High throughput functional epitope mapping: Revisiting phage display platform to scan target antigen surface

Antibody engineering must be accompanied by mapping strategies focused on identifying the epitope recognized by each antibody to define its unique functional identity. High throughput fine specificity determination remains technically challenging. We review recent experiences aimed at revisiting the oldest and most extended display technology to develop a robust epitope mapping platform, based on the ability to manipulate target-derived molecules (ranging from the whole native antigen to antigen domains and smaller fragments) on filamentous phages. Single, multiple and combinatorial mutagenesis allowed comprehensive scanning of phage-displayed antigen surface that resulted in the identification of clusters of residues contributing to epitope formation. Functional pictures of the epitope(s) were thus delineated in the natural context. Successful mapping of antibodies against interleukin-2, epidermal growth factor and its receptor, and vascular endothelial growth factor showed the versatility of these procedures, which combine the accuracy of site-directed mutagenesis with the high throughput potential of phage display.     

小麦6B染色体微切割及其不同片段的DNA文库构建

用Nd:YAG激光微束将处于丝分裂中期的小麦(Triticum aestivum L.)6B染色体微切割为四段,并用微细玻璃针将每个片段分别回收.将分离的染色体片段DNA用Sau3A接头介导的多聚酶链式反应(LA-PCR)分别扩增.Southem杂交证明4个特定区域的DNA确实来自于小麦基因组.用一系列(42对引物)位于6B染色体上的微卫星序列对微切割的染色体片段的PCR产物进行了验证.结果表明,获得的染色体片段的PCR产物来自于小麦6B染色体.将6B染色体4个片段的第二轮PCR产物克隆到pGET-vector中,建立了4个染色体特定区域的基因组文库,命名为R1、R2、R3和R4,分别包含2.1×105、2.74×105和2.93×105个重组子克隆.每个文库均随机挑选150个克隆进行质粒的小量制备和酶切验证.结果显示;插入片段大小在300～1800之间,平均大小为820～870bp,其中43%～48%的克隆为低/单拷贝序列,42%～47%为中/高拷贝序列.本研究为详细分析植物单染色体的不同片段的分子遗传学研究提供了基础.     

正常人体淋巴细胞cDNA文库的构建

应用GIBCOBRL建库试剂盒建立了正常人体淋巴细胞cDNA文库。取新鲜的正常人外周血,分离出淋巴细胞,进行体外培养,提取总RNA,纯化mRNA,并将其反转录成cDNA,与SalI和NotI接头连接后插入λZipLox载体,体外包装后转染到Y1090宿主菌中,进行滴度测试及文库扩增。构建的正常人淋巴细胞cDNA文库含2-6×106重组子,克隆效率为5×1012重组子/g cDNA,插入片段长度约为1~5kb。扩增后的文库浓度为3×107重组子/μl,将文库稀释到10-6时所产生的噬菌斑密度最为适宜。试验结果表明,该库符合标准,所构建的正常人淋巴细胞cDNA文库为进一步筛选目的基因、制作基因芯片等提供了有效的工具。 Abstract:A lymphocyte cDNA library of normal human was constructed in order to obtain specific gene and prepare lymphocyte gene chips to detect the relative genes between psychiatric diseases and immunity.The lymphocyte was abstracted from fresh normal human blood and cultured in vitro.Total RNA of lymphocyte was extracted from the cultured cells and then mRNA was extracted further.Moreover,single-strand cDNA and double-strand cDNA were synthesized in turn.The double-strand cDNAs were ligated to SalI and NotI adaptor,which were later ligated to arms of λZipLox.Ligated-cDNAs were packed in vitro,and then infected E.coli Y1090.Titering the phage and amplifying the library.The lymphocyte cDNA library consisted of 2-6×106 recombinants with the length of 1~5kb and the cloning efficiency was 5×1012 recombinants/g cDNA.The amplified library was 3×107recombinants/μl in concentration and the number of bacteriophage plagues was the most suitable in density after it was diluted to 10-6 in concentration.The constructed cDNA library of normal human lymphocyte would be helpful to further detecting target genes and preparing gene chips etc.     

