+10 Gz重复暴露大鼠脑差异表达基因cDNA文库的构建

目的构建＋10Gz重复暴露大鼠脑差异表达基因的消减cDNA文库。方法本实验用SD大鼠，分别提取暴露组与对照组的总RNA，并分离纯化mRNA，应用抑制性消减杂交技术分离＋10GI重复暴露大鼠脑差异表达基因eDNA片段并建立消减eDNA文库；利用PCR对随机挑选的75个白色菌落进行插入片段的验证，对其中70个克隆进行eDNA斑点杂交验证。结果所构建的eDNA文库扩增后包含约400个白色克隆和100个兰色克隆，随机挑选75个白色克隆入质粒载体后共获得70个阳性克隆。结论应用抑制性消减杂交技术成功构建了＋10Gz重复暴露大鼠脑差异表达基因消减eDNA文库，为进一步筛选和克隆脑损伤相关基因奠定了基础。     

人毛乳头细胞凝集性生长差异表达基因cDNA文库的构建

目的 构建毛乳头细胞凝集性生长状态下差异表达基因的消减cDNA文库。方法 分别提取凝集性生长和非凝集性生长状态下毛乳头细胞中的总RNA，应用SMART cDNA合成技术和抑制性消减杂交技术分离毛乳头细胞凝集性生长状态下差异表达基因的cDNA片段并建立消减cDNA文库；利用PCR对随机挑选的100个白色菌落进行插入片段的验证，对其中证实有插入片段的30个克隆进行cDNA斑点杂交验证。结果 所构建的消减cDNA文库扩增后得到320个阳性克隆，随机挑选的100个阳性克隆中95％的均有长度在100—600bp的插入片段，cDNA斑点杂交验证显示27个(90．0％)克隆为阳性。结论 结合SMART cDNA合成技术，应用抑制性消减杂交成功地构建了毛乳头细胞凝集性生长差异表达基因消减cDNA文库，为进一步筛选和克隆毛乳头细胞凝集性生长相关基因奠定了基础。     

上海市外来人员肺结核病分布与临床特征

目的：了解上海市外来人口中肺结核病人的分布和临床特征。方法：对1996年1～12月全市外来人口活动性肺结核病报告登记资料进行分析。结果：外来人员肺结核病人总数达2279人，青壮年占总病例65％以上；男性病例为主；主要来源于江苏、浙江、安徽、四川、江西等省；登记病人来沪暂居地主要集中在市区及城乡地区，外来人口结核病管理重点在市区及城乡地区。病人分型以Ⅲ型为主。查痰率仅60．1％，但排菌率高达40％。外来人。的病例发现工作及治疗管理难度较大，目前登记病例规则化疗仅占10．4％。结论：有必要通过有关部门的共同协作，拟定切实可行的外来人口结核病管理办法。     

Intracellular antibodies for proteomics

The intracellular antibody technology has many applications for proteomics studies.

The potential of intracellular antibodies for the systematic study of the proteome has been made possible by the development of new experimental strategies that allow the selection of antibodies under conditions of intracellular expression. The Intracellular Antibody Capture Technology (IACT) is an in vivo two-hybrid-based method originally developed for the selection of antibodies readily folded for ectopic expression. IACT has been used for the rapid and effective identification of novel antigen–antibody pairs in intracellular compartments and for the in vivo identification of epitopes recognized by selected intracellular antibodies. IACT opens the way to the use of intracellular antibody technology for large-scale applications in proteomics. In its present format, its use is however somewhat limited by the need of a preselection of the input phage antibody libraries on protein antigens or by the construction of an antibody library from mice immunized against the target protein(s), to provide an enriched input library to compensate for the suboptimal efficiency of transformation of the yeast cells. These enrichment steps require expressing the corresponding proteins, which represents a severe bottleneck for the scaling up of the technology.

We describe here the construction of a single pot library of intracellular antibodies (SPLINT), a naïve library of scFv fragments expressed directly in the yeast cytoplasm in a format such that antigen-specific intrabodies can be isolated directly from gene sequences, with no manipulation whatsoever of the corresponding proteins. We describe also the isolation from SPLINT of a panel of intrabodies against a number of different proteins.

The application of SPLINT on a genome-wide scale should help the systematic study of the functional organization of cell proteome.     

Localization of hepatitis B surface antigen epitopes present on variants and specifically recognised by anti-hepatitis B surface antigen monoclonal antibodies

Small hepatitis B surface antigen (HBsAg) is considered to be the best marker for the diagnosis of Hepatitis B virus infection. However, HBsAg variants with mutations within the "a" determinant may be poorly or not detected by diagnostic assays. Three anti-HBsAg monoclonal antibodies (6H6B6, 27E7F10, and 2G2G10), directed against conformational epitopes, were tested for their ability to detect the wild-type HBsAg as well as variant forms and their respective epitopes were localised on the HBsAg sequence by using the phage-displayed peptide library technology. Whereas 6H6B6 did not detect mutations T123N, S143L, D144A and G145R, 27E7F10 binding was affected by mutations P120T and G145R. In contrast, 2G2G10 reacted strongly with all tested variants including variant with the G145R mutation. Part of the 6H6B6 epitope was located in the major hydrophilic region (MHR) at residues 101-105, the 27E7F10 epitope (residues 214-219) was located near the C-terminal end of the antigen and the 2G2G10 epitope at residues 199-208, within the theoretical fourth transmembrane helix. The 2G2G10 epitope localisation brings information about the HBsAg structure and the validity of established topological models. Finally, 2G2G10 is a valuable tool for HBsAg variant detection that is used as capture phase in a new bioMérieux diagnostic assay, which is currently in development.     

