mediated host immune responses toward the transgene-expressing cells. To decrease this potential immunogenicity, a 24-amino acid Gly-Ala (GA) stretch derived from Epstein-Barr virus nuclear antigen-1 (EBNA1) and known to overcome proteasomal degradation was fused to a trans-dominant Gag variant (sgD1). To determine the capacity of this fusion polypeptide to repress viral replication, PM-1 cells were transduced with sgD1 and GAsgD1 transgenes, using retroviral gene transfer. Challenge of stably transfected permissive cell lines with various viral strains indicated that N-terminal GA fusion even enhanced the inhibitory properties of sgD1. Further studies revealed that the GA stretch increased protein stability by blocking proteasomal degradation of Gag proteins. Immunization of BALB/c mice with a DNA vaccine vector expressing sgD1 induced substantial Gag-specific immune responses that were, however, clearly diminished in the presence of GA. Furthermore, recognition of cells expressing the GA-fused transgene by CD8(+) T cells was drastically reduced, both in vitro and in vivo, resulting in prolonged survival of the transduced cells in recipient mice.     

蛋白转导结构域-bcr/abl融合基因的构建和表达

目的 构建含蛋白转导结构域（PTD）与慢性粒细胞白血病（CML）bcr/abl融合基因片段的质粒，并在大肠杆菌中表达。方法 PCR扩增的CML bcr/abl基因片段，经DNA测序后，与合成编码PTD的DNA片段一起手稿质粒pET-16b，构建表达载体pEPb，转化大肠杆菌并进行了PTD－bcr/abl蛋白的诱导表达和纯化。结果 跨越bcr/abl断裂点的523bp的目的片段被有效地扩增，DNA序列分析表明所构建的含PTD－bcr/abl融合基因的质粒与设计相同。PTD－bcr/abl融合蛋白在转化大肠杆菌获得了高效表达并纯化。结论 成功地获得了PTD－bcr/abl融合蛋白片段的基因表达产物，为进一步研究CML的免疫治疗奠定了基础。     

Functional expression of chimeric Fab of an anti-CD40L mAb: Vector design and culture condition optimization

CD40L is an important costimulatory molecule in the induction of the humoral and cell-mediated immune responses. 4F1, a specific murine antagonistic monoclonal antibody against human CD40L molecule, is a promising candidate biomedicine for autoimmune diseases, transplantation rejection and anti-angiogenesis therapy of cancer. To avoid the mAb induced thromboembolism as a consequence of platelet surface FcγR activation, we attempted to construct a chimeric Fab of 4F1 to minimize its side effects for potential clinical use. A chimeric version of anti-CD40L Fab was generated by transferring mouse variable regions into a human framework. Our study indicated that 4F1 could be simply and rapidly converted to chimeric Fab which could be expressed in bacteria and purified in reasonable quantities. This chimeric antibody maintained its bioreactivity to human CD40L.