针对单克隆抗体MGb1的重组抗独特型抗体的筛选与鉴定

目的 利用噬菌体呈现技术筛选针对抗胃癌单克隆抗体（简称单抗）MGb1的重组抗独特型抗体（抗-Id）,为研制胃癌重组抗-Id瘤苗提供候选分子。方法 以单克隆抗体MGb1免疫Balb／c小鼠,取脾分离mRNA。RT-PCR分别扩增抗体VL和VH cDNA,经linker DNA连接形成ScFv DNA。将scFv DNA与载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7感染后,获得重组噬菌体抗体ScFv文库。以单抗MGb1对文库进行4轮淘选后,随机挑取克隆经ELISA筛选呈现抗-Id scFv的噬菌体单克隆,进而经竞争ELISA对其所属抗-Id类型进行初步鉴定。结果 VL和VH cDNA分别约为320和340bp,ScFv DNA约为750bp。抗体ScFv文库经四轮淘选后,在随机筛检的50个克隆中得到18个呈现抗-Id ScFv的噬菌体单克隆。在18个克隆中,有4个呈现β或γ型抗-Id ScFv。结论 经重组噬菌体抗体技术成功地筛选到了针对单抗MGb1的噬菌体呈现型抗-Id ScFv,从而为进一步获得能诱导抗胃癌免疫的抗-Id ScFv奠定了基础。     

Screening for antibodies against intact cancer cells with a naïve large phage antibody library

Large phage antibody libraries are expected to be an efficient technology for obtaining humanized anti-tumor antibodies. However, few reports have been concerned about the selection strategies for intact cancer cells as targets. In this study, a 2x1011 large na?ve scFv library from blood B lymphocytes of normal adult donors and neonatal cord blood was constructed using the LoxP-cre system. Three human esophageal cancer cell lines were equally mixed for use as the target. These intact cells were divided into two groups, PF and NF, according to the treatment method of the cells (fixed with 2% PFA or not, respectively). Positive phage antibodies were identified following 4 panning cycles of adhesion, elution and amplification. Using a cell ELISA assay, it was found that the additional procedure of directly infecting XL1-Blue bacteria with the cell pellet following washing with an acidic solution can effectively decrease the loss of positive phage, yielding a positive screening rate of 11.6% (61/525). Most of the phage antibodies displayed binding activity with all three esophageal cancer cell lines. Moreover, other phages (such as NFc53a and NFc70a) appeared to be specific for certain cell lines. Regarding the method used to treat the target cells, both the positive screening rate and the genetic diversity of the antibody variable region were significantly higher in the NF group (12.9 and 71.4%, respectively) than in the PF group (10.5 and 14.2%, respectively). Immunohistochemical analysis demonstrated that the scFv phages have good specificity for esophageal cancer. This technology is helpful for developing small molecular tracers and targeted therapies for malignant tumors.     

人源性抗乳腺癌单链抗体库的筛选与初步鉴定

目的:本研究旨在已构建的大容量人源性抗乳腺癌噬菌体单链抗体库的基础上,筛选出高亲和力的特异性单链抗体(scFv)并对抗体基本特性进行初步鉴定。方法:以人乳腺癌细胞系MCF-7为靶标,经过4轮淘洗,筛选出高亲和力的特异性抗乳腺癌scFv,并对其结构序列进行分析;通过ELISA和Western blot方法,鉴定scFv的亲和力和特异性,以及其蛋白的基本表达情况。结果:成功构建具有高亲和力的抗乳腺癌单链抗体库,获得scFv的长度约为750 bp,ELISA证实所得抗体对乳腺癌细胞具有良好的亲和力和高度的特异性,IPTG诱导表达及Western blot结果显示,scFv为相对分子质量(Mr)30 000的可溶性蛋白。结论:本研究在已构建的大容量抗乳腺癌单链抗体库的基础上,筛选获得了高亲和力的抗乳腺癌单链抗体库。研究结果为进一步获得可应用于临床诊断和治疗的乳腺癌靶向性抗体奠定了良好的基础。     

A novel approach for immunization,screening and characterization of selected scFv libraries using membrane fractions of tumor cells

