人源性老年痴呆单链噬菌体抗体库的构建

目的 构建人源性老年痴呆(AD)单链噬菌体抗体库,为进一步筛选AD相关抗原的人源性抗体奠定基础.方法 从200 ml AD病人的外周血中分离B淋巴细胞,提取总RNA,经逆转录合成总cDNA.以PCR技术,利用特定的引物分别扩增出人抗体的重链和轻链可变区基因片段(VH、VL),并分别克隆入噬菌粒pDAN5中,构建出含人单链抗体可变区(scFv)基因序列的pDAN5克隆载体,电转化感受态大肠杆菌XLI-Blue后,经辅助噬菌体M13K07超感染后回收全部重组噬菌体,构建成初级噬菌体抗体库.从抗体库中随机挑选数个克隆,提取质粒,PCR扩增目的片段后,用内切酶BstN Ⅰ消化,每个克隆经消化后的DNA指纹印迹用琼脂糖凝胶电泳分析,评价抗体库的多样性.结果 所有抗体VH和VL基因片段均得到了扩增并成功克隆及转化,经辅助噬菌体感染后,构建成初级抗体库.BstN Ⅰ消化后的DNA指纹图谱显示各克隆抗体基因各不相同,经计算构建的噬菌体抗体库容量为1.5×106.结论 利用噬菌体抗体库技术成功构建了AD病人的单链可变区噬菌体抗体库.     

特异性抗肝癌噬菌体单链抗体库的构建及鉴定

噬菌体单链抗体（ＳｃＦｖ）弥补了传统ＭｃＡｂ制备繁琐、抗原性强、穿透力弱等不足,为ＨＣＣ临床和基础研究提供新的手段。我们用基因工程技术和噬菌体表面呈现技术构建和筛选特异性抗ＨＣＣ噬菌体ＳｃＦｖ库。 １．材料与方法：（１）抗ＨＣＣ噬菌体ＳｃＦｖ库的构建：应用ＨＣＣ细胞常规免疫ＢＡＬＢ／Ｃ小鼠,从其脾脏提取ｔＲＮＡ并纯化ｍＲＮＡ,逆转录合成ｃＤＮＡ,ＰＣＲ扩增抗体轻链和重链可变区,应用ｌｉｎｋｅｒ－（Ｇｌｙ４Ｓｅｒ）３装配ＳｃＦＶ基因,在其５’端和３’端分别引入内切酶ＳｆｉＩ和ＮｏｔＩ酶切位点,连接…     

抗甲状腺刺激性免疫球蛋白单链抗体噬菌体抗体库的初建

目的 建立抗甲状腺刺激性免疫球蛋白单链抗体噬体抗体库,为进一步获得功能性抗体可变区基因,制备单链抗体和单链抗体－辣根过氧化物酶融合蛋白,用于甲状腺自身免疫性疾病的治疗和检测打下基础,方法 利用ＲＴ－ＰＣＲ技术从分泌抗甲状腺刺激性免疫球蛋白的杂交瘤细胞中扩增出轻重链可变区基因,与噬粒表达载体ｐ３ＳＣＭＨ连接,转化大肠杆菌,以甲状腺刺激性免疫球蛋白阳性患者血清ＩｇＧ为抗原对表达的噬菌体抗体进行筛选,结     

人源性阿尔茨海默病噬菌体单链抗体库的构建

目的构建人源性阿尔茨海默病(AD)噬菌体单链抗体(scFv)库,为筛选β淀粉样蛋白(Aβ1-42)的人源性特异性抗体奠定基础。方法采集18例AD患者的外周血40ml,提取总RNA,应用RT-PCR法得到人抗体可变区重链(VH)和可变区轻链(VL)基因。将VH和VL由连接肽连接得到scFv片段,将所得片段双酶切后,克隆至pCANTAB5E噬菌体载体,大肠埃希菌TG1感受态细胞经电击转化,辅助噬菌体M13K07拯救后构建scFv型噬菌体抗体库。结果总RNA经逆转录PCR扩增VH和VL可变区基因的凝胶电泳显示,PCR产物长度分别为360bp和300bp,其连接形成的scFv片段长度为750bp,最终构建了库容为2.4×109的scFv库。BstNⅠ酶切鉴定构建的scFv库,经凝胶电泳可见,酶切片段长度差异性大,显示scFv库具有良好的多样性。结论成功构建人源性AD噬菌体scFv库,为进一步筛选Aβ特异性抗体,继而为AD的治疗研究奠定基础。     

