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A Simplified PCR-SSP Method for hla-a2 Subtype in a Population of Wuhan,China

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Cellular & Molecular Immunology 2006年第6期3卷 453-458页

作者： Bing Liang Lijun Zhu Zhihui Liang Xiufang Weng Xiaoling Lu Cai＇e Zhang Hui Li Xiongwen Wu Department of Immunology Tongjl Medical College Huazhong University of Science ＆ Technology Wuhan China

hla-a2 is the most frequent hla-A allele in all ethnic populations, and an important restriction element for peptide presentation to T cells in infectious disease and cancer. However, the hla-a2 supertype consisting of up to 75 subtypes, mutation studies and analyses using cytotoxic T lymphocytes suggest the functional relevance of subtype-specific differences in hla-a2 molecules for peptide binding and T-cell recognition. Therefore, it is necessary for T-cell response study to discriminate the hla-a2 subtypes and to understand the profile of hla-a2 allellc distribution in a given population. In this study, we developed a simple, robust approach based on the nested polymerase chain reaction using sequence-specific primers （PCR-SSP） to discriminate 17 hla-a2 subtypes which cover the most hla-a2 alleles （〉 99% allele frequency） reported in Chinese, using 15 combinations of 19 allelic specific primers. In the first round of PCR, 3 combinations of 5 primers were used to determine whether the tested sample was hla-a2 positive, meanwhile the subtypes of hla-A＊0209 and hla-A＊0215N were determined for the variant position of these two subtypes is in exon 4 instead of exon 2, 3. Samples of hla-a2 positive were subtyped in the second round of PCR, using PCR products of the first round as templates. This strategy was applied to test the samples of 78 random hla-a2 positive individuals for their hla-a2 subtypes. Those samples were screened for hla-a2 positive by the first round PCR-SSP from 154 healthy blood donors in Wuhan, China. The subtyping results were verified by using flow cytometric analysis （FCM） with hla-a2 specific monoclonal antibody BB7.2 and DNA sequencing. The typing results of the samples show 50.7% random individuals in the population carry hla-a2, hla-A＊0201 ranks the first （allele frequency = 15.5%）, followed by A＊0207 （5.8%）, A＊0206 （4.7%）, A＊0203 （2.6%）, A＊0210 （0.7%）, and these 5 alleles account for 99.0% hla-a2 subtypes of allele frequency. Our study indicates that the developed typing method is simpl

关键词： hla-a2 PCR-SSP subtype

Combined Transfection with EBV-Specific Epitopes and hla-a2 genes is More Effective than Separate Transfection in Promoting CTL Lysis against Nasopharyngeal Carcinoma

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Cellular & Molecular Immunology 2004年第3期1卷 229-234页

作者： WeijunDing ChoylenFong LaboratoryofMicrobiologyandImmunology ChengduUniversityofTraditionalChineseMedicine37#ShierQiaoStreetChengdu610072China LaboratoryofImmunotherapy DivisionofCellularandMolecularResearchNationalCancerCentreofSingaporellHospitalDrive169610.Singapore

To augment specific cytotoxic T lymphocyte(CTL)lysis is a promising strategy for cancer therapy,in this study, we examined the boosting effect of CTLs upon autologous lymphoblastoid B cell lines(LCLs)transfected with diverse plasmids,to explore the possible CTL-based immunotherapy of nasopharyngeal carcinoma(NPC). FCM analysis displayed rather high ratio(>30%)of successfully transfected LCLs by utilizing the DMRIE-C kit.CTL assays demonstrated that substantially higher ratio of CTL specific lysis was observed upon the LCLs transfected with both expression vectors encoding EBV-specific epitopes and their presentation molecule hla-a2,in contrast with those transfected separately.By transfecting the vector encoding hla-a2 alone,only the LCLs of hla-a2^+ donors elicited markedly higher CTL lysis.CTL assays also showed that there existed no marked differences upon transfection by either different vectors(pcDNA3,pNGVL3 or pNGVL3-hFlex),or different EBV-derived peptides(LMP_2Pepl or LMP_2Pep2),or with or without the doubled DNA sequence encoding peptides.This study indicated a promising immunotherapy strategy on NPC through boosting and eliciting the EBV-specific CTL activation by transferring vectors encoding both EBV-specific epitopes and their presentation molecule hla-a2 into autologous LCL,the presentation cells of MHC/peptide tetrameric complex.Cellular & Molecular Immunology.2004;1(3):229-234.