吉林双阳型梅花鹿sentrin/SUMO的发现

为了确定梅花鹿未知的编码区cDNA,我们利用TaKaRa公司的cDNA 合成试剂盒及PCR cDNA文库试剂盒,构建了吉林双阳型梅花鹿子宫PCR cDNA文库。将文库的PCR产物克隆入pGEM-Teasy载体并进行测序后,应用BLAST网络服务对测得的序列在GenBank数据库中进行同源性比较。结果显示构建的PCR cDNA文库中包含有不同长度的cDNA片段,而且自该文库中我们发现了一与人sentrin-1/SUMO-1（small ubiquitin-related modifier 1）高度同源的全编码区cDNA序列。此序列已在Genbank登录,登录号为AF 242526。这说明我们自梅花鹿子宫PCR cDNA文库中发现了梅花鹿的sentrin/SUMO基因。 Abstract: In order to identify unknown encoding cDNAs of Cervus nioppon Temminck (sika deer),we constructed a cDNA library of uterus from Jilin-Shuangyang Cervus nippon Temminck using PCR cDNA library kit.PCR products of the library were cloned into pGEM-Teasy vectors and the cDNAs were sequenced and analyzed by nucleotide homology comparison against GenBank Database using the BLAST network service.The results showed that the cDNA library contained cDNA fragments of different lengths and a full length encoding cDNA highly homologous to human sentrin-1/SUMO-1 (small ubiquitin-related modifier 1) was identified.The cDNA was deposited in GenBank under the accession number AF 242526.These show that Cervus nippon Temminck-derived sentrin/SUMO gene has been discovered from PCR cDNA library of uterus from Cervus nippon Temminck.     

HA结合肽的筛选与序列分析

利用噬菌体随机十二肽库对羟基磷灰石进行结合肽筛选,经4轮生物淘洗和选择、噬菌体扩增和DNA测序,获得一组多肽序列,其中含有较高比例的脯氨酸、亮氨酸、谷氨酸、谷氨酰胺以及疏水氨基酸。经Blast分析、CLUSTALW多重序列比对推得羟基磷灰石结合肽可能的结合基序为VLPP、LP(X)₆PL/X、PP(X)_4~6P,(X为任意氨基酸)。认为脯氨酸等氨基酸的高比例特性以及结合基序特征可以很好地解释羟基磷灰石的生物可利用性和安全性。在生物体中未发现结合肽有价值的相似DNA编码序列和多肽、蛋白质序列。噬菌体单克隆反筛功能测定进一步确定了相关序列对羟基磷灰石的结合能力。     

Construction of a Diverse Fab Expression Library from Autoimmunized Mice Based on an Improved Preparation of Cloning Arms from Bacteriophage Vectors: A New Library with Potential for Screening of Biocatalysts

This paper describes an improved procedure for the preparation of cloning arms from vectors Lc1 and Hc2 for the construction of cDNA libraries in phage δ for expression in E. coli of the combinatorial Fab fragments of the immunoglobulin repertoire from autoimmunized mice. It was found that annealing, dialysis and dephorphorylation after the restriction enzyme digestion of vectors were critical for the high efficient preparation of cloning arms. The construction of Fab cDNA libraries involves the use of primer-directed PCR amplification products of total RNA from autoimmunized mice. Fab production was confirmed by the immunoassay and sequence analysis. The partial sequenced genes for Fab fragments were different from each other in the FR4, CDR3 and FR3 regions, indicating a diverse nature of the library. This library may be used to screen for catalytic Fab's.     

Advances in plant chromosome genomics

Next generation sequencing (NGS) is revolutionizing genomics and is providing novel insights into genome organization, evolution and function. The number of plant genomes targeted for sequencing is rising. For the moment, however, the acquisition of full genome sequences in large genome species remains difficult, largely because the short reads produced by NGS platforms are inadequate to cope with repeat-rich DNA, which forms a large part of these genomes. The problem of sequence redundancy is compounded in polyploids, which dominate the plant kingdom. An approach to overcoming some of these difficulties is to reduce the full nuclear genome to its individual chromosomes using flow-sorting. The DNA acquired in this way has proven to be suitable for many applications, including PCR-based physical mapping, in situ hybridization, forming DNA arrays, the development of DNA markers, the construction of BAC libraries and positional cloning. Coupling chromosome sorting with NGS offers opportunities for the study of genome organization at the single chromosomal level, for comparative analyses between related species and for the validation of whole genome assemblies. Apart from the primary aim of reducing the complexity of the template, taking a chromosome-based approach enables independent teams to work in parallel, each tasked with the analysis of a different chromosome(s). Given that the number of plant species tractable for chromosome sorting is increasing, the likelihood is that chromosome genomics – the marriage of cytology and genomics – will make a significant contribution to the field of plant genetics.