人源性抗-HBc单链噬菌体抗体库的构建

目的 构建人源性单链噬菌体抗体库，为筛选人源性抗—HBc单链抗体奠定基础。方法 利用逆转录—聚合酶链反应(RT—PCR)和噬菌体表面展示技术，直接从乙肝病毒核心抗体(抗—HBc)阳性患者淋巴细胞中提取总RNA，逆转录成cDNA；合成全套人抗体可变区引物扩增抗体可变区基因，并将重、轻链可变区基因进行拼接装配成单链抗体(ScFv)基因，重组于噬菌粒载体叶pHEN1，转化抑制型大肠埃希菌E.coliTG1，以辅助噬菌体援救后，构建成人源性单链噬菌体库。结果 成功地构建了人源性抗—HBc单链噬菌体库，库容量达10^6。结论 利用RT—PCR和噬菌体表面展示技术可以成功构建人源性单链抗体库，并达到建库标准，可进一步从中筛选人源性单链抗体。     

抗HBV Pre S2单链噬菌体抗体基因在噬菌体载体中的克隆及初步表达

目的 利用基因重组技术构建ＨＢＶ前Ｓ２单链抗体基因并使其在大肠杆菌中永生化和表达活性的抗ＨＢＶ前Ｓ２单链抗体（ＳｃＦｖ），方法和结果 利用基因重组技术，在构建抗ＨＢＶ前Ｓ２３Ｂ９ＳｃＦｖ基因的基础上，在此基因中引入限制性同切酶位点，克隆到噬菌体表达载体－ｐＣＡＮＴＡＢ５噬菌粒中，经亲和筛选得到２株阳性重组克隆，诱导表达产物在特异性阻断ＥＬＩＳＡ实验中具有一定阻断亲本３Ｂ９单抗与ＨＢＶ前Ｓ２抗原的结     

抗-HBs的人源性噬菌体抗体库的构建

目的构建含抗－ＨＢｓ的人源性噬菌体抗体库。方法用ＲＴ－ＰＣＲ技术,从自然感染的抗－ＨＢｓ阳性的两名献血员的外周血淋巴细胞中,扩增出人抗体轻链Ｋ基因和重链Ｆｄ基因,将二基因先后克隆人ＰＣｏｍｂ３Ｈｓｓ载体中,转化大肠杆菌,再用辅助噬菌体感染。结果构建了库容量为１５×１０５的轻链基因抗体库,轻链基因插人率为５７％和库容量为５×１０５的人Ｆａｂ体库,轻、重链基因插人率为５０％。经辅助噬菌体感染,得到噬菌体滴度为５×１０１４ＣＦＵ／ｍｌ的人源性噬菌体抗体库。结论成功构建了合抗－ＨＢｓ的人源性噬菌体抗体库,为进一步筛选ＨＢｓＡｇ的人Ｆａｂ噬菌体抗体奠定了基础。     

从人源性噬菌体抗体库中筛选人抗HBsAg的Fab噬菌体抗体

目的从人源性噬菌体抗体库中筛选人抗HBsAgFab噬菌体抗体。方法用固相化HBsAg对所构建的噬菌体抗体库进行三轮淘筛,从中筛选人抗HBsAgFab噬菌体抗体。结果第三轮淘筛后洗脱下来的噬菌体,较第一轮增加80倍,含有抗体轻链基因和重链基因的克隆,也由淘筛前的17%增至100%,经ELISA证实,筛选到的抗HBsAg噬菌体抗体对HBsAg具有良好的结合活性,而与牛血清白蛋白和HAVAg等无关抗原均不反应。结论HBsAg对抗体库的淘筛,富集了表面呈现抗HBsAg的单克隆抗体的噬菌体。     