Isolation of cell-surface specific antibodies prerequisites the functional expressing of antigens on intact cells, which are maintained routinely by cell culturing. However, long-term culturing of tumor cells could alter their antigen expression patterns and stable fixation of whole cells is not guaranteed on plastic surfaces during stringent screening procedures. We prepared functional breast cancer cell-membrane fractions that express surface molecules in their native conformation. Specific binding phages were isolated from phage antibody libraries constructed from the spleen messenger RNA of mice immunized with breast cancer cell-membrane fractions. After negative selection on non-mammary carcinoma cells and four rounds of positive selection on breast carcinoma cell lines, phage antibodies were enriched that bound specifically to breast cancer cell lines as confirmed by phage enzyme linked immunosorbent assay (ELISA) using 96-well plates coated with breast cancer cell membranes. The isolated phage antibodies were highly specific for the breast cancer cell line 8701-BC but not on other carcinoma such as the Hodgkin-derived cell line L540Cy as demonstrated by ELISA and flow cytometry. This report describes a rapid and more versatile method for isolating antibody fragments compared to whole cell screening procedures. One single membrane preparation can be stored for at least 15 months at -80 degrees C and used to immunize mice or for screening of antibody libraries. The selection and screening strategy used should be generally applicable to identify novel cell-surface antigens and their corresponding antibodies.     

Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools

We defined phage genera by measuring genome relationships by the numbers of shared homologous/orthologous proteins. We used BLAST-based tools (CoreExtractor.vbs and CoreGenes) to analyze 55 fully sequenced bacteriophage genomes from the NCBI and EBI databases. This approach was first applied to the T7-related phages. Using a cut-off score of 40% homologous proteins, we identified three genera within the T7-related phages, redefined the phi29-related phages, and introduced five novel genera. The T7- and phi29-related phages were given subfamily status and named "Autographivirinae" and "Picovirinae", respectively. Our results confirm and refine the ICTV phage classification, enable elimination of errors in public databases, and provide a straightforward tool for the molecular classification of new phage genomes.     

中和抗肾小球基底膜抗体的单链抗体的筛选与鉴定

目的 制备人抗肾小球基底膜(GBM)抗体的特异性人源化单链可变区抗体.方法 采用噬菌体表面展示技术,获得一个与人抗GBM抗体结合活性较强的单链可变区抗体片段的阳性克隆,并对该克隆进行DNA序列测定分析.结果 对噬菌体单链可变区抗体库经过3轮筛选后,与第1轮相比富集了137倍.噬菌体抗体与人抗GBM抗体的结合活性其中有35株克隆ELISA的吸光度较高.对这些噬菌体抗体进行交叉反应后,确定其中有10株交叉反应较弱.确定1株(C31)阳性克隆提取质粒,进行DNA序列测定,大小为750 bp,并符合人源化单链可变区抗体的序列结构.结论 应用噬菌体展示技术成功获得人-抗GBM抗体的单链可变区抗体基因,为临床上治疗Goodpasture综合征奠定实验基础.     

噬菌体抗体库技术筛选结肠癌单抗MC3的抗独特型抗体

目的：获得结肠癌单抗MC3的噬菌体呈现型抗独特型抗体（anti-Id）。方法：分离经单抗免疫鼠脾脏的mRNA,经RT－PCR分别扩增抗体VH和VLDNA,进而连接形成ScFv DNA。将ScFv DNA与载体pCANTAB5E的连接产物转化于大肠杆菌TG1,经M13KO7挽救后获得噬菌体呈现型ScFv文库。以MC3对文库进行四轮筛选后,随机挑取克生经ELISA筛选呈现anti-Id ScFv的噬菌体克隆。结果：VH、VL和ScFv DNA分别约为340、320和750bp。抗体ScFv文库经四轮筛选后,在随机筛检的50个克隆中得到15个呈现antii-Id ScFv的菌体单克隆。结论：用噬菌体抗体库技术成功地获得了单抗MC3的anti-Id ScFv,从而为进行结肠癌重组anti-Id疫苗作用的研究提供了候选分子。     

