PSMA mimotope isolated from phage displayed peptide library can induce PSMA specific immune response

Prostate-specific membrane antigen (PSMA) is a cell surface glycoprotein expressed predominantly in prostate secretory acinar epithelium and prostate cancer cells as well as in several extraprostatic tissues.Mouse monoclonal antibody 4G5 specific to the extracellular domain of PSMA was used to screen two phage displayed peptide libraries (9aa linear and 9aa cys library).Three 4G5-reactive phagotopes were identified.Sequence analysis of isolated clones demonstrated that the interaction motif“VDPA/SK” has high homology to 719-725aa on PSMA.Immunohistochemical staining of the prostate cancer sample with the psma-mimic phagotope(mimotope) immunized serum antibodies demonstrate that the mimotope isolated from the phage displayed peptide libraries can induce PSMA specific immune response in vivo.     

Topological mimicry and epitope duplication in the guanylyl cyclase C receptor.

Guanylyl cyclase C (GCC) is the receptor for the gastrointestinal hormones, guanylin, and uroguanylin, in addition to the bacterial heat-stable enterotoxins, which are one of the major causes of watery diarrhea the world over. GCC is expressed in intestinal cells, colorectal tumor tissue and tumors originating from metastasis of the colorectal carcinoma. We have earlier generated a monoclonal antibody to human GCC, GCC:B10, which was useful for the immunohistochemical localization of the receptor in the rat intestine (Nandi A et al., 1997, J Cell Biochem 66:500-511), and identified its epitope to a 63-amino acid stretch in the intracellular domain of GCC. In view of the potential that this antibody has for the identification of colorectal tumors, we have characterized the epitope for GCC:B10 in this study. Overlapping peptide synthesis indicated that the epitope was contained in the sequence HIPPENIFPLE. This sequence was unique to GCC, and despite a short stretch of homology with serum amyloid protein and pertussis toxin, no cross reactivity was detected. The core epitope was delineated using a random hexameric phage display library, and two categories of sequences were identified, containing either a single, or two adjacent proline residues. No sequence identified by phage display was identical to the epitope present in GCC, indicating that phage sequences represented mimotopes of the native epitope. Alignment of these sequences with HIPPENIFPLE suggested duplication of the recognition motif, which was confirmed by peptide synthesis. These studies allowed us not only to define the requirements of epitope recognition by GCC:B10 monoclonal antibody, but also to describe a novel means of epitope recognition involving topological mimicry and probable duplication of the cognate epitope in the native guanylyl cyclase C receptor sequence.     

人癌胚抗原单链抗体基因的构建和筛选

从分泌抗癌胚抗原(carcinoembryoni antigen, CEA)单抗的杂交瘤细胞株C50中提取总RNA, 逆转录成cDNA, PCR扩增分别得到抗体轻、重链可变区基因, 再利用两对PCR引物合成和扩增得到全单链抗体基因. 将含轻、重链可变区序列的DNA片段克隆于含噬菌体基因Ⅲ的噬菌粒pCANTAB5. 重组克隆在噬菌体表面表达基因Ⅲ与单链抗体的融合蛋白. 表达具抗原结合活性的单链抗体的重组噬菌体可以通过亲和筛选的方法筛选得到并富集. 利用该方法我们可以从许多分泌不同抗体的杂交瘤细胞RNA中快速克隆和筛选功能性抗体可变区基因.     

龋齿DNA疫苗(PGJAP)中污染大肠杆菌噬菌体的检测方法的建立

龋齿DNA疫苗工程菌采用的大肠杆菌DH-5α在生产过程中极易污染大肠杆菌噬菌体,所以应对原始菌种、主菌种和工作菌种及大量生产时的发酵液作大肠杆菌噬菌体检测。用大肠杆菌噬菌体VCSM13为标准噬菌体株,对大肠杆菌C3000和DH-5α分别作噬菌斑检测和pfu值计算,验证并确定以VCSM13作为标准噬菌体株,C3000作为检测菌株,对龋齿DNA疫苗原始菌种、主菌种(第一代)、工作菌种(2007001)和其发酵液(200703)分别作噬菌体检测,并建立了检测大肠杆菌噬菌体的直接噬菌斑法。结果显示VCSM13在DH-5α的噬菌斑计数为76,pfu/ml为7.6×1013,C3000的噬菌斑计数为81,pfu/ml为8.1×1013,龋齿DNA疫苗的原始菌种、主菌种、工作菌种和发酵液,噬菌斑计数全部为0。Pfu也为0。阳性对照为74,pfu/ml是7.4×1013,阴性对照为0。通过对阳性对照样本作增殖法试验及挑斑接种验证后,证明此法操作简单,灵敏度高。     