噬菌体抗体库的构建及人源抗内毒素类脂A抗体的筛选

目的寻找一种治疗内毒素血症及其并发症较为有效的途径.方法从感染革兰阴性杆菌J7患者体内含有内毒素类脂A抗体的淋巴细胞中提取mRNA,经反转录再从IgG重链Fd两端及轻链通用引物分别扩增Fab基因片段,依次插入到噬菌体载体pCOMR3中,电穿孔转入大肠杆菌XL-blue,经辅助病毒VCSM13感染,重组噬菌体溶源裂解.结果Fab抗体表达于噬菌体CPⅢ的N端,噬菌体抗体库的容量为4.8×106,筛选出抗内毒素类脂A抗体,经LPS蛋白对噬菌体抗体库进行3轮淘选,使抗内毒素类脂A的特异性抗体得到100倍的富集.结论通过直接和竞争ELISA实验筛选出3株结合活性好的特异性抗体,为下一步应用研究奠定了基础.     

丙型肝炎病毒非结构蛋白3人源单链抗体的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（ＨＣＶ）非结构蛋白（ＮＳ）３的人单链抗体（ＳｃＦｖ）,以解决鼠单抗蝗免疫原性问题,为ＨＣＶ的基因治疗研究开辟新途径。方法 采用噬菌体表面展示技术,以重组的ＨＣＶＮＳ３为包被抗原,从噬菌体单链楞变区抗体库中经过５轮“吸附－洗脱－扩增”筛选过程,获得抗原结合活性较强的ＨＣＶＮＳ３人单链抗体（ＳｃＦｖ片段）阳性克隆,并对其进行免疫体及序列测定。结果 筛选出来的ＳｃＦｖ片段     

An hepatitis B virus surface antigen specific single chain of variable fragment derived from a natural immune antigen binding fragment phage display library is specifically internalized by HepG2.2.15 cells

Hepatitis B virus surface antigen (HBsAg), a specific antigen on the membrane of hepatitis B virus (HBV)-infected cells, provides a perfect target for therapeutic drugs. In order to mediate successful targeted delivery of these therapies, it is essential to have antibodies that recognize HBsAg with high specificity and affinity. In this report, we constructed a natural immune antigen binding fragments (Fab) antibody phage display library against HBsAg and after three rounds of panning, five Fab fragments with significant HBsAg binding ability were selected and analysed. DNA sequencing revealed that all the light chains had the same sequence, while all the Fd genes exhibited different sequences. For further application, all of the Fab antibodies were reconstructed into single chain antibodies (scFvs) and expressed in Escherichia coli BL21 cells. Indirect enzyme-linked immunosorbent assay analysis demonstrated that all five scFvs maintained a high affinity for HBsAg and could bind HBsAg on the membrane of HBV-infected cells. Indirect fluorescent staining analysis revealed that one of the scFvs (scFv15) could be internalized into HBsAg-positive HepG2.2.15 cells through clathrin-mediated endocytosis pathway. The internalizing scFv15 antibody would have great potential for the targeted delivery of therapeutics to HBV-infected cells.     

噬菌体展示技术构建人源性抗乙型肝炎表面抗原单链抗体库

[目的]应用噬菌体展示技术构建人源性抗乙型肝炎(乙肝)表面抗原(HBsAg)单链抗体(ScFv)库。[方法]提取经免疫具有乙肝表面抗体的正常人淋巴细胞总RNA,采用反转录、触减PCR扩增人免疫球蛋白G(IgG)重链可变区基因(VH)和轻链可变区基因(VL),用Linker连接肽经重叠PCR将VH和VL基因连接成VH-Linker-VL形式的ScFv基因。将ScFv与pCANTAB-5E载体通过相同的酶切位点连接后,转化大肠杆菌TG1,经M13K07辅助噬菌体拯救,构建成全套人源性抗HBsAg ScFv库。[结果]成功构建了人源性抗HBsAg噬菌体ScFv库。[结论]人源性HBsAg噬菌体ScFv库的构建,为进一步筛选特异性高,具有治疗作用的乙肝抗体奠定基础。     