抗HBsAg/RBC双特异噬菌体抗体的构建

目的 通过不同外壳蛋白的展示，构建抗HBsAag/RBC双特异噬菌体抗体。方法通过DNA重组技术，将抗HBsAg Fab与噬菌体基因8融合，抗RBC ScFv与噬菌体基因3融合，这2种融合基因以不同的启动子驱动，并克隆于同一噬菌体表达载体上，得到双特异噬菌体抗体表达载体，转化大肠杆菌后获取噬菌体抗体上清，用ELISA和红细胞凝集试验检测其双特异活性。结果ELISA和RBC凝集实验证明，本方法可形成双特异噬菌体抗体，可使含有HBsAg的RBC悬液产生凝集。用展示性能得到提高的蛋白8变种取代野生型蛋白8可提高双特异噬菌体抗体的活性。结论 HBsAb和RBC两种抗体分子能够通过不同噬菌体外壳蛋白同时展示于同一噬菌体表面，形成双特异噬菌体抗体。可用于人外周血HBsAg的快速血凝法检测。     

用噬菌体肽库筛选IBDV单克隆抗体的模拟表位

目的:筛选法氏囊病毒(IBDV)单克隆抗体所针对的模拟抗原表位。方法:以纯化的3株IBDV单克隆抗体为靶分子用噬菌体12肽库筛选相应抗原模拟表位;应用间接和竞争ELISA鉴定所筛选的阳性样品;最后对与抗体高亲和结合的噬菌体进行测序分析。结果:经过四轮淘洗,随机挑取30个克隆经间接ELISA鉴定,发现其中22个克隆结合活性较高。进一步应用竞争ELISA,获得14个噬菌体克隆,其抑制率高达50%以上。对这些噬菌体进行DNA测序,并分析比对了IBDV相应序列,确定了3个抗原模拟表位。结论:通过噬菌体随机肽库成功筛选出3个IBDV模拟表位,为进一步研究IBDV抗原性质奠定了基础。     

Screening of scFv-displaying phages recognizing distinct extracellular domains of EGF receptor by target-guided proximity labeling method

We recently constructed the scFv-displaying phage library with extremely high repertoire and have successfully utilized for screening scFv antibodies against various proteins, polysaccharides and glyco-lipids. Here, we developed a new screening strategy to isolate scFv antibodies against cell surface EGF receptor (EGFR). For this, we applied two slightly different methods of "target-guided proximity labeling," such as Proximity selection (ProxiMol) method and a new sulfo-SBED labeling method with the aide of monoclonal anti-human EGFR antibody B4G7 as a guide molecule. ProxiMol method relies on the Biotin-labeling of scFv-displaying phages that bound to the target in a vicinity of 100? from the guide molecule, whereas sulfo-SBED method transfers Biotin to scFv-displaying phages, which bound to the target in a distance of 20 ?. After two rounds of panning on the EGFR-overexpressing A431 cells starting from approx. 1 × 1012 pfu, 47 each of Biotin-labeled scFv-displaying phages were recovered using Streptoavidin-coated magnetic beads, and among them total 11 scFv-phages were found to be definitely positive for binding to A431 cell surface by ELISA assay. Restriction mapping and sequencing analysis of these scFv-phage DNAs revealed that they encode 4 different scFv-nucleotide sequences in total. Immuno-fluorescent microscopy provided evidence that these 4 scFv antibodies bind specifically to EGFR on the A431 cells, showing slightly different staining patterns. Thus, "target-guided proximity labeling" methods were powerful for isolating scFv-displaying phages that recognize distinct extracellular domains of the target receptor. This novel screening strategy could be applicable to many other cell surface antigens and receptors.     

抗丙型肝炎病毒非结构蛋白NS3单链抗体的制备及免疫组织化学研究

目的 研制抗丙型肝炎病毒（HCV）非结构蛋白NS3的人源噬菌体单链抗体,并探讨其在临床诊断中的应用价值。方法 以重组的丙型肝炎病毒非结构蛋白NS3为固相抗原,利用亲和筛选的原理,从噬菌体抗体库中经过5轮“吸附－洗脱－扩增”的筛选过程及酶联免疫吸附试验（ELISA）、斑点免疫杂交试验和DNA序列分析,获得HCV NS3的人源单链抗体;用该抗体与不同来源的HCV NS3抗原进行反应;对10例石蜡包埋的丙型肝炎患者肝组织进行免疫组化鉴定。结果 ELISA结果表明,所制备的HCV NS3人源单链抗体能与不同来源的HCV NS3抗原特异性结合（吸光度A值为1.38）;免疫组化结果表明,该抗体能够特异性识别丙型肝炎患者肝组织HCV NS3抗原,与正常肝组织及乙型肝炎病毒（HBV)的表面抗原均无交叉反应。结论 此法制备的单链抗体亲和性好,特异性强,且制备方法简便,周期短,可用于HCV NS3病原的检测。     

