生物素化的HLA-A*2402-抗原肽复合物的制备

目的 制备生物素化的HLA-A＊2402-抗原肽复合物。方法 将原核高效表达的sHLA-A＊2402-BSP融合蛋白及β2m和HLA-A＊2402限制性抗原肽EB病毒BRLF1蛋白中的九肽NH2-TYPVLEEMF-COOH进行稀释、复性、折叠，形成HLA—A＊2402-抗原肽复合物单体，并在BirA酶作用下进行生物素化，使生物素结合到HLA-A＊2402-抗原肽复合物中H链C端的BSP序列上形成生物素化的sHLA—A＊2402-抗原肽复合物单体。利用特异性单克隆克体（W6／32和兔抗人β2微球蛋白抗体）及链霉亲合素进行ELISA和Westem blot，检测稀释复性和生物素化的折叠产物。结果 折叠复合物中，主要含有HLA—A＊2402-肽复合物单体及β2两种成分，其中HLA-A＊2402-肽复合物单体可生物素化。结论 成功制备出生物素化的HLA—A＊2402-抗原肽复合物单体，为进一步构建四聚体及制备人工抗原提呈细胞奠定了基础。     

HLA-A*0206-BSP和-A*0207-BSP融合基因的构建与表达

采用RT-PCR技术从HLA-A*0206和-A*0207阳性个体的PBMC中分别克隆出HLA-A*0206和-A*0207基因的全长cDNA序列,构建HLA-A*0206和-A*0207克隆载体。再利用PCR技术从构建的克隆载体中扩增HLA-A*0206和-A*0207的α链(重链)胞外段序列,分别经双酶切置换本室保存的HLA-A*0201-BSP重组体中的HLA-A*0201胞外段序列,使HLA-A*0206和-A*0207与BirA酶底物肽(BirA substrate peptide,BSP)序列融合,构建HLA-A*0206-BSP和-A*0207-BSP融合基因的表达载体,经限制性酶切和DNA测序证实。然后将该表达载体转化E.coliBL21(DE3)后获得表达产物,通过体外稀释复性,初步纯化的表达产物通过ELISA和Western blot检测证明能够与β2微球蛋白(HLA I类分子轻链)及HLA-A2限制性抗原肽(HBV core 18-27)折叠形成具有HLA I类分子天然构象的抗原肽/HLA-A2复合物单体。为进一步构建HLA-A*0206和-A*0207四聚体,探讨相应HLA-A2亚型的功能特点提供了物质基础。     

HBV抗原肽-HLA-A*2402复合物四聚体的构建及初步检测应用

目的：构建2种HBV抗原肽-HLA-A＋2402复合物四聚体，并初步用该2种四聚体检测针对不同抗原肽特异性的细胞毒性T细胞（CTL）。方法：原核高效表达HLA-A＋2402-BSP和β2m蛋白后，分别与乙型肝炎病毒抗原肽P01756-764和Corell7-125在体外复性折叠成可溶性的HLA-A＋2402抗原肽复合物单体，经BirA酶作用并通过凝胶过滤层析法纯化复合物单体，分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成2种HBV抗原肽四聚体。最后进行流式细胞术检测。结果：Dot-ELISA和ELISA检测显示获得了2种具有天然构象的生物素化的HBV抗原肽-HLA-A＋2402复合物单体。结论：构建的2种四聚体可以检测乙型肝炎感染者体内特异性的CTL。     

负载HCMV pp65抗原肽的HLA-A*1101-GPI四聚体的制备和鉴定

为体外复性制备负载人巨细胞病毒(HCMV)pp65抗原肽的HLA-A*1101-GPI四聚体,研究优化HLA-A*1101重链与生物素化酶底物肽融合蛋白(HLA-A*1101-BSP)在大肠杆菌中的诱导表达的温度、时间和诱导剂IPTG浓度,并以抗HLA-A*0201抗血清进行免疫印迹鉴定HLA-A*1101-BSP的表达水平。将初步纯化的HLA-A*1101-BSP与β2-微球蛋白(β2m)和HCMV抗原肽pp6516-24(GPISGHVLK,简称GPI)一起利用稀释法进行重折叠复性,获得可溶性HLA-A*1101-GPI单体,经生物素化和纯化后,与Streptavidin-PE结合成四聚体。流式细胞仪分析显示HLA-A*1101-GPI四聚体具有与特异性细胞毒T细胞(CTL)的结合活性,表明成功获得可溶性HLA-A*1101-GPI四聚体,为研究HLA-A*1101限制性CTL的免疫应答打下基础。     