人源性抗日本血吸虫噬菌体抗体库的构建及初步鉴定

目的利用噬菌体抗体展示技术构建人源性抗日本血吸虫Fab段噬菌体抗体库并进行初步鉴定。方法RT-PCR从对血吸虫感染有一定抗性成年人外周血淋巴细胞中扩增出入IgG轻链和重链Fd段基因片段，将其克隆入PComb3中．构建人源性Fab段噬菌体抗体库。并用限制性内切酶酶切、序列分析、DOT-BLOT以及SDS-PAGE等方法对噬菌体抗体库进行初步鉴定。结果噬菌体抗体库的滴度为6．2×10^11／L。库容量为1．2×10^7。酶切鉴定结果显示噬菌体抗体库轻重链重组率达到66．6％，测序结果表明克隆入PComb3的是人免疫球蛋白轻链和重链基因,DOT-BLOT实验证明该抗体库表达了人源性的抗体，而SDS-PAGE证实了免疫球蛋白Fab段的可溶性表达。结论成功地构建了人源性抗日本血吸虫Fab段噬菌体抗体库，为筛选人源性抗日本血吸虫的抗体、研究这些抗体的特性和功能奠定了基础。     

Platelet-reactive IgG antibodies cloned by phage display and panning with IVIG from three patients with autoimmune thrombocytopenia

Autoimmune thrombocytopenic purpura (AITP) is a severe disease in children with a still unknown aetiology. It is not known why AITP can either be transient and self limiting or become chronic. The beneficial use of intravenous immunoglobulins (IVIG) in certain groups of AITP patients has been proven. It is, however, not clear how IVIG functions. To analyse patient-derived monoclonal IgG platelet autoantibodies that interact with IVIG in an anti-idiotypic manner, the combinatorial antibody phage display system was applied. From three different patients a large number of clones specifically reacting with IVIG molecules were derived. Many of these IVIG binders also reacted strongly with platelets in ELISA and FACS, in contrast to IVIG binders derived from a healthy individual. The heavy and light chain variable regions were sequenced and compared with each other and with databases. In all three AITP patients clones with a striking complementarity-determining region (CDR) sequence homology to each other and to many of the known anti-platelet antibodies were observed. Selected Fab-phages representing the characteristic variable regions that occurred in the investigated patients with AITP may now be used to clone potentially regulatory anti-idiotypes from healthy donors by phage display.     

Preparation of single chain variable fragment of MG(7) mAb by phage display technology

AIM: To develop the single chain variable fragment of MG MG(7)murine anti-human gastric cancer monoclonal antibody using the phage display technology for obtaining a tumor-targeting mediator. METHODS: mRNA was isolated from MG MG(7) producing murine hybridoma cell line and converted into cDNA. The variable fragments of heavy and light chain were amplified separately and assembled into ScFv with a specially constructed DNA linker by PCR. The ScFvs DNA was ligated into the phagmid vector pCANTAB5E and the ligated sample was transformed into competent E. Coli TG1. The transformed cells were infected with M13K07 helper phage to form MG MG(7) recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by means of bacterial colony count and restriction analysis. After two rounds of panning with gastric cancer cell line KATO III of highly expressing MG(7)-binding antigen, the phage clones displaying ScFv of the antibody were selected by ELISA from the enriched phage clones. The antigen-binding affinity of the positive clone was detected by competition ELISA. HB2151 E. Coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the MG(7) ScFv. ELISA assay was used to detect the antigen-binding affinity of the soluble MG(7) ScFv. Finally, the relative molecular mass of soluble MG(7) ScFv was measured by SDS-PAGE. RESULTS: The V(H), V(L) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 X 10(6) and 8 of 11 random clones were recombinants. Two phage clones could strongly compete with the original MG(7) antibody for binding to the antigen expressed on KATO III cells. Within 2 strong positive phage clones, the soluble MG(7) ScFv from one clone was found to have the binding activity with KATO III cells. SDS-PAGE showed that the relative molecular weight of soluble MG(7) ScFv was 32. CONCLUSION: The MG(7) ScFv was successfully produced by phage antibody technology, which may be useful for broadening the scope of application of the antibody.     

