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Enhancement of binding activity of soluble human CD40 to CD40 ligand through incorporation of an isoleucine zipper motif

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Acta Pharmacologica Sinica 2006年第3期27卷 333-338页

作者： xian-hui he~(2,3) li-hui xu~4 yi liu~(2,5) ~2key laboratory of ministry of education for tissue transplantation and immunology,jinan university,guangzhou 510632,china.~4 Institute of Bioengineering,jinan university,guangzhou 510632.china key laboratory of ministry of education for tissue transplantation and immunology Jinan University Guangzhou China Institute of Bioengineering Jinan University Guangzhou China

Aim:To investigate the effect of incorporation of an isoleucine zipper(IZ)motifinto CD40 on binding activity of CD40 for the CD40 ligand(CD40L).Methods:Prokaryotic expression vectors for 2 soluble CD40 derivatives,shCD40His andshCD40IZ containing an IZ dowain,were constructed and expressed in Escheri-chia *** recombinant proteins were purified to homogeneity after refoldingfrom inclusion *** molecular weights in solution of shCD40His andshCD40IZ were compared by size-exclusion chromatography,and their bindingactivity for CD40L on Jurkat T cells was determined by flow ***:shCD40His and shCD40IZ were *** of them possessed significantbinding activity for the cognate ligand CD40L expressed on the cell ***40IZ had much higher binding activity to its ligand(CD40L)than ***,size-exclusion chromatography demonstrated thatshCD40IZ existed in high molecular mass forms that were most likely to be trimersin ***:Incorporation of an IZ motif into CD40 enhances itsbinding activity for CD40L through trimerization of the CD40 derivative.

关键词： soluble CD40 isoleucine zipper trimerization binding activity CD40L

Cloning and Identification of Two Novel Splice Variants of Human PD-L2

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Acta Biochimica et Biophysica Sinica 2004年第4期36卷 284-289页

作者： xian-hui he#*,yi liu&#,li-hui xu1, and Yao-ying ZENG* key laboratory of tissue transplantation and immunology, ji’nan university, ministry of education, 601 Huangpu Road West, guangzhou 510632, china 1Institute of Bioengineering, ji’nan university, 601 Huangpu Road West, guangzhou 510632, china key laboratory of tissue transplantation and immunology Ji'nan University Ministry of Education601 Huangpu Road West Guangzhou 510632 China Institute of Bioengineering Ji'nan University601 Huangpu Road West Guangzhou 510632 China

PD-L2, a newly identified member of B7 family, plays a role in down-regulating T cell responses. The common PD-L2 mRNA (type I) is the splicing product containing all 6 exons. We report here the identification of two human PD-L2 splice variants in activated leukocytes. One splice variant (type II) is generated through splicing out exon 3 encoding Igconstant-like domain; it retains all other regions without a frame shift. The other variant (type III) is created by splicing out exon 3 to an alternative acceptor site 5 bp downstream of the canonical acceptor site, leading to a frame shift. Consequently, the translated protein should be a soluble form. Furthermore, type I isoform is expressed on the plasma surface whereas type II isoform showed a pattern of intracellular membrane distribution in the transiently transfected K562 cells. In addition, the expression patterns of PD-L2 splice variants are variable in different individuals and distinct cellular status. These results suggest that PD-L2 expression may be controlled by posttranscriptional regula- tion through alternative splicing, and modulation of PD-L2 isoform expression may influence the outcome of immune response.

关键词： costimulatory molecules PD-L2 splice variant leukocytes

Procedure for preparing peptide-major histocompatibility complex tetramers for direct quantification of antigen-specific cytotoxic T lymphocytes

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World Journal of Gastroenterology 2005年第27期11卷 4180-4187页

作者： xian-hui he li-hui xu yi liu key laboratory of ministry of education of china for tissue transplantation and immunology Jinan UniversityGuangzhou 510632 Guangdong Province China Institute of Bioengineering Jinan University Guangzhou 510632 Guangdong Province China Department of Dermatology First Affiliated HospitalZhengzhou University Zhengzhou 450052 Henan Province China

AIM： To establish a simplified method for generating peptide-major histocompatibility complex （MHC） class I ***： cDNAs encoding the extracellular domain of human lymphocyte antigen （HLA）-A＊0201 heavy chain （A2） and β2-microglobulin （132m） from total RNA extracted from leukocytes of HLA-A2＋ donors were doned into separate expression vectors by reverse transcription-polymerase chain reaction. The recombinant A2 and 132m proteins were expressed in ~/a oo/i^uain BL21（DE3） and recovered from the inclusion body fraction. Soluble A2 proteins loaded with specific antigen peptides were refolded by dilution from the heavy chain in the presence of light chain 132m and HLA-A2-restricted peptide antigens. The refolded A2 monomers were biotinylated with a commercial biotinylation enzyme （BirA） and purified by low pressure anion exchange chromatography on a Q-Sepharose （fast flow） *** tetramers were then formed by mixing A2 monomers with streptavidin-PE in a molar ratio of 4：1. Flow cytometry was used to confirm the expected tetramer staining of CD8^＋ T ***： Recombinant genes for HLA-A＊0201 heavy chain （A2） fused to a BirA substrate peptide （A2-BSP） and mature β2m from HLA-A2＋ donor leukocytes were successfully doned and highly expressed in E. coli, Two soluble monomeric A2-peptide complexes were reconstituted from A2-BSP in the presence of 132m and peptides loaded with either human cytomegalovirus pp65495-503 peptide （NLVPMVATV,NLV; designated as A2-NLV） or influenza virus matrix protein Mp58-66 peptide （GILGFVFTL, GIL; designated as A2-GIL）. Refolded A2-NLV or A2-GIL monomers were biotinylated and highly purified by single step anion exchange column chromatography. The tetramers were then formed by mixing the biotinylated A2-NLV or A2-GIL monomers with streptavidin-PE, leading to more than 80% multiplicationas revealed by SDS-PAGE under non-reducing, unboiled conditions. Flow cytometry revealed that these tetramers could specifically bind to CD8^＋