三种常见HBV抗原肽-HLA-A*0201复合物四聚体的构建及初步检测应用

目的 体外构建三种常见HBV抗原肽-HLA-A*0201复合物四聚体,并初步用该三种四聚体对抗原肽特异性的细胞毒性T细胞(CTL)进行了初步检测.方法 原核高效表达的HLA-A*0201-BSP和β2m蛋白,分别和三种HBV抗原肽(HBVCore18-27,Env335-343,Po1575-583)体外复性折叠成可溶性的HLA-A*0201抗原肽复合物单体,经BirA酶作用并通过凝胶过滤层析法纯化复合物单体,分别将复合物单体与藻红蛋白标记的链霉亲和素按一定比例耦合构建成相应的三种抗原肽四聚体.最后进行流式细胞仪检测.结果 Dot-ELISA和ELISA检测显示获得了三种具有天然构象的生物素化的HBV抗原肽-HLA-A*0201复合物单体.构建的三种四聚体均可以检测到相应特异性的CTL,在自限性感染患者体内针对乙肝核心抗原(core18-27)的CTL细胞频数(0.18%)高于针对聚合酶抗原(po1575-583)(0.08%)和包膜抗原(env335-343)(0.06%).结论 成功构建了三种具有完整构象的生物素化HBV抗原肽-HLA-A*0201复合物单体,所构建的三种四聚体都可以检测到抗原特异性的CTL.     

sHLA-A*0201-抗原肽四聚体的构建与功能检测

目的：构建有功能的sHLA-A＊0201-抗原肽四聚体，建立一套HLA Ⅰ类分子／抗原肽的四聚体制备技术。方法：利用基因工程及体外折叠技术构建sHLA-A＊0201-LMP2A426-434复合物分子，并在BirA酶的作用下生物素化。与荧光标记的亲和素衍生物以分子数4：1结合而形成HLA-A＊0201-LMP2A426-434四聚体。获得的四聚体对长期混合淋巴细胞培养中增殖的抗原特异性CTL进行检测，同时用细胞毒实验验证。结果：荧光标记的亲和素与生物素化的HLA-A＊0201-抗原肽复合物分子正确结合，制备的四聚体能够与长期混合淋巴细胞培养出的特异性T细胞结合。结论：从结构与功能上证实成功地获得有功能的HLA-A＊0201-抗原肽复合物四聚体。为进一步研究T细胞识别机制与功能奠定了基础，也为过程复杂的HLA-抗原肽四聚体制备提供了可行的免疫学监控方法。     

BirA酶基因表达载体的构建、原核表达及表达产物的活性鉴定

目的:构建生物素-蛋白连接酶(BirA酶)基因的表达载体,并在大肠杆菌BL-21(DE3)中表达具有生物学活性的BirA酶。方法:用PCR法扩增BirA酶基因。将PCR产物克隆入pGEX-4T-2中构建BirA酶-GST融合蛋白基因的重组表达载体pGEX-BirA。经测序验证后,在大肠杆菌BL-21(DE3)中诱导表达,表达产物采用谷胱苷肽-琼脂糖层析柱进行纯化。以带有生物素酶底物肽(BirAsubstratepeptide,BSP)的HLA-A2-肽复合物为底物,用ELISA和Westernblot鉴定表达产物的生物素化活性。结果:构建了pGEX-BirA原核表达质粒,并在大肠杆菌BL-21(DE3)中诱导表达Mr为61300的BirA酶-GST融合蛋白,表达产物经谷胱苷肽-琼脂糖层析柱纯化后,得到N端带有GST标签的BirA酶蛋白。ELISA和Westernblot的结果显示,表达产物能使HLA-A2-肽复合物生物素化。结论:成功地制备了具有生物学活性的Bi-rA酶,为研究蛋白质分子的相互作用提供了有效的制剂。     

