人源性肺癌噬菌体展示单链抗体库的构建

目的:构建人源性肺癌噬菌体单链抗体库,为筛选肺癌相关抗原的抗体奠定基础。方法:提取肺癌转移淋巴结总RNA,用RT-PCR技术扩增人抗体重链可变区(VH)和轻链可变区(VL)基因,在体外将VH和VL连接成单链抗体(ScFv)基因,并克隆到噬菌粒载体pCANTAB 5E中,电转化至感受态的大肠杆菌TG1,经辅助噬菌体超感染,形成噬菌体单链抗体库,采用限制性内切酶鉴定其多样性。结果:从肺癌转移淋巴结中成功提取RNA,逆转录PCR扩增出人可变区基因,连接形成单链抗体,最终构建了库容为1.2×108的抗人肺癌单链抗体库。BstNⅠ酶切法证明构建的抗体库具有良好的多样性。结论:成功地构建了噬菌体展示的抗人肺癌单链抗体库,为进一步筛选肺癌相关蛋白的可溶性抗体奠定了基础。     

抗人肺癌噬菌体展示可溶性ScFv的筛选与鉴定及其序列测定

目的:制备抗人肺癌单链抗体(single chain Fv ferment antibody,ScFv),并对抗体生物学特性进行初步研究.方法:以人肺腺癌细胞系A2为抗原,对5 F-11杂交瘤细胞噬茵体抗体库进行富集和筛选.以人肺腺癌细胞系A2和正常人淋巴细胞为抗原,进行酶联免疫吸附试验,从富集后的噬菌体抗体库中筛选出只与A2细胞结合的阳性克隆.筛选的噬茵体克隆转染大肠埃希茵HB2151,得到可溶性单链抗体分泌克隆.可溶性抗体分泌克隆测序.应用 ELISA、竞争性ELISA、SDS-PAGE及蛋白质印迹法对其中的2A7-1克隆进行初步鏊定.结果:以肺腺癌细胞A2为抗原进行了4轮富集.进一步筛选得到18个仅识别A2细胞而与人淋巴细胞无反应的融合抗体分泌克隆.转染大肠埃希茵HB2151后筛选到能与A2细胞特异结合的可溶性抗体分泌克隆2A7-1.竞争性ELISA结果显示,5F-11能强烈抑制2A7-1与A2细胞的结合.SDS-PAGE蛋白质印迹法显示得到大小约为30×103的单链抗体.结论:通过噬菌体抗体技术成功分离到了鼠单抗5F-11的可溶性ScFv分泌克隆,为进一步的抗体应用研究奠定了基础.     

人源抗肝癌单链抗体库的构建及初步鉴定

目的构建人源抗肝癌单链抗体基因噬菌体表面呈现文库。方法利用噬菌体表面呈现技术,构建基因文库,经过panning筛选富集后,用ELISA方法检验抗原结合活性。结果从30个噬菌体克隆中筛选到8个具有肝癌细胞株SMMC7721结合活性的阳性克隆。结论从外周血淋巴细胞中获取可变区基因,利用噬菌体抗体库技术制备人源抗肝癌单链抗体的策略是可行的。     

全人源肝癌噬菌体单链抗体的筛选及特异性鉴定

目的对全人源肝癌噬菌体单链抗体库进行鉴定,筛选肝癌抗体,同时对抗体的活性及特异性进行鉴定。方法PCR鉴定阳性重组菌TG1中人肝癌ScFv的插入率。先以人成纤维细胞吸附后再以体外培养的肝癌细胞SMMC7721为抗原对所建抗体库进行3轮“吸附洗脱扩增”的亲和筛选。将筛选后的ScFv进行PCR鉴定及双酶切鉴定;通过ELISA法及FCM鉴定其与人肝癌细胞及正常细胞的结合活性。结果ScFv基因插入率为70%。在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮得到提高,第3轮为第1轮的214倍。筛选后的ScFv进行PCR鉴定及双酶切鉴定,均可检测到目的基因。ELISA分析结果显示18个克隆与SMMC7721呈阳性反应,阳性率为90%,15个克隆与成纤维细胞有交叉反应。得到3株肝癌单链抗体。ScFv的FCM鉴定表明,以正常胎肝细胞L02为对照,ScFv与肝癌结合比率为41.3%。特异性鉴定表明,其与肝癌细胞结合活性明显高于正常细胞。结论利用噬菌体抗体库技术结合减数筛选得到了肝癌噬菌体单链抗体及其基因,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性。     

