抗原肽HPV18 E77-15诱导产生特异性细胞毒T细胞的实验研究

目的探讨HLA—A2限制性表位HPV18E77—15的免疫原性。方法对抗原肽HPV18E7-15进行T2结合分析;通过体外细胞培养技术抗原肽诱导特异性CTL扩增;采用RPE标记的表位特异性五聚体和FITC标记的CD8＋抗体通过流式细胞仪检测抗原肽所诱导产生的特异性CTLs。结果抗原肽HPVl8E77—15可将T2细胞表面HLA—A＊0201分子的表达提高2．6倍;在流式细胞仪检测中,抗原肽HPV18E77—15刺激诱导产生CD8＋五聚体＋双阳性的抗原肽特异性CTLs为1．87％。结论抗原肽HPV18E77—15能够诱导HLA—A2阳性正常人外周血淋巴细胞产生特异性CTLs,表明HPV18E77-15具有一定的免疫原性。     

QLFATINSTL（93-102aa）是隐球菌抗原MP98CTL优势表位

目的鉴定隐球菌抗原MP98中的HLA—A＊0201限制性CD8^＋CTL表位。方法应用数据库SYFPEITHI预测隐球菌MP98中可能存在的HLA—A＊0201限制性CD8^＋CTL表位,经流式细胞术分析各抗原肽与HLA-A＊0201的亲合力,经时间分辨荧光法检测外周血单个核细胞（PBMC5）对各抗原肽产生的增殖反应,经细胞毒性实验研究各抗原肽诱导的特异性T细胞的细胞毒杀伤活性,逐步鉴定MP98的HLA—A＊0201限制性CD8^＋CTL表位。结果位于MP98氨基酸序列肽3（93—102aa）与HLA—A＊0201分子具有较高的亲合力,并都能刺激HLA—A＊0201阳性个体PBMC增殖,并诱导产生具有特异性杀伤活性的CTL。结论肽3QLFATINSTL（93—102aa）是抗原MP98上HLA—A＊0201限制性CD8^＋CTL的优势表位,可作为隐球菌疫苗设计的候选表位。     

淋巴瘤相关抗原肽刺激所获细胞毒性T细胞克隆的特征

目的研究抗肿瘤细胞毒性T淋巴细胞(CTL)克隆识别靶细胞的肽特异性和人类白细胞抗原(HLA)限制性,以及其赖以识别抗原肽的T细胞受体(TCR)基因表达序列的分子特征。方法利用体外CTL刺激扩增体系,应用免疫球蛋白重链可变区(IgHV)框架区上的B淋巴瘤相关抗原九肽(IgHV1QLVQSGAEV和IgHV3SLYLQMNSL)负荷的抗原递呈细胞(APC),刺激正常HLAA0201供者的外周血单个核细胞(PBMC),每周1次共4次,用流式细胞仪检测体外培养细胞的免疫表型的变化,并用肽/主要组织相容性基因复合体(MHC)四聚体方法检测肽特异性的CTL增殖情况。应用酶联免疫吸附实验(ELISA)检测CTL与不同的靶细胞共同孵育时释放干扰素γ(IFNγ)的能力。应用逆转录多聚酶链反应(RTPCR)和T细胞受体(TCR)基因指纹谱型图方法检测培养前后的淋巴细胞的克隆变化,并用直接测序的方法得到CTL克隆的TCR基因序列。结果B淋巴瘤相关抗原肽4次刺激体外培养的PBMC后CD8+CTL大量增殖,CD4/CD8明显下降(0.10vs1.43,P<0.05)。IgHV1QLVQSGAEV/HLAA0201四聚体和CD8双阳性的肽特异性CTL数量(49.38%)比刺激前(0.04%)明显上升。ELISA检测IFNγ的分泌表明其识别靶细胞是肽特异性和HLA限制性的。TCR基因指纹谱型图显示抗原肽反复刺激获得的CTL的TCR表达集中于个别基因家族,呈克隆性增殖。结论应用IgHV基因框架区来源的B淋巴瘤相关抗原九肽可以使特异性CTL克隆增殖,这些克隆以肽特异性和HLA限制性的方式识别靶细胞。了解这些CTL的作用和TCR识别相关抗原肽的分子结构,将有助于确定体内T细胞和B细胞相互调节的机制。     