HCV HLA-A2限制性复合多表位基因的构建、克隆表达及其免疫特性分析

目的:构建丙型肝炎病毒(HCV)HLA-A2限制性复合多表位基因的原核表达载体,表达纯化,并观察其免疫原性。方法:分别合成HCV HLA-A2限制性多表位基因、人泛素基因,串联后得到融合基因Ub-Mep,克隆入原核表达质粒pRSET-A,转化E.coliBL21,IPTG诱导融合蛋白表达,薄层扫描分析表达蛋白组成;可溶性分析后用Ni2+-NTA凝胶亲和层析柱纯化、透析并浓缩融合蛋白;Western blot分析纯化蛋白的特异性和抗原性;免疫小鼠分析其免疫原性。结果:成功构建复合多表位抗原基因的原核表达质粒pRSET-Ub-Mep,目的基因可高效表达,表达产物主要以包涵体形式存在,Ni2+-NTA纯化可获得目的蛋白,纯化蛋白具有良好的抗原性和免疫原性。结论:成功构建HCV HLA-A2限制性复合多表位基因并进行原核表达,表达的多表位基因抗原具良好的免疫原性,为进一步的HCV A2限制性复合多表位诱导的细胞免疫应答研究奠定基础。     

HBc-HLA-A*1101-β2m复合物单体及其四聚体的制备和鉴定

目的制备HBc-HLA-A ＊ 1101-β2m复合物单体和四聚体,并对其特异性进行了鉴定。方法原核高效表达的HLA-A ＊ 1101-BSP和β2m蛋白,在抗原肽（乙型肝炎病毒的核心蛋白HBc88-96）的存在下,体外复性折叠成可溶性的HLA-A ＊ 1101抗原肽复合物单体,经BirA酶作用并通过凝胶过滤层析法纯化复合物单体;然后将此复合物单体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;最后用流式细胞仪检测分析该四聚体结合特异性CTL的能力。结果Western blot和Dot-ELISA检测显示所制备的HLA-A ＊ 1101抗原肽复合物单体具有天然构象,且可被生物素化;流式细胞仪分析显示所制备的四聚体可与HLA-A11^＋供者的抗原特异性CTL结合。结论成功制备了具有完整构象的生物素化HLA-A ＊ 1101抗原肽复合物单体;此四聚体可以特异检测抗原特异性的细胞毒性T细胞。     

人肿瘤抑素相关T21RGD肽的表达与纯化

目的 构建引入RGD序列人肿瘤抑素相关21肽（T21RGD）―内含肽―几丁质结合蛋白融合表达载体，实现目的肽的几丁质珠亲和层析一步纯化。方法 人工设计合成T21RGD肽基因， 经重组定向克隆插入到原核表达载体pTYB2，酶切和测序鉴定正确后转化入大肠杆菌BL21（DE3），IPTG诱导其融合表达，对表达蛋白进行几丁质珠亲和层析，DTT诱导柱上裂解以获得T21RGD肽。经Trcine-SDS-PAGE和反相高效液相色谱(RP-HPLC)对纯化产物进行鉴定。结果 质粒酶切和测序结果显示，含T21RGD肽基因按正确方向和序列插入质粒,融合蛋白表达量达菌体蛋白总量的50％以上，Trcine-SDS-PAGE显示了T21RGD肽目的条带与预期相符，反相高效液相色谱测定峰纯度达97％。结论 成功构建T21RGD肽融合表达载体，实现了融合蛋白在大肠杆菌中高效表达和T21RGD肽几丁质珠亲和层析一步纯化，为进一步研究T21RGD肽抗肿瘤活性和作用机理奠定基础。     