关键词： Cloning, Molecular HLA-A Antigens HLA-A2 Antigen Humans Recombinant Fusion Proteins Research Support, Non-U.S. Gov't T-Lymphocytes, Cytotoxic beta 2-Microglobulin

加载HCMV抗原肽的HLA-A*0201单体及其四聚体制备和鉴定

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生物工程学报 2004年第3期20卷 382-388页

作者：何贤辉徐丽慧刘毅蔡小嫦曾耀英组织移植与免疫教育部重点实验室暨南大学广州510632 暨南大学生物工程研究所广州510632 暨南大学第一附属医院皮肤科广州510632 郑州大学第一附属医院皮肤科郑州450052

细胞毒T淋巴细胞 (CTL)在控制病原体感染以及抗肿瘤过程中发挥重要作用 ,因而特异性CTL的检测相当重要 ;而过去检测CTL的方法都是间接的 ,最近发展起来的四聚体技术则是直接检测抗原特异性CTL的有效而特异的方法 ,成为目前研究T细胞免... 详细信息

细胞毒T淋巴细胞 (CTL)在控制病原体感染以及抗肿瘤过程中发挥重要作用 ,因而特异性CTL的检测相当重要 ;而过去检测CTL的方法都是间接的 ,最近发展起来的四聚体技术则是直接检测抗原特异性CTL的有效而特异的方法 ,成为目前研究T细胞免疫应答的关键技术。报道一种简化的四聚体制备程序 ,利用该程序成功制备加载人巨细胞病毒 (HCMV)抗原肽的HLA_A2四聚体 ,具有特异性结合CTL活性。HLA_A 0 2 0 1重链基因是通过RT_PCR方法从HLA_A2 + 供者白细胞中克隆 ,进而以PCR方法构建在羧基端融合生物素化酶BirA底物肽 (BSP)的HLA_A 0 2 0 1(A2 )重链胞外区原核表达载体 ,A2重组蛋白在大肠杆菌中得到高表达 ,主要以包涵体形式存在。加载抗原肽的可溶性A2单体是A2胞外区在轻链 β2 微球蛋白和HLA_A2限制性HCMVpp6 5 495_50 3抗原肽 (NLVPMVATV ,NLV)存在时通过稀释法复性获得 ,以BirA对其进行生物素化 ,然后以阴离子交换树脂纯化 ,得到的纯化A2_NLV单体与Streptavidin_PE按 4 :0 8比例混合形成四聚体 ,结合程度在 85 %以上 ,流式细胞仪分析显示该四聚体具有与HLA_A2 + 供者的特异性CTL结合活性。总之 ,这种简化的四聚体制备程序 ,不仅有利于该技术的推广 ,为特异性T细胞免疫研究建立必要的技术平台。

关键词：人类白细胞抗原四聚体细胞毒T淋巴细胞包涵体人巨细胞病毒

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人β_2-微球蛋白基因克隆及其在大肠杆菌中的高效表达

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生物工程学报 2004年第1期20卷 99-103页

作者：何贤辉徐丽慧刘毅曾耀英组织移植与免疫教育部重点实验室(暨南大学) 广州510632

β2 微球蛋白 (β2 m)是主要组织相容性复合体 (MHC)Ⅰ类分子的轻链部分 ,为制备MHCⅠ类分子四聚体的必要成分。根据已报道的序列设计特异引物 ,利用RT PCR方法从人白细胞中克隆了 β2 m基因 ,并构建了成熟β2 m的原核表达载体 ,在大... 详细信息

β2 微球蛋白 (β2 m)是主要组织相容性复合体 (MHC)Ⅰ类分子的轻链部分 ,为制备MHCⅠ类分子四聚体的必要成分。根据已报道的序列设计特异引物 ,利用RT PCR方法从人白细胞中克隆了 β2 m基因 ,并构建了成熟β2 m的原核表达载体 ,在大肠杆菌中得到高效表达。表达的β2 m大部分在包涵体中 ,经洗涤、变性和复性 ,并以强阴离子交换柱层析纯化 ,获得SDS PAGE纯的人重组 β2 m ,Western印迹法分析表明该蛋白具有与抗人天然 β2 m抗体反应的特性。此工作为制备MHCⅠ类分子四聚体奠定基础。

关键词： β2-微球蛋白基因克隆大肠杆菌表达基因克隆重组蛋白包涵体

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