结核杆菌抗原Ag85C的HLA-A*0201限制性CD8+CTL表位的预测及鉴定

目的：预测及鉴定结核杆菌（Mycobacterium tuberculosis，Mtb）抗原Ag85C的HLA-A＊0201限制性CD8^＋CTL表位，为基于表位的结核疫苗研究提供依据。方法：应用SYFPEITHI数据库预测结核杆菌抗原Ag85C序列中可能存在的HLA-A＊0201限制性T细胞表位，利用T2细胞株分析各预测的抗原肽与HL-A＊0201分子的亲合力，选取高亲合力肽诱导特异性CTL细胞，检测各高亲合力肽刺激其特异性CTL细胞分泌的IFN-γ水平、在体外的CTL增殖反应以及细胞杀伤毒性，逐步鉴定出Ag85C的HLA-A＊0201限制性CD8^＋CTL表位。结果：SYFPEITHI数据库从Ag85C序列中预测到14条能够结合HLA—A＊0201分子的抗原肽，其中3个抗原肽（170～178aa、317～325aa和144～153aa）显示与T2细胞上HLA-A＊0201分子有高结合力，而抗原肽FLTREMPAWL（144～153aa）能够诱导大多数HLA—A＊0201阳性结核患者及PPD（＋）健康志愿者产生特异性CTL细胞，并分泌大量的IFN-γ，能够诱导CTL体外发生增殖，能够产生特异性杀伤活性。结论：成功鉴定出抗原肽FLTREMPAWL（144～153aa）结核杆菌抗原Ag85C的HLA-A＊0201限制性CD8^＋CTL表位，可作为结核疫苗设计的候选表位，为进一步研发新型有效的抗结核疫苗奠定了基础。     

基于GIS的中国人群乙肝多表位疫苗的预测

目的预测可能的HLA Ⅰ类限制性HBV特异性CTL表位组合在中国人群的理论免疫应答率,旨在指导乙型肝炎（HBV）多表位疫苗的研制和应用。 方法利用“中国多表位疫苗设计的HLA Ⅰ积累表型频率空间预测系统”计算HLA A、B限制性HBV特异性CTL表位组合在中国人群中的累积表型频率（CPF）,得出理论免疫应答率,并绘制CPF预测等值线图,比较结果。 结果HLA A或B单一超型组合针对中国人群的理论免疫应答率普遍偏低。HLA A和B超型组合针对中国人群的理论免疫应答率相对较好。 结论根据不同HLA组分组合设计的多表位疫苗（或DNA疫苗）在中国各地人群中的免疫效果不同,根据中国人HLA分布特点,进行疫苗理论免疫应答率预测研究,可以指导制备适合中国人群的HBV特异性CTL多表位疫苗,应用前景广阔,有重要的指导意义。     

1型糖尿病人来源胰岛素原特异性T细胞克隆的建立及表型鉴定

[目的]研究1型糖尿病相关的胰岛素原(Proinsulin,PI)T细胞表位。[方法]采用有限稀释法建立糖尿病病人胰岛素原抗原特异性的T细胞克隆,~(51)Cr释放细胞毒性试验测定T细胞激活的HLA限制性和PI肽段中最小的抗原表位,用流式细胞仪分析其表型及表面肿瘤坏死因子凋亡诱导配体(tumor necrosis factor-related apopto-sis-inducing ligand,TRAIL)的表达。[结果]建立了PI抗原特异性的CD4~+ T细胞克隆TWHPI-1,CD8~+ T细胞克隆TWH PI-2。前者为HLA DRB4 0101限制,PI最小抗原表位在PI(79～87),74.2％细胞表达 TRAIL。后者为HLA A1限制。PI最小抗原表位在PI(78～86),94.9％细胞表达TRAIL。[结论]HLA DRB4 0101-Al可能参与IDDM的致病过程,TRAIL可能是两株T细胞克隆引起胰岛细胞死亡的因子之一。     