Construction and Functional Test of HLA-A＊2402-Peptide Tetramer

HLA-A~*2402 is one of the most frequent HLA-A allele in Asian population.To construct HLA-A~*2402-peptidetetramers,the transmembrane and intracellular segments of HLA-A~*2402 cDNA were replaced with BSP sequenceto form a fusion gene of sHLA-A~*2402-BSP.The sHLA-A~*2402-BSP fusion protein and β2m were high-levelexpressed as insoluble aggregates in E.coli,and refoided to form an HLA-A~*2402-peptide monomeric complex bydilution method in the presence of an antigenic peptide.The HLA-A~*2402-peptide monomeric complex wasbiotinated and tetramized to prepare HLA-A~*2402-peptide tetramer.Then using the HLA-A~*2402-peptidetetramers to detect antigen-specific cytotoxic T lymphocyte(CTL)induced by artificial antigen presenting cell(aAPC)in vitro.The results showed that HLA-A~*2402-peptide tetramer was prepared correctly,and functional indetecting antigen-specific CTL in vitro,HLA-A~*2402-peptide monomeric and its multimeric complexes areexpected to provide a powerful tool for studying mechanisms of immune-related diseases in Asian populations.Cellular & Molecular Immunology.2005;2(2):145-149.     

HLA-A*0203-BSP的表达和复性及其四聚体的鉴定

目的：优化诱导条件大批量表达生物素化酶BirA底物肽（BSP）与HLA-A＊0203重链胞外域的融合蛋白（HLA—A＊0203、BSP），并制备负载HLA-A＊0203限制性EB病毒抗原肽EBNA3 596-604的四聚体（HIA—A}0203／SVR）。方法：以HLA—A＊0203-BSP原核表达载体转化E．coli BL21（DE3）菌株，优化诱导条件进行大批量重组蛋白的表达。通过稀释法复性可溶性HLA-A＊0203／SVR单体，然后以BirA对其进行生物素化，并以阴离子交换树脂纯化。将纯化的HLA-A＊0203／SVR单体与荧光素标记的链亲和素按4：1的比例混合形成四聚体，通过对特异性CTL进行染色验证其结合活性。结果：当IPTG的浓度为0．4mmol／L，于37℃诱导过夜后，融合蛋白的表达最多。该重组蛋白相对分子质量（Mr）为34003，与HLA—A＊0203-BSP的理论Mr相一致。该重组蛋白以包涵体形式存在于沉淀部分，约占菌体总蛋白的30％。负载抗原肽的可溶性HLA-A＊0203／SVR单体是在同时存在HLA-A＊0203，BSP、β2微球蛋白及HLA-A＊0203限制性抗原肽SVR的情况下通过稀释法复性而获得。该单体生物素化并纯化后与荧光素标记的链亲和素按4：1的比例混合后即形成四聚体。流式细胞术（FCM）分析证实，该四聚体具有与HLA—A2^＋供者特异性CTL结合的活性。结论：HLA—A＊0203-BSP融合蛋白在优化条件下获得高效表达。以此蛋白制备的HLA-A＊0203／SVR四聚体具有与HLA-A2^＋供者特异性CTL结合的活性，为研究HLA—A＊0203个体EB病毒特异性CTL的免疫应答打下了基础。     

sHLA-A*020-1抗原肽复合物的构建

以原核表达的sHLA A 0 2 0 1 BSP为重链 ,β2 m为轻链 ,与人工合成的EBV抗原肽LMP2A (4 2 6 4 34、NH2 CLGGLLTMV COOH )利用稀释法进行共折叠复性 ,形成可生物素化的可溶性HLA A 0 2 0 1抗原肽复合物。并利用仅与天然构象HLAI类分子结合的单抗W6 / 32 ,通过Dot ELISA和Westernblot对折叠产物的构象进行鉴定 ,证实复性后成功获得了天然构象的sHLA A 0 2 0 1 抗原肽复合物。为进一步组装可溶性HLA/抗原肽四聚体以及制备人工抗原提呈细胞奠定基础。     

Preparation and Identification of HLA-A＊1101 Tetramer Loading with Human Cytomegalovirus pp65 Antigen Peptide