Unifying classical and molecular taxonomic classification: analysis of the Podoviridae using BLASTP-based tools

We defined phage genera by measuring genome relationships by the numbers of shared homologous/orthologous proteins. We used BLAST-based tools (CoreExtractor.vbs and CoreGenes) to analyze 55 fully sequenced bacteriophage genomes from the NCBI and EBI databases. This approach was first applied to the T7-related phages. Using a cut-off score of 40% homologous proteins, we identified three genera within the T7-related phages, redefined the φ29-related phages, and introduced five novel genera. The T7- and φ29-related phages were given subfamily status and named “Autographivirinae” and “Picovirinae”, respectively. Our results confirm and refine the ICTV phage classification, enable elimination of errors in public databases, and provide a straightforward tool for the molecular classification of new phage genomes.     

构建预设CDR3基因噬菌体抗体库筛选抗人整合素ανβ3 mAb的人源化Fab

目的:构建预设CDR3基因的噬菌体抗体库,通过抗原表位导向选择方法筛选抗人整合素ανβ3单克隆抗体(mAb)人源化Fab。方法:将鼠mAbLCDR3重组到人轻链可变区文库中并与人/鼠嵌合重链Fd基因配对构建杂合噬菌体抗体库,用固相人整合素ανβ3抗原筛选人源化轻链基因。再用所获人轻链基因与移植有鼠mAbHCDR3的人重链Fd基因配对构建人源噬菌体抗体库,筛选人源化Fab。结果:分别构建了库容为2.1×106和2×107的杂合噬菌体抗体库和人源噬菌体抗体库,筛选到3株人源Fab克隆。经间接ELISA及竞争抑制ELISA证实,能特异结合整合素ανβ3抗原,其中人源D5株Fab克隆的基因序列表明,人轻链可变区基因属VKIII亚群,人重链可变区基因属VH1亚群。结论:利用噬菌体抗体库技术,成功地进行了鼠抗人整合素ανβ3mAbE10人源化的改造,为进一步临床应用奠定了基础。     

BSA-Y-DOTA免疫噬菌体Fab抗体库的构建及鉴定

目的 :构建钇 十二烷四乙酸 (Y DOTA)免疫噬菌体Fab抗体库。方法 :将牛血清白蛋白 (BSA)与DOTA交联 ,并与金属Y鳌合制备成BSA Y DOTA ,用其免疫BALB/c小鼠。检测抗血清的滴度后 ,分离抗体阳性的小鼠脾淋巴细胞 ,提取总RNA。利用RT PCR扩增全套重链Fd和轻链基因 ,依次插入经改造的噬菌体载体pComb3M的相应酶切位点 ,构建成Fab噬菌体抗体库。用酶切、序列测定及ELISA等方法 ,对重组率、多样性及Fab的展示情况进行鉴定。结果 :成功得制备了BSA Y DOTA交联物 ,并获得较好的免疫效果。免疫小鼠的全套重链Fd片段和轻链均得到正确扩增 ;Fd片断和轻链基因均插入到载体pComb3M中 ;Fab抗体库的库容量达 8× 10 7;重组率约为90 % ,且具有良好的抗体基因多样性。另外 ,Fab片段也被展示于噬菌体表面。结论 :成功地构建了半抗原Y DOTA的Fab噬菌体抗体库 ,为筛选Y DOTA特异性抗体奠定了基础     

人源性噬菌体抗体库的构建及抗amylin抗体的初步筛选

目的:构建人源性噬菌体抗体库,从中筛选胰淀素(amylin)单克隆抗体(mAb),测定其特异性及抗原结合活性。方法:从正常健康人的外周血淋巴细胞中提取总RNA,用RT-PCR方法扩增人免疫球蛋白Fd段和轻链基因,构建噬菌体抗体库。酶切和PCR鉴定后,阳性克隆进行DNA测序分析。用人amylin抗原对抗体库进行筛选富集,将得到的阳性克隆进行Phage-ELISA鉴定,结果进行统计学分析。结果:最终构建的抗体库库容约为0.8×108,酶切鉴定显示有插入片段,抗体库重组率为70%。阳性克隆进行DNA测序证实所获基因为人免疫球蛋白可变区基因。以amylin抗原进行3轮筛选,抗体库得到特异性富集。阳性克隆进行Phage-ELISA检测证实有良好的抗胰淀素抗原的特异性。结论:成功构建了一个人源性噬菌体抗体库,为从中获得人源抗amylin的mAb奠定了实验基础。     