乙型肝炎病毒c抗原特异性T细胞系的建立及CTL克隆的筛选

目的：建立乙型肝炎病毒（HBV）抗原特异性T细胞系，并筛选得到抗原肽特异性细胞毒性T淋巴细胞（CTL）克隆。方法通过流式细胞染色方法鉴定出1例急性乙型肝炎患者的人类白细胞分化抗原（HLA）为HLA‐A2型，分离患者外周血单个核淋巴细胞（PBMCs）；用重组人HBVc抗原（HBcAg）刺激PBMCs建立抗原特异性T细胞系；用HBcAg抗原肽刺激和有限稀释方法，筛选出HBcAg抗原肽特异性CTL克隆；以固相酶联免疫斑点技术（ELISPOT）和乳酸脱氢酶（LDH）细胞毒试验对HBcAg特异性T细胞系和CTL克隆进行功能鉴定。结果建系后HBcAg特异性T细胞明显得到富集，有限稀释后获得15株CTL克隆，其中14株表现出明显的HBcAg抗体肽特异性，包括抗原肽Core18‐27特异性CTL克隆8株，抗原肽Core107‐115特异性CTL克隆4株，另外2株CTL克隆对2条HBcAg抗原肽有交叉反应性。结论成功建立HBcAg抗原特异性T细胞系和CTL克隆，为后续试验奠定基础。     

人食管癌Eca-109细胞Mr小于3000膜蛋白肿瘤抗原肽的筛选

目的:寻找被T细胞识别的食管癌细胞抗原肽,为食管癌免疫治疗奠定基础。方法:应用pH3.3的枸橼酸磷酸盐缓冲液间隔24h多次酸洗贴壁生长的Eca109细胞,获得人主要组织相容性复合体Ⅰ类分子(HLAⅠ)类分子结合的多肽,经超过滤,反相HPLC色谱层析得到不同组分多肽,DC递呈抗原肽刺激特异性细胞毒T淋巴细胞(CTL)反应,51Cr杀伤实验检测CTL对肿瘤细胞杀伤作用。结果:Mr小于3000的混合多肽经过反相HPLC可获得50多个组分,混合肽、P17、P29和P41组分体外诱导实验表明可以诱导出较强的CTL反应。结论:酸洗法能有效获得HLAⅠ类分子结合的多肽抗原,Mr小于3000的多肽成分中3个组分存在能引起特异性的抗肿瘤CTL免疫反应的肿瘤抗原,具有潜在的免疫治疗作用。     

肿瘤抗原MAGE-n HLA-A2限制性细胞毒性T细胞表位的预测及合成鉴定

目的　采用表位重建技术 ,对预测MAGE n抗原HLA A2限制性CTL表位结合活性进行研究。方法　用SYFPEITHI超基序法远程预测系统和量化基序多项式法结合的预测方法 ,筛选新的MAGE n抗原HLA A2限制性CTL表位作为候选表位。对预测的抗原表位进行多肽合成 ,并用HPLC进行纯化 ,质谱法 (MS)鉴定。候选表位与HLA A2位点的亲和力及结合稳定性用竞争结合抑制实验、T2结合稳定性实验及MHC 肽复合体稳定性实验测定。结果　用表位预测法筛选MAGE n抗原 5个HLA A2限制性CTL表位为候选表位。QLVFGIEVV(15 9 16 7)、IMPKTGFLI(195 2 0 3)和FLIIVLVMI(2 0 1 2 0 9) 3个表位具有较高的HLA A2结合力 (IC50 <15 μM)和结合稳定性 (DT5 0 >6h) ,可作为MAGE n抗原HLA A2限制性CTL候选表位进行进一步研究。结论　表位预测结合表位重建方法可提高肿瘤抗原CTL表位研究的效率。MAGE n抗原HLA A2限制性CTL表位经免疫学鉴定后 ,可望用于新型肝癌治疗性多肽疫苗的设计研究 ,为临床肝癌特异性治疗奠定基础。     