全人源肝癌噬菌体单链抗体的筛选及特异性鉴定

目的：对全人源肝癌噬菌体单链抗体库进行鉴定，筛选肝癌抗体，同时对抗体的活性及特异性进行鉴定。方法：PCR鉴定阳性重组菌TG1中人肝癌ScFv的插入率。先以人成纤维细胞吸附后再以体外培养的肝癌细胞SMMC-7721为抗原对所建抗体库进行3轮“吸附-洗脱-扩增”的亲和筛选。将筛选后的ScFv进行PCR鉴定及双酶切鉴定；通过ELISA法及FCM鉴定其与人肝癌细胞及正常细胞的结合活性。结果：ScFv基因插入率为70％。在亲和筛选过程中，肝癌噬菌体单链抗体得到富集，收获率逐轮得到提高，第3轮为第1轮的214倍。筛选后的ScFv进行PCR鉴定及双酶切鉴定，均可检测到目的基因。ELISA分析结果显示18个克隆与SMMC-7721呈阳性反应，阳性率为90％，15个克隆与成纤维细胞有交叉反应。得到3株肝癌单链抗体。ScFv的FCM鉴定表明，以正常胎肝细胞L-02为对照，ScFv与肝癌结合比率为41．3％。特异性鉴定表明，其与肝癌细胞结合活性明显高于正常细胞。结论：利用噬菌体抗体库技术结合减数筛选得到了肝癌噬菌体单链抗体及其基因，且筛选后的抗体片段与人肝癌细胞有特异性的结合活性。     

全人源抗MLAA-34单链抗体的筛选和鉴定

目的:抗体药物治疗是目前癌症治疗中很有前景的一种方法,利用前期实验得到的MLAA-34纯化蛋白作为抗原,在噬菌体抗体库中进行筛选得到抗MLAA-34的单链抗体,进行测序并鉴定其亲和力。方法:包被纯化的MLAA-34抗原蛋白于96孔板上,封闭过夜,加入1×1012 cfu噬菌体抗体库,4 ℃结合4 h,洗脱,洗脱后的噬菌体感染大肠杆菌TG1扩增,经过3轮淘选,淘选得到的克隆再进行Phage-ELISA的鉴定,筛选得到阳性克隆。结果:纯化的MLAA-34作为抗原,经过在噬菌体抗体库中的3轮固相筛选,噬菌体的回收得到富集,洗脱的噬菌体产率显示回收率逐级增加,富集倍数约1 000倍。最后一轮筛选后挑选出188个重组噬菌体克隆,His标签蛋白作为对照蛋白,应用Phage-ELISA筛选出33个阳性克隆。结论:利用得到的MLAA-34蛋白为目标抗原,对全人源噬菌体抗体库进行淘选,应用Phage-ELISA法挑选出和MLAA-34抗原结合力的阳性噬菌体,为抗MLAA-34抗体的制备奠定了基础。     

嵌合轻链为模板导向筛选人源性抗肝癌抗体重链Fd片段

目的:利用噬菌体抗体库和导向选择技术,以抗HAb18G嵌合抗体轻链为模板,筛选人源性抗肝癌抗体Fd片段.方法:利用RT-PCR自肝癌患者外周血淋巴细胞中扩增全套人抗体重链Fd基因片段,克隆入含嵌合L链的展示载体pComb3X,建立人-鼠杂合Fab噬菌体抗体库.然后,利用大肠杆菌表达的非融合HAb18GE为抗原进行亲和筛选,利用pⅢ融合抗体的形式进行克隆鉴定,并对筛选出的杂合抗体进行初步的功能检测.结果:构建成功库容量为2×107PFU的杂合人-鼠噬菌体抗体库,利用非融合表达的HAb18GE进行6轮筛选.ELISA及流式细胞仪检测,其中7个克隆呈特异性阳性反应.其中克隆AP6-2和AP6-7亲和力较高,测序显示其基因序列相同,且与亲本抗体恒定区序列相同,属IgG2亚类,可变区属VH3家族.结论:通过本实验,利用嵌合轻链为模板,进行Fd片段替换,成功筛选到人源性抗体Fd片段.     