丙型肝炎病毒非结构蛋白4A人源单链可变区抗体的筛选与鉴定

目的 利用噬菌体表面展示技术,从半合成人源化单链可变区抗体库中筛选、鉴定丙型肝炎病毒（ＨＣＶ）非结构蛋白ＮＳ４Ａ的单链可变区抗体（ＳｃＦｖ）及其编码基因,为抗ＨＣＶ的细胞内免疫基因治疗研究开辟新途径。方法 采用噬菌体表达展示技术,以重组的ＨＣＶ非结构蛋白ＮＳ４Ａ为包被抗原,从噬菌单链可变区抗体库中经过３轮“吸附－洗脱－扩增”筛选过程,获得抗原结合活性较强的ＨＣＶ ＮＳ４Ａ人单链可变区抗体段阳性克隆     

SLE患者血清中病原体相关噬菌体7肽的检测

目的用噬菌体7肽库筛选系统性红斑狼疮（SLE）患者血清特异性抗体,测序分析其实际意义。方法先用30例正常人混合血清与噬菌体7肽库反应,未与正常人白清结合的7肽再与30例SLE患者混合血清结合,获得SLE血清特异性结合的噬菌体克隆。用患者混合血清进行Dot—ELISA实验鉴定获得的噬菌体克隆,进一步用SLE患者及正常人血清各12例筛选阳性噬菌体的混合克隆,确定阳性噬菌体克隆与个体血清之间的结合情况,并对最终鉴定的噬菌体克隆进行测序与比对分析。结果混合的阳性噬菌体克隆与SLE患者个体血清反应阳性率明显高于其与正常人血清的反应率;序列分析显示阳性噬菌体克隆的抗原表位与杆菌、球菌、弧菌等微生物有同源性,与裂殖酵母属、链球菌属、立克次（氏）体属等有100％同源性,与人类抗原表位无关。结论SLE患者血清中存在与病原体抗原表位结合的抗体成分,提示SLE可能与病原体感染有关。     

Conservation of the N-terminus of some phage tail proteins

Summary. To study the interaction between lipopolysaccharide and protein, a comparative approach was employed using seven Salmonella enterica serovar Typhimurium typing phages as the protein model systems. This interaction has been studied in detail in the Salmonella enterica serovar Typhimurium phage P22 system and involves only the viral tailspike protein. Similarity between these phages and phage P22 was monitored in this Report by assaying restriction endonuclease digestions, capsid size, reactivity to the P22 tailspike protein monoclonal antibody, mAb92, which reacts with the N-terminus of the P22 tail protein and the ability to produce a PCR fragment using primers made to the ends of the P22 tailspike gene. The data indicate that tailspike similarity exists between most of these phages and a scheme reclassifying them is presented and that the N-terminus of the P22 tailspike protein may be a motif for many phage systems and may serve as a aid in the taxonomy of phages. The data suggest a classification scheme in which the N-terminus of some tailspike proteins (head-binding region in some tail proteins) may play a critical element role in the classification of Salmonella viruses.     

Characterization of a newly discovered Mu-like bacteriophage, RcapMu, in Rhodobacter capsulatus strain SB1003

The α-proteobacterium Rhodobacter capsulatus is a model organism for the study of bacterial photosynthesis and the bacteriophage-like gene transfer agent. Characterization of phages that infect Rhodobacter is extremely rare, and scarce for the α-proteobacteria in general. Here, we describe the discovery of the only functional Mu-like transposing phage to have been identified in the α-proteobacteria, RcapMu, resident in the genome-sequenced R. capsulatus SB1003 strain. RcapMu packages ~ 42 kb of total DNA, including < 3 kb of host DNA with no conserved motifs, indicative of replicative transposition with little insertion site preference. The phage genome contains 58 ORFs with comparable organization to known transposable phages. Shotgun proteomics of purified RcapMu particles detected all proteins with predicted structural functions as well as seven hypothetical proteins. Overall, comparison of RcapMu to enterobacteria phage Mu and other Mu-like phages revealed only regional homology to these phages, providing further evidence for the promiscuous, modular nature of bacteriophage evolution.