人类白细胞抗原—抗原肽复合物特异性细胞毒性T细胞的诱生及克隆

为建立一种有效诱导及克隆人类白细胞抗原（HLA）－抗原肽复合物特异性细胞毒性T细胞（CTL）的方法,用EB病毒转化的B淋巴母样细胞系细胞作为刺激细胞,采用长期混合淋巴细胞培养法诱导自身外周血T细胞库中抗原肽－HLA复合物特异性CTL增殖、分化,并用有限稀释法对之进行克隆。通过细胞毒实验证实获得的CD3^ 、CD8^ T细胞克隆具有高度抗原特异性并受某些HLA型别的限制。     

ENDOGENOUS EXPRESSION AND HLA STABILIZATION ASSAY OF PLASMODIUM FALCIPARUM CTL EPITOPEMINIGENE IN HUMAN HLA－A2. 1 AND HLA－BS1 CELLS

Objective. To evaluate the Plasmodium falciparum CTL epitope vaccines in HLA class I allele specific human cell lines that have high frequency among Chinese population. Methods. Synthesized oligonucleotides encoding for P.f. CTL epitope genes, constructed eukaryotic expression plasmids, transfected the minigenes into HLA class I allele specific human cell lines and identified endogenous expressing of the minigenes by RT-PCR and HLA stabilization assay. Results. Two mini-genes encoding Plasmedium falciparum CTL epitopes were designed and cloned, respectively, into an eukaryotic expressing vector to form TR26 which was restricted to HLA-B51,SH6 which was restrictedto HLA-A2.1, and TS, which had the two aforementioned mini-genes fused in tandem. All of these CTL epitope genes were transfected and endogenously expressed in respective cell lines containing appropriate HLA molecules.The obviously increased expressions of HLA class I molecules were detected in the transfected cell lines. It was demonstrated that the two discrete Plasmedium falciparum epitope genes were effectively processed and presented, and the close proximity of the two epitope genes in one chain as in mini-gene TS did not interfere with the process-ing and presenting of each epitope gene in corresponding cell line. Conclusion. A successful expression and presentation of multiple CTL epitope mini-gene in MHC class I allele specific human cell lines were demonstrated by an in vitro assay, which could be corresponding to the vaccina-tion of CTL vaccines in people with different MHC I molecules. This work also suggested the possibility of constructing a multiple CI~ epitope plasmodium falciparum DNA vaccine that could cover most of Chinese population.     

ENDOGENOUS EXPRESSION AND HLA STABILIZATION ASSAY OF PLASMODIUM FALCIPARUM CTL EPITOPE MINIGENE IN HUMAN HLA- A2.1 AND HLA- B51 CELLS

INTRODUCTION Malaria is responsible for 300～ 500 million morbidity per year in the endemic tropical and subtropical areas, and Plasmodium falciparum, the causative agent of the most virulent form of human malaria infections, causes about 3 million deaths. The increasing incidence of resistance to most antimalarial drugs has stimulated researches aimed at controlling this disease by vaccination. Many studies have demonstrated the critical role played by CD8＋ cytotoxic T lymphocytes (C…     

Induction of cytotoxic T lymphocytes from the peripheral blood of a hepatocell ular carcinoma patient using melanoma antigen- 1 (MAGE- 1) peptide