全人源食管癌噬菌体单链抗体库的筛选及特异性鉴定

目的：我们应用噬菌体抗体库技术构建了食管癌相关的人源单链抗体文库，本文目的在于对该抗体库进行鉴定，筛选食管癌抗体，同时对抗体的活性进行检测。方法：PCR鉴定TG1中食管癌单链抗体scFv的插入率；1．5％琼脂糖凝胶电泳鉴定Sfi I和NotI双酶切质粒的结果；先以人正常食管上皮细胞吸附后再以食管癌细胞Eca109为抗原对所建抗体库进行4轮的亲和筛选；将阳性重组噬菌体克隆感染Ecoli HB2151进行可溶性抗体表达及经亲和柱层析纯化；用SDS—PAGE测定该抗体的相对分子量；用Western blot鉴定该抗体；用ELISA法、免疫组化法鉴定该抗体与人食管癌细胞的结合的特异性。结果：seFv基因插入率为91．7％；酶切后检测到目的基因片段。在亲和筛选过程中，食管癌噬菌体单链抗体得到富集，收获率逐轮得到提高，第4轮为第1轮的141倍；SDS—PAGE与Western blot结果显示抗体的相对分子量为左右和30kd条带染色；在Ecoli HB2151中实现了单链抗体的可溶性表达。免疫组织化学结果提示可溶性抗体仅染食管癌组织，而肝癌组织和胃癌组织不染色。免疫细胞化学结果表明此可溶性抗体可使Eca109细胞染色。ELISA测定结果显示可溶性抗体具有较高的免疫活性，能与Eca109细胞结合，而不与胃癌BGC-823和NHEEC结合。结论：利用噬菌体抗体库技术的阴性、阳性筛选得到了食管癌噬菌体单链抗体，且筛选后的抗体片段与人食管癌细胞有特异性的结合活性。     

人源噬菌体抗体对Peroxiredoxin I高表达肺腺癌细胞增殖的抑制作用

背景与目的：研究表明过氧化物酶Peroxiredoxin I （Prx I）与癌症的发展有密切关系。我们已通过噬菌体展示技术构建了肺腺癌相关的人源单链抗体库。本研究对该库进行筛选,得到抗PrxI肺腺癌单链抗体,并检测其对肺腺癌细胞A549增殖的抑制作用。方法：PCR法检测TG1中scFv基因插入率,1％琼脂糖凝胶电泳鉴定蹄I和Not I双酶切质粒的结果,以A549细胞及在肺癌中高表达的抗氧化蛋白PrxI为靶抗原分别对抗体库进行3轮筛选富集。将阳性克隆用IPTG诱导表达并进行检测。放射性核素计数法测定细胞单链抗体内摄水平,MTT法及流式细胞术检测单链抗体对A549细胞的增殖抑制和凋亡情况,免疫印迹法检测抗体作用A549细胞后PrxI的表达水平。结果：scFv基因插入率为77％,双酶切鉴定检测到目的条带。在亲和筛选过程中．肺腺癌单链抗体得到富集,收获率逐轮提高,第6轮为第1轮的180倍。ELISA法检测到在随机选取的10个克隆中．有6个与A549细胞呈阳性反应,阳性率60％。SDS-PAGE及ELISA检测证实得到人源抗Prx I肺腺癌单链抗体。被A549细胞内摄的单链抗体介导了细胞的凋亡以及细胞内PrxI蛋白表达水平的下降。结论：从噬菌体抗体库中筛选获得具有较高特异性的抗PrxI肺腺癌单链抗体。单链抗体与肺腺癌细胞有特异性亲和力,并能有效抑制其增殖。     

全人源抗乳腺癌噬菌体单链抗体库的构建

目的:利用噬菌体展示技术构建全人源性抗乳腺癌单链抗体库。方法:从临床获取未化疗乳腺癌病人外周血样30份,分离出单个核细胞(PBMC),提取总RNA,用RT-PCR技术逆转录获得cDNA,并扩增出全套人抗体重链(VH)和轻链(VL)基因,经重叠延伸PCR(SOE-PCR),在体外将两者连接成单链抗体(scFv)基因片段,将该片段用Sfi I和Not I酶切后克隆至pCantab5E噬菌体载体,电转化TG1感受态菌,收集培养后平板上的菌落,即构建初级噬菌体单链抗体库。结果:得到长度约为360bp和340bp的VH和VL,拼接后得到的scFv长度约为750bp;经PCR初步鉴定插入率约为80%,BstN 1多样性酶切检验,酶切图谱呈多样性。经测序验证,最终获得库容约为2.4×106pfu/ml初级单链抗体库。结论:本研究获得了全人源抗乳腺癌噬菌体单链抗体库,为下一步筛选抗人乳腺癌细胞特异性单链抗体奠定了基础。     