MHC/peptide tetramer technology has been widely used to study antigen-specific T cells, especially for identifying virus-specific CD8^＋ T cells in humans. The tetramer molecule is composed of HLA heavy chain, β2-microglobulin （β2m）, an antigenic peptide, and fluorescent-labeled streptavidin. To further investigate the HLA-A＊1101-restricted CD8^＋ T cell responses against human cytomegalovirus （HCMV）, we established an approach to prepare HLA-A＊1101 tetramer complexed with a peptide from HCMV. The cDNA encoding HLA-A＊1101 heavy chain was cloned and the prokaryotic expression vector for the ectodomain of HLA-A＊1101 fused with a BirA substrate peptide （HLA-A＊1101-BSP） at its carboxyl terminus was constructed. The fusion protein was highly expressed as inclusion bodies under optimized conditions in Escherichla coli. Moreover, HLA-A＊1101-BSP protein was refolded in the presence of β2m and an HCMV peptide pp6516.24 （GPISGHVLK, GPI）. Soluble HLA-A＊1101-GPI monomer was biotinylated and purified to a purity of 95%, which was subsequently combined with streptavidin to form tetramers at a yield of 〉 80%. The HLA-A＊1101-GPI tetramers could bind to virus-specific CD8^＋ T cells, suggesting soluble HLA-A＊1101-GPI tetramers were biologically functional. This study provides the basis for further evaluation of HLA-A＊1101-restricted CD8^＋ T cell responses against HCMV infection.     

High Level Expression of HLA-A＊0203-BSP Fusion Protein in Escherichia coli and Construction of Soluble HLA-A＊0203 Monomer and Tetramer Loaded with Epstein-Barr Virus Peptide

Major histocompatibility complex （MHC） tetramer technology is critical for characterization of antigen-specific T cells. In the present study we reported the successful generation of HLA-A＊0203 tetramer loaded with Epstein- Barr virus EBNA3596-604 peptide （SVRDRLARL, SVR）. Prokaryotic expression vector for the ectodomain of the heavy chain of HLA-A＊0203 fused with a BirA substrate peptide （HLA-A＊0203-BSP） was constructed and the expression conditions of the fusion protein in Escherichia coli （E. coli） were optimized. The fusion protein was highly expressed in inclusion bodies within E. coil It was then refolded in the presence of 132-microglobulin and SVR peptide to form a soluble HLA-A＊0203-SVR monomer. After biotinylation with BirA, the monomer was purified by anion-exchange chromatography and its purity was up to 95%. The tetramer was then formulated by mixing the biotinylated monomer with streptavidin-PE at a ratio of 4：1. Flow cytometry showed that this tetramer could specifically react with antigen-specific CD8^＋ T cells, indicating that it was biologically functional. These results provide a foundation for further characterization of antigen-specific CD8^＋ T cells from HLA-A＊0203 subjects.     

Improved generation of HLA class I/peptide tetramers

As a tetrameric major histocompatibility complex (MHC) class I-peptide complex (tetramer) is capable of detecting antigen-specific cytotoxic T lymphocyte (CTL) by flow cytometry, significant information about the generation of in vivo immunity can be obtained. It is, however, difficult to make a soluble wild type of MHC class I heavy chain by the prokaryotic expression system. Therefore, we developed a new method for making soluble mutant HLA-A*2402 heavy chain. In this method, signal sequences were deleted, and the codon was changed to silent mutated nucleotide sequences that bacteria could use as preferable codon. When purified mutant HLA-A*2402 molecules were examined for the protein generation by SDS-polyacrylamide gel electrophoresis (PAGE) and western blotting using anti-HLA class I monoclonal antibody (mAb) as compared with wild type, a large amount of mutant heavy chain could be detected. In contrast, the expression of wild-type stable HLA-A*2402 heavy chain molecule was not detected in this system. Consequently, by using mutant HLA-A*2402/peptide tetramers, CTL precursors (CTLp) that specifically recognize antigenic peptide derived from the X;18 chromosomal translocations of synovial sarcoma were detected in patients' PBL.