Objective To investigate the possibility of using melanoma antigen- 1 (MAGE- 1) peptide as a tumor vaccine to treat hepatocellular carcinoma (HCC). Methods The expressions of MAGE- 1 in 8 HCC cell lines and in liver cancer tissue from a patient were detected using RT- PCR. The type of human leucocyte antigen Ⅰ (HLAⅠ) of both 8 HCC cell lines and peripheral blood mononuclear cells of the patient was detected using a microcytotoxicity method to screen out target cell lines for the cytotoxicity assay. Peripheral blood mononuclear cells from the HCC patient pulsed with an MAGE- 1 peptide (NYKCRFPEI) were used as antigen presenting cells. Autogenous peripheral blood mononuclear cells were stimulated with antigen presenting cells every 7 days for 4 times to elicit cytotoxic T lymphocytes. The phenotype of effector cells was analyzed using flow cytometry. The cytotoxicity of effector cells was detected with a lactate dehydrogenase releasing assay. Results The expressions of both MAGE- 1 and HLA- A24 were detected in BEL7405 cell line which were used as the positive target cell line in the cytotoxicity assay. The expression of MAGE- 1 alone was detected in HLE, BEL7402, BEL7404, QGY7703 and SMMC7721 cell lines, and the expression of neither MAGE- 1 nor HLA- A24 was shown in QGY 7701 and HpG2 cell lines. The last 7 cell lines could be used as negative target cell lines in the cytotoxicity assay. Peripheral blood mononuclear cells expanded 32 folds during 28- day culture. The ratio of CD3[ + T cells in creased by 16% (from 54% to 70%), and the ratio of CD8[ + T cells increased by 20% (from 36% to 56%) during 28- day culture. When the ratio of effector cells to target cells was 10∶1,effector cells exhibited 62. 5% cytotoxicity against autogenous lymphoblasts pulsed with the peptide (NYKCRFPEI) of MAGE- 1 antigen, 40. 25% cytotoxicity against BEL7405 cells, compared with 17. 88% cytolysis observed against autogenous lymphoblasts, 19. 55% against HLE cells, and 1. 6% against QGY7701 cells. When the ratio of effector cells to target cells was 3. 3∶1, the cytotoxicity of effector cells against the peptide pulsed autogenous lymphoblasts was 53. 6%, which was much higher against autogenous lymphoblasts, HLE cells and QGY7701 cells at 15. 6%, 13% and 1%, respectively. Conclusion The results demonstrate that cytotoxic T lymphocytes with the ability to specifically lyse target cells expressing both MAGE- 1 and HLA- A24 could be successfully induced by the MAGE- 1 peptide NYKCRFPEI in vitro. This indicates that a good result might be anticipated if this peptide is used as a tumor vaccine to treat HLA- A24 HCC patients.     

丙型肝炎病毒NS3区C末端HLA-11限制性CTL表位的作用

目的 研究丙型肝炎病毒（ｈｅｐａｔｉｔｉｓ Ｃ ｖｉｒｕｓ,ＨＣＶ）非结构３区（ＮＳ３）Ｃ末端ＨＬＡ－１１限制的细胞Ｔ细胞（ＣＴＬ）表位在ＨＣＶ感染中的作用。方法 血清学方法检测３例患的ＨＬＡ分型,他们均存在ＨＬＡ－Ａ１１表型,以ＨＬＡ－１１结合基序为依据,预测了ＨＬＡ－１１限制的ＣＴＬ表位。２例慢性患５ａ内３个时间点和３ａ内２个时间点及转阴患的血清样本,用反转录ＰＣＲ扩增出ＨＣＶ基因片段,     

稳定表达结核分枝杆菌38抗原的EL-4细胞株的建立及鉴定

将结核分枝杆菌的38抗原基因片段经PCR方法扩增并插入到pcDNA3真核表达载体中，通过脂质体转染EL-4细胞，经G418筛选，用RT-PCR方法和ELISA方法检测整合和表达情况。结果成功地构建了pcDNA3—38抗原重组质粒，转染细胞中可检测到38抗原的存在，PCR扩增也证实38抗原基因已稳定整合于EL-4细胞的染色体中。本实验为今后在小鼠体内检测结核DNA疫苗激发的细胞毒性T淋巴细胞(CTL)反应奠定了基础。     

MAGE-12的HLA-A2限制性细胞毒性T淋巴细胞表位肽的预测及其三维结构构建

[目的]从理论上分析预测肿瘤抗原MAGE- 12(melanoma antigen- 12)的HLA-A2(histocompatility leukocyte antigen-A2)限制性细胞毒性T淋巴细胞(cytotoxic T lymphocyte,CTL)表位肽并构建其三维结构.[方法]以肿瘤特异性抗原MAGE-12为研究目标,采用超基序法、量化基序法、多项式法、延展基序法与三维结构构建相结合的CTL表位预测方法.[结果]预测出的MAGE-12的表位中有4个符合HLA-A2限制性CTL表位要求.[结论]预测出的4个HLA-A2限制性CTL表位为MAGE-12的表位的可能性较大,经后续实验筛选、鉴定后,可用于基于MAGE-12的肿瘤治疗性多肽疫苗的设计研究.     