人源性大肠癌自然致敏噬菌体抗体Fab呈现库的构建与筛选

目的构建大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,初步筛选相关抗大肠癌抗体。方法取大肠癌病人转移淋巴结,提取淋巴结总 RNA,逆转录 PCR 扩增重链 Fd 和 k 轻链 cDNA。依次将 PCR 产物插入载体 pComb3的相应部位,构建人源性大肠癌噬菌体 Fab 基因库,并应用噬菌体表面呈现技术对该抗体库进行淘选及鉴定。结果所选2种 Ig 亚类的重链 Fd 片段、2种 k 轻链 cDNA 得到扩增。Fd 片段和 k 轻链均插入 pComb3的重组率为40%,Fab 噬菌体表达库容达1.48×10~6。经3轮淘选,抗体库得到约120倍的富集。3轮抗体库的点印记免疫染色均显示有 Fab 表达:酶联免疫吸附实验显示其与大肠癌抗原有结合活性。结论成功构建了大肠癌病人自然致敏淋巴结抗体 Fab 段噬菌体呈现库,利用噬菌体抗体库技术,可筛选相关抗大肠癌抗体。     

变形链球菌重组表面蛋白(rA-P)人源单链抗体的筛选及其活性检测

目的从PDNA5噬菌体抗体库中筛选变形链球菌重组表面蛋白的人源噬菌体单链抗体,将其表达制备非融合单链抗体,并探讨其生物学特性。方法用变形链球菌重组表面蛋白rA-P包被免疫试管,对pDAN5噬菌体抗体库进行5轮亲和免疫筛选制备抗rA-P蛋白的噬菌体单链抗体,并将其感染大肠杆菌HB2151,用IPTG诱导表达非融合性单链抗体,非融合性单链抗体包涵体复性后用Western blot检测其生物活性。结果筛选得到13株抗rA-P蛋白的噬菌体单链抗体;用HB2151表达的非融合性单链抗体分子量为30kD,表达量达菌体蛋白的30%,Western blot检测显示,复性后的非融合性单链抗体能与rA-P蛋白发生特异性抗原抗体反应,证明表达的非融合性单链抗体有生物活性。结论成功地利用噬菌体抗体库技术筛选、表达制备有生物活性的抗变链菌表面蛋白的人源单链抗体。     

Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer.     

Monoclonal antibodies to rabbit hepatocyte myosin that cross-react with human liver myosin

We prepared polyclonal and monoclonal antibodies against myosin purified from rabbit hepatocytes. Immunoblotting analyses revealed that the polyclonal antibody and four (HM1, HM2, HM3 and HM4) of five monoclonal antibodies reacted with myosin heavy chain. Chymotryptic cleavage of myosin yielded a 130 kDa fragment comprising the tail portion of the myosin heavy chain and a 67 kDa fragment comprising the ATP-binding active site of the myosin head. All active antibodies reacted with epitopes localized in the 130 kDa fragment. Monoclonal antibodies HM3 and HM4 and the polyclonal antibody reacted strongly with myosin heavy chains from a human liver homogenate prepared from a surgically resected liver specimen. Immunocytochemical analyses showed that myosin is localized along the plasma membrane as well as around the bile canaliculi in both rabbit and human hepatocytes. Immunocytochemical analyses on liver blocks obtained from those patients who suffered various types of diseases accompanying cholestasis clearly indicated a marked increase in pericanalicular myosin. This altered myosin localization is analogous to that observed in phalloidin-treated liver. Thus, myosin localization, determined using these antibodies, can provide a valid morphological basis for diagnosing the pathological state of the patient liver.