关键词： NPC LMP2 LCL hla-a2 CTL

Screening of hla-a2 restricted CTL epitopes using molecular simulation

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Chinese Science Bulletin 2003年第18期48卷 1962-1966页

作者： GENG Miao WU Yuzhang LIN Zhihua JIA Zhengcai ZHOU Wei ZOU Liyun Institute of Immunology PLA Third Military Medical UniversityChongqing 400038China College of Bioengineering Chongqing Institute of TechnologyChongqing 400050China

A new technology has been established to screen CTL epitope by using molecular simulation and mo-lecular dynamics. By using this technology, four peptides of MAGE-2 were screened and further identified by peptide binding assay and cytotoxic assay. Results showed that three candidate peptides had higher affinity to hla-a2 molecule and were able to pulse CTL activities in vitro in human PBMC. The results indicated that identification of CTL epi-topes with computer assistant could overcome the compli-cated experimental steps, particularly the peptide elution step, and could directly observe peptide-MHC-Ⅰ compound binding pattern and could be used to screen CTL epitopes quickly.

关键词： CTL epitope hla-a2 identification computer assis-tant molecular simulation.

人乳头瘤病毒E6/E7混合CTL表位的筛选与免疫效应研究

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中文科技期刊数据库（全文版）医药卫生 2024年第1期 115-120页

作者：张汶歌姚德生广西医科大学附属肿瘤医院妇瘤科广西南宁530000

验证有效的人乳头瘤病毒(HPV)E6、E7蛋白的hla-A*0201限制性细胞毒性T淋巴细胞(CTL)表位,比较混合表位和单个表位的体外免疫效应差异,为宫颈癌复合多肽疫苗研究提供基础。方法对宫颈癌相关抗原 HPV E6、E7蛋白CTL表位进行筛选,采用hla... 详细信息

验证有效的人乳头瘤病毒(HPV)E6、E7蛋白的hla-A*0201限制性细胞毒性T淋巴细胞(CTL)表位,比较混合表位和单个表位的体外免疫效应差异,为宫颈癌复合多肽疫苗研究提供基础。方法对宫颈癌相关抗原 HPV E6、E7蛋白CTL表位进行筛选,采用hla-a2分子结合力实验检测T2细胞的表面平均荧光强度(MIF),采用酶联免疫斑点法(ELISOPT)检测IFN-γ斑点数,采用乳酸脱氢酶(LDH)释放实验检测吸光度(OD)。结果对已报道的 11条表位多肽进行验证,经过T2细胞肽结合力绑定实验、ELISPOT实验,结果表明,多肽P2 TIHDIILECV、P3 FAFRDLCIV、P5 YMLDLQPET、P9 ELTEVFEFA、P11 TLQDIVLHL均具有高免疫反应性,与健康志愿者相比,5条多肽能够刺激宫颈癌患者外周血CTL产生特异性反应,由混合表位诱导的效应细胞对肿瘤细胞的杀伤作用明显优于单个表位的作用。结论验证了5条多肽序列具有免疫反应性,明确了混合表位体外免疫效应优于单个表位,为进一步开展宫颈癌复合多肽疫苗的研究奠定了基础。

关键词：宫颈癌人乳头瘤病毒 E6蛋白 E7蛋白 hla-a2 限制性 CTL 表位

DNA TYPING SYSTEM FOR hla-a2 ALLELES BY POLYMERASE CHAIN REACTION WITH SEQUENCE- SPECIFIC PRIMERS

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Chinese Medical Sciences Journal 2001年第3期16卷 161-164页

作者：张庆瑞翟宁耿龙宋芳吉 Department of Dermatology the 202 Hospital of PLA Key Laboratory of Immunodermatology the First Hospital of China Medical University

Objective. To establish a PCR- SSP method for discriminating as many hla- A* 02 alleles, which could easily be introduced into a routine laboratory. Methods. In this study we typed hla- A* 02 polymorphisms by a sequence- specific primer (SSP) method, which involved round 1 and round 2 PCR reactions to detect 17 hla- A* 02 alleles (they are hla- A* 0201- 0217 alleles) covering exon 2 and exon 3. Results. We have found that DNA sample concentration and purity were the most important variables in determining the quality of the results. For identifying correct band size, the size marker used was important. We noticed that different PCR machines performed differently. By this method, we detected 20 hla- A* 02 positive genomic DNA samples and found 4 kinds of hla- A* 02 alleles. They were hla- A* 0201, 0203, 0206 and 0210. Conclusion. The hla- A* 02 PCR- SSP method was proven to be a reliable and easily applicable typing method. Our results suggest that the SSP described here provides an optimal hla- A* 02 typing technique that may be useful in selecting donor- recipient pairs in bone marrow transplantation between unrelated individuals.