人源单克隆抗人免疫缺陷病毒1型抗体Fab段基因的获得

应用噬苏体抗体库技术有效地筛选出了多株抗ＨＩＶ－１人源单克隆抗体。以逆转录聚合酶链反应（ＲＴ－ＰＣＲ）从ＨＩＶ－１感染者外周血淋巴细胞中扩增抗体轻重链可变区基因,插入载体ｐＣＯＭＢ３,建立噬菌体抗体库。分别以ＨＩＶ－１ｇｐ１２０和ｇｐ１６０为固相抗原,经过多轮筛选,从中获得了多株抗ＨＩＶ－１ｇｐ４１、ｇｐ１２０和ｇｐ１６０的单克隆抗体Ｆａｂ段基因。抗ＨＩＶ特异性噬菌体抗体随抗体库的筛选高度富集,抗     

从混合的噬菌体抗体库中直接筛选高亲和性的抗肿瘤坏死因子-α人源单克隆抗体

采用噬菌体抗体库技术,从未经免疫的混合人噬菌体抗体库直接筛选抗ＴＮＦα的人源单克隆抗体,３种不同的检测手段表明,经过５轮的筛选,能够与ＴＮＦα结合的噬菌体抗体得到了有效的富集。对所获得的噬菌体抗体克隆进行的初步鉴定表明,７个噬菌体抗体克隆能有效地与人重组ＴＮＦα结合,并且这种结合具有特异性,由这７个噬菌体抗体克隆制备的抗体Ｆａｂ片段也能有效地与ＴＮＦα结合。由此,我们通过噬菌体抗体技术直接获得了ＴＮＦα的人源单克隆抗体,这为进一步制备具有临床应用前景的人源抗ＴＮＦα单克隆抗体打下了基础。     

A型肉毒毒素表位多肽免疫和人源噬茵体单链抗体文库的构建

采用表位合成多肽免疫获得编码人源抗体的基因材料,抗体轻重链可变区基因通过剪接重叠延伸(SOE)技术进行组装后,亚克隆入噬菌体粒中构建免疫库。以重组制备的A型肉毒毒素保护性抗原为配体筛选结合子,ELISA鉴定,获得具有较高抗原结合力的噬菌体克隆,进行基因测序和家族定位分析。结果显示,表位合成多肽免疫,成功构建了库容大于10^8的重组抗体库。体外3轮免疫淘筛,筛选到人源A型肉毒抗毒素克隆,基因结构分析表明,其中ScFvB17全长750bp,可编码250个氨基酸残基：VH属于VH4家族,369bp,编码123个氨基酸残基;Vκ属于κ链Ⅱ家族,336bp,编码112个氨基酸残基。基因的同源分析表明,这是一株特异的单链抗体新基因。     

Construction of recombinant monoclonal antibodies from a chicken hybridoma line secreting specific antibody

The chicken is a useful animal for the development of the specificantibodies against the mammalian conserved proteins. We generated twotypes of recombinant chicken monoclonal antibodies (mAbs), using a phagedisplay technique from a chicken hybridoma HUC2-13 which secreted themAb to the N-terminal of the mammalian prion protein (PrP). Althoughthe mAb HUC2-13 is a useful antibody for the prion research, thehybridoma produces a low level of antibody production. In order to producea large amount of the mAb, we have constructed a single chain fragmentvariable region (scF_V) mAb by using the variable heavy(V_H) and light (V_L)genes which were amplified by using the two primer pairs and theflexible linker. The two phage display mAbs (HUC2p3 and HUC2p5)expressed on a M13 filamentous phage and their soluble type mAbs(HUC2s3 and HUC2s5) were reacted with the PrP peptide antigen in theELISA. In the Western blot analysis, the mAbs HUC2p3 and HUC2s3 wereas reactive to PrP^c from mouse brains as the mAb HUC2-13 was. The nucleotide sequences of V_H and V_L genes from HUC2-13 and the two cloneswere identical except for only one residue. These results indicate that themethods presented here provide an effective tool for the improvement ofthe low levels of antibody production in the chicken hybridoma system.     

核受体PPARγ结合小肽的筛选及其功能

为筛选与核受体过氧化物酶体增殖物激活受体γ(PPARγ)结合的功能短肽,在大肠杆菌BL21(DE3)中表达PPARγ配体结合域(LBD)的融合蛋白,并利用Ni2+-NTA离子交换树脂对表达蛋白进行纯化.以此纯化蛋白为靶,采用固体包被法对噬菌体展示随机十二肽库及环七肽库进行亲和筛选.经ELISA法鉴定特异结合的高亲和力阳性噬菌体单克隆并测序.同时利用PPARγ的配体rosiglitazone与噬菌体小肽进行竞争性结合抑制实验.最终获得与PPARγ-LBD高亲和力的十二肽3个,环七肽5个,分别含LXXLL和DXXRW(其中X为非特异氨基酸残基)保守序列.Rosiglitazone不影响噬菌体小肽与靶蛋白的结合,说明获得与配体rosiglitazone结合位点不同的目的肽.