Enhanced expression of HLA class I molecules in human hepatocellular carcinoma cells transduced with gamma-interferon gene.

OBJECTIVE To investigate the expression of exogenous gamma-interferon gene in human hepatocellular carcinoma cells following retroviral transduction and the effect on the expression of surface HLA class I molecules.

METHODS Retroviral vector pLXSN was used to introduce human gamma-interferon (IFN-gamma) gene into four different human hepatocellular carcinoma cell lines (HCC). The G418-resistant colonies were isolated and cloned. The integration and expression of IFN-gamma gene were determined by PCR and RT-PCR analysis. A bioassay method was used to test the amount of IFN-gamma secreted by gene modified HCC cells. The expression of HLA class I molecules in HCC cells were analyzed by flow cytometry using indirect fluorescence staining.

RESULTS Four different HCC cell lines were successfully transduced with human IFN-gamma gene using retroviral vector. The integration and expression of IFN-gamma gene were shown only in the transduced cells. All four genetically modified HCC cells can secrete varied amount of IFN-gamma and demonstrate a significant up-regulation of surface HLA class I antigens. One specific HLA class I antigen, HLA-A2, has almost the same degree of increase as that of the total HLA class I molecules after transduction with IFN-gamma gene.

CONCLUSIONS Gene modification with IFN-gamma gene can significantly enhance the expression of HLA class I molecules in HCC cells and may increase its immunogenicity. These gene modified tumor vaccines can be helpful in tumor biotherapy.

     

重组人乳头瘤病毒CTL表位疫苗的构建与表达

目的：构建人乳头瘤病毒的细胞毒性T淋巴细胞（CTL）表位疫苗并在大肠杆菌中表达。方法：应用多轮PCR 的方法合成CTL表位基因序列,将其亚克隆入pET28a表达载体中,并在大肠杆菌BL21 (DE3) 中表达重组蛋白质,用Western blotting对重组蛋白进行鉴定。结果：采用计算机表位预测的方法选取HPV6、11、16型的CTL表位,通过5轮PCR 人工合成人乳头瘤病毒CTL表位疫苗的基因片段HPVepi,用亚克隆的方法构建了重组表达质粒pET28a-Tat-HPVepi,并在大肠杆菌高效表达了该重组蛋白质,经Western blotting鉴定显示出特异性条带。结论：结合计算机表位预测和PCR的方法构建并在大肠杆菌中高效表达了一种人乳头瘤病毒CTL表位疫苗。     

胞浆表达绿色荧光蛋白的伯氏疟原虫的构建和筛选

目的:为了追踪疟原虫发育及侵染的情况,构建及筛选胞浆稳定表达绿色荧光蛋白（GFP）的伯氏疟原虫。方法:构建含有伯氏疟原虫230p基因和GFP基因的重组质粒pL0035-GFP,质粒线性化后通过电转染转入野生型伯氏疟原虫;基于双交换同源重组原理,利用乙胺嘧啶筛选获得在230p基因处插入GFP的重组疟原虫;有限稀释法筛选表达GFP的单克隆重组伯氏疟原虫;PCR鉴定重组疟原虫基因型,荧光显微镜和流式细胞术检测GFP表达情况。结果:PCR及DNA测序结果表明GFP基因成功整合到伯氏疟原虫基因230p;荧光显微镜可观察到重组疟原虫胞浆呈绿色荧光,流式细胞术检测到绿色荧光信号。结论:成功构建胞浆表达绿色荧光蛋白的伯氏疟原虫。