关键词：脱氧核糖核酸类型人白细胞抗原等位基因聚合酶链反应 PCR-SSP hla-a2

Identification and structural definition of two discrepant immunogenic hla-a2-restricted CTL epitopes with identical primary anchor residues derived from transmembrane domain of SARS-CoV membrane protein

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生物物理学报 2009年第S1期25卷 33-34页

作者： George Fu Gao CAS Key Laboratory of Pathogenic Microbiology and Immunology Institute of MicrobiologyChinese Academy of Sciences(CAS)

Severe acute respiratory syndrome (SARS) that emerged in China with highly contagious and life-threatening character in November 2002 still remains potential risk of future

关键词： SARS CD8+ T cell epitope hla-a2 structure

Identification of the epitopes on HCV core protein recognized by hla-a2 restricted cytotoxic T lymphocytes

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World Journal of Gastroenterology 2001年第4期7卷 583-586页

作者： Hong-Chao Zhou De-Zhong Xu Xue-Ping Wang Jing-Xia Zhang Ying-Huang Yong-Ping Yan Yong Zhu Bo-Quan Jin Department of Epidemiology,the Fourth Military Medical University,Xi’an 710033,Shaanxi Province,ChinaDepartment of Immunology,the Fourth Military Medical University,Xi’an 710033,Shaanxi Province,China Department of Immunology the Fourth Military Medical University Xi'an 710033 Shaanxi Province China. juliu@***

AIM To identify hepatitis C virus (HCV) core protein epitopes recognized by hla-a2 restricted cytotoxic T lymphocyte (CTL).METHODS Utilizing the method of computer prediction followed by a 4 h 51 Cr-release assay confirmation.RESULTS The results showed that peripheral blood mononuclear cells (PBMC) obtained from two hla-a2 positive donors who were infected with HCV could lyse autologous target cells labeled with peptide 'ALAHGVFAL (core TS0-158)'.The rates of specific lysis of the cells from the two donors were 37.5% and 15.8%,respectively. Blocking of the CTL response with anti-CD4 mAb caused no significant decrease of the specific lysis.But blocking of CTL response with anti-CD8 mAb could abolish the Iysis.CONCLUSION The peptide (core 150 - 158 ) is the candidate epitope recognized by hla-a2 restricted CTL.

关键词： hepatitis C virus cytotoxic T lymphocyte hla-a2 epitope

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

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中华医学杂志 2003年第12期83卷 1080-1083页

作者：董海龙隋延仿叶菁李增山曲萍张秀敏陈广生禄韶英第四军医大学病理教研室西安710032

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... 详细信息

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 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。

关键词：肿瘤抗原 MAGE-n hla-a2 细胞毒性 T细胞表位重建技术免疫学鉴定肝癌

hla-a2肽四聚体的构建及其在乙、丙型肝炎中的初步应用

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中华医学杂志 2004年第21期84卷 1818-1822页

作者：朴文花何豫席宏丽孙新婷张恒辉徐京航赵鸿许文燮李在琉王贵强延边大学医学院北京大学第一医院感染疾病科 100034

目的　分别构建乙型肝炎病毒 (HBV)和丙型肝炎病毒 (HCV)特异的hla a2肽四聚体 ,为检测乙、丙型肝炎患者体内特异性的细胞毒T细胞 (CTL)提供直接、有效的方法 ,指导临床用药。方法　分别构建含有hla a2 BSP(含有 15个可供识别生物素化... 详细信息

目的　分别构建乙型肝炎病毒 (HBV)和丙型肝炎病毒 (HCV)特异的hla a2肽四聚体 ,为检测乙、丙型肝炎患者体内特异性的细胞毒T细胞 (CTL)提供直接、有效的方法 ,指导临床用药。方法　分别构建含有hla a2 BSP(含有 15个可供识别生物素化的氨基酸位点 )和 β2微球蛋白(β 2m)基因的原核表达载体 ,并进行表达、复性、鉴定及纯化。再分别将HBV和HCV特异性短肽与hla a2 BSP和 β 2m蛋白在体外进行耦合 ,该复合物经纯化浓缩后进行生物素化。生物素化的产物经纯化浓缩后形成单体 ,此单体再与藻红蛋白标记的链霉亲和素按一定比例耦合构建成四聚体 ,最后进行流式细胞仪检测。结果　获得了高效、稳定的pBV2 2 0 hla a2 BSP和 pBV2 2 0 β 2m原核表达载体 ,表达量分别占菌体的 4 6 %和 2 6 %左右 ;纯度达 90 %以上。构建的四聚体可成功的检测急、慢性乙型和丙型肝炎患者中特异性的CTL ,急性HBV感染时CTL占 1 84 % ,慢性HBV感染时CTL占0 0 2 %～ 0 6 8% ,慢性HCV感染的CTL占 0 0 2～ 0 72 %。结论　高效、稳定的pBV2 2 0 hla a2 BSP和 pBV2 2 0 β 2m原核表达载体大量的表达可用于构建各种肽特异性的四聚体复合物 ;在体外构建的MHC Ⅰ类分子四聚体 ,为特异性细胞毒T细胞的检测提供有效的工具。

关键词： hla-a2 四聚体 CTL 丙型肝炎特异性体外 HCV 原核表达载体纯化复性