A3--a novel colon and pancreatic cancer reactive antibody from a primate phage library selected using intact tumour cells

The identification of novel tumour-associated antigens (TAAs) is pivotal for progression in the fields of tumour immunotherapy and diagnosis. In the present study, we have developed, based on flow cytometric evaluation and use of a mini-library composed of specific antibody clones linked to different antibiotic resistance markers, methods for positive and subtractive selection of phage antibodies employing intact cells as the antigen source. An scFv phage library (2.7 x 10(7)) was constructed from a primate (Macaca fascicularis) immunised with pooled human colon carcinomas. This library was selected for 3 rounds by binding to Colo 205 colon adenocarcinoma cells and proteolytic elution followed by phage amplification. Several antibodies reactive with colon carcinomas and with restricted reactivity to a few epithelial normal tissues were identified by immunohistochemistry. One clone, A3 scFv, recognised an epitope that was homogeneously expressed in 11/11 of colon and 4/4 pancreatic carcinomas studied and in normal tissue restricted to subtypes of epithelia in the gastrointestinal tract. The A3 scFv had an apparent overall affinity approximately 100-fold higher than an A3 Fab, suggesting binding of scFv homodimers. The cell surface density of the A3 epitope, calculated on the basis of Fab binding, was exceptionally high, approaching 3 million per cell. We also demonstrate efficient T-cell-mediated killing of colon cancer cells coated with A3 scFv fused to the low MHC class II binding superantigen mutant SEA(D227A). The identified A3 molecule thus represents a TAA with properties that suggest its use for immunotherapy of colon and pancreatic cancer.     

用不同方法从大容量噬菌体抗体库中筛选抗完整食管癌细胞的单链抗体

目的:初步探索从天然的单链大容量噬菌体抗体库中筛选肿瘤细胞抗体的技术策略.方法:通过两种方法,即2%多聚甲醛固定细胞筛选法(PF)及活细胞直接筛选法(NF),经过4轮"吸附-洗脱-扩增"的淘筛过程,对食管癌KYSE-170细胞进行筛选,得到抗食管癌细胞的噬菌体抗体,完善了噬菌体抗体库筛选完整细胞的特异性抗体的技术策略.并且进一步制备了其可溶性抗体.结果:活细胞NF组的筛选阳性率高于2%多聚甲醛固定PF组,筛选特异性抗体的阳性率分别为25.00%和12.50%,两组的差异具有统计学意义.结论:利用单链大容量抗体库可以采用不同的筛选方法成功制备与肿瘤细胞结合的噬菌体抗体,为今后的研究和应用奠定基础.     

Screening and Identification of Peptides Specifically Targeted to Gastric Cancer Cells from a Phage Display Peptide Library

Background: Gastric cancer is the second most common cancer among the malign cancer types. Inefficiency oftraditional techniques both in diagnosis and therapy of the disease makes the development of alternative and noveltechniques indispensable. As an alternative to traditional methods, tumor specific targeting small peptides can be usedto increase the efficiency of the treatment and reduce the side effects related to traditional techniques. The aim of thisstudy is screening and identification of individual peptides specifically targeted to human gastric cancer cells usinga phage-displayed peptide library and designing specific peptide sequences by using experimentally-eluted peptidesequences. Methods: Here, MKN-45 human gastric cancer cells and HFE-145 human normal gastric epithelial cellswere used as the target and control cells, respectively. 5 rounds of biopannning with a phage display 12-peptide librarywere applied following subtraction biopanning with HFE-145 control cells. The selected phage clones were establishedby enzyme-linked immunosorbent assay and immunofluorescence detection. We first obtain random phage clonesafter five biopanning rounds, determine the binding levels of each individual clone. Then, we analyze the frequenciesof each amino acid in best binding clones to determine positively overexpressed amino acids for designing novelpeptide sequences. Results: DE532 (VETSQYFRGTLS) phage clone was screened positive, showing specific bindingon MKN-45 gastric cancer cells. DE-Obs (HNDLFPSWYHNY) peptide, which was designed by using amino acidfrequencies of experimentally selected peptides in the 5th round of biopanning, showed specific binding in MKN-45cells. Conclusion: Selection and characterization of individual clones may give us specifically binding peptides, butmore importantly, data extracted from eluted phage clones may be used to design theoretical peptides with betterbinding properties than even experimentally selected ones. Both peptides, experimental and designed, may be potentialcandidates to be developed as useful diagnostic or therapeutic ligand molecules in gastric cancer research.     