应用噬菌体肽文库筛选McAb F3特异性结合肽

目的：应用噬菌体展示肽文库筛选可与抗汉坦病毒囊膜蛋白单抗F3特异性结合的配体肽。方法：采用protein-A亲和层析纯化的F3单抗为筛选配基，对噬菌体展示的随机12肽文库进行生物亲和淘洗，夹心ELISA、竞争ELISA鉴定筛选克隆的结合特性，并进一步对阳性克隆进行序列测定和分析。结果：通过3轮生物淘洗，能被抗体捕获的噬菌体克隆为91．7％（11／12）；ELISA测定显示筛选到的噬菌体短肽能与F3单抗特异性结合；序列分析表明7个阳性克隆氨基酸序列相同，均为－MHGPTKNQMWHT，同源性分析显示该序列与HTNV／SEOV M蛋白G2区第750－759位氨基酸有较高的同源性，结论：获得了具有良好结合活性的模拟表位肽，为基于表位水平的HFRSV（肾病综合征出血热病毒）特异性分子多肽疫苗的设计提供了重要依据。     

Isolation of DNA from the centromere of human chromosome 7 by microdissection

Centromeres remain the least characterized regions of human chromosomes because they have a very high content of repetitive DNA. Here, we describe a micro-dissection library from the centromeric region of human chromosome 7 and its use for generating sequence tagged sites (STSs). The library contains about 1500 clones with an average insert size of 150 bp and only about 15% of the clones harbour repetitive human DNA. Seven clones hybridizing to alphoid DNA were found to correspond to a fragment of the D7Z2 alphoid array on chromosome 7, thus confirming the origin of the library. A number of clones not containing known repetitive DNA were used to generate STSs that identified yeast artificial chromosomes (YACs) and in turn allowed the STSs to be placed on the physical map. One STS is located between the two Genethon genetic markers closest to the centromere on the q side. Another STS was located 3–4 cM away in 7q11.2, while a third identified YACs containing both low-copy and alphoid sequences that are not yet mapped but are clearly centromeric. The library therefore comprises a collection of sequences from the centromeric region of chromosome 7 that can be used to generate STSs and to map the entire centromeric region.This revised version was published online in November 2005 with corrections to the Cover Date.     

抗SARS-CoV病毒N蛋白的单链抗体(scFv)筛选

目的筛选出抗SARS-CoV病毒N蛋白的单链抗体。方法利用原核表达所获得的SARS病毒N蛋白,筛选人源单链抗体噬菌体展示库,经特异性的检测,以期得到抗SARS-CoV病毒N蛋白的特异单链抗体。结果获得了8个抗SARS病毒N蛋白的候选克隆。经测序,获得了编码抗体可变区的基因序列,并进行了原核表达。结论筛选得到的抗SARS-CoV病毒N蛋白单链抗体具有高度的特异性,可以用作临床实验或研究SARS病毒过程中快速检测SARSN蛋白或SARS病毒粒子的候选抗体。     

用噬菌体表面展示技术筛选与肝癌细胞系结合的抗体模拟肽

目的:从噬菌体展示肽库中,筛选可与肝癌细胞特异性结合的抗体模拟肽。方法:通过生物淘选使噬菌体富集。利用ELISA法,鉴定噬菌体单克隆原种的亲和性,并进行统计学分析。通过竞争ELISA,分析筛选所得抗体模拟肽的结合位点,并进一步分析抗体模拟肽的序列组成。结果:随着淘选次数的增加,出现噬菌体的富集。ELISA的结果显示,相对于正常肝细胞,筛选所得环状7肽对肝癌细胞系SMMC7721和BEL7402均有良好的结合活性(P<0.05),且与SMMC7721细胞的结合活性明显优于与BEL7402细胞的亲和性(P<0.05)。在α=0.01的水平上,7肽单克隆噬菌体原种可明显与scFv竞争结合SMMC7721细胞(0.005相似文献   

差异筛选肝癌凋亡细胞cDNA文库的简便方法

目的差异筛选人肝癌凋亡细胞ｃＤＮＡ文库。方法消减杂交和点杂交相结合的噬菌斑原位杂交法筛选ｃＤＮＡ文库，首先采用（－）ｃＤＮＡ探针杂交，挑取（－）的噬菌斑克隆，再用（－）和（＋）两种ｃＤＮＡ探针与初筛出的噬菌斑克隆杂交的差异筛选方法。结果得到４个充分孤立的噬菌斑克隆，其插入片段长度为１．５ｋｂ左右。结论该方法简便、快速，是差异筛选ｃＤＮＡ文库的一种较为可行的简便方法。