Screening peptides binding specifically to colorectal cancer cells from a phage random peptide library

The aim of this study was to screen for polypeptides binding specifically to LoVo human colorectal cancer cells using a phage-displayed peptide library as a targeting vector for colorectal cancer therapy. Human normal colorectal mucous epithelial cells were applied as absorber cells for subtraction biopanning with a c7c phage display peptide library. Positive phage clones were identified by enzyme-linked immunosorbent assay and immunofluorescence detection; amino acid sequences were deduced by DNA sequencing. After 3 rounds of screening, 5 of 20 phage clones screened positive, showing specific binding to LoVo cells and a conserved RPM motif. Specific peptides against colorectal cancer cells could be obtained from a phage display peptide library and may be used as potential vectors for targeting therapy for colorectal cancer.     

鼻咽癌人源抗独特型单链抗体的制备及筛选

背景与目的:抗独特型抗体作为肿瘤抗原替代物可用于肿瘤治疗,这已在临床试验中得到证实。但由于目前所使用的抗独特型抗体多为鼠源性,用于人体可产生人抗鼠抗体反应,从而影响疗效。本实验拟构建噬菌体人源抗独特型抗体库,并从中筛选出能模拟鼻咽癌相关抗原的β型抗独特型单链抗体scFv(Ab2βscFv),以解决鼠源性抗独特型抗体用于临床所产生的人抗鼠抗体反应。方法:体外致敏并用EB病毒(Epstein-Barrvirus,EBV)转化鼻咽癌患者的外周血单个核细胞(peripheralbloodmononuclearcell,PBMC),用RT-PCR分别扩增VH和VL基因并连接成scFv基因,将scFv基因与载体fUSE5连接后,转化大肠杆菌MC1061,构建噬菌体呈现型scFv库。在用单抗FC2对文库进行4轮筛选后,用SandwichELISA和结合抑制法从中筛选出β型Ab2scFv。结果:用单抗FC2体外致敏并经EBV转化的10例鼻咽癌患者的PBMC中,8例有鼻咽癌抗独特型抗体产生。经PCR分别扩增出5种VH(γ、μ)和7种VL(κ、λ)基因,经连接组成14种scFv基因。在与载体连接后,导入大肠杆菌MC1061,得到库容为1.5×108的初级噬菌体抗独特型抗体库。经富集筛选后,从中随机挑取270个克隆进行ELISA筛选,得到91个Ab2scFv单克隆,阳性率为33.7%。再用结合抑制法从中初步筛选出5个可能为β型的Ab2scFv。结论:联     

Specificity rescue and affinity maturation of a low-affinity IgM antibody against pro-gastrin-releasing peptide using phage display and DNA shuffling.

The objectives of the present study were to use phage display to rescue the specificity of an IgM antibody and by the use of DNA shuffling to construct a sublibrary from which mutants with higher affinity could be selected. As a test system, a hybridoma cell line producing low-affinity IgM against recombinant pro-gastrin-releasing peptide (ProGRP) was chosen as starting material for construction of a single-chain Fv (scFv) phage library. One clone, pGRP5, demonstrating affinity for the antigen, was selected for the study. Random mutations were introduced into the scFv sequence by DNA shuffling, and mutants with raised affinity were selected by biopanning against recombinant ProGRP. Clones with affinities improved by approximately two orders of magnitude were selected after the first round of DNA shuffling. An additional 8- to 9-fold rise in affinity was demonstrated in mutants after the second round of mutagenesis. Sequence analysis demonstrated changes primarily in the complementarity-determining region (CDR) H1, CDR L1 and CDR H3 as compared to the original clone. Thus, by the use of phage display in combination with DNA shuffling, the specificity of an IgM antibody was rescued and the affinity was raised almost three orders of magnitude.     

抗胃癌单抗3H11的单链抗体表达载体的构建和表达

目的构建抗胃癌鼠单抗３Ｈ１１的单链抗体（ＳｃＦｖ）,以利于临床应用。方法采用噬菌体抗体库技术和ＰＣＲ介导的定位点突变技术,构建和改造抗胃癌ＳｃＦｖ基因。结果从３Ｈ１１杂交瘤制备抗体库,以胃癌细胞系（ＭＧＣ８０３）进行筛选,未获阳性克隆。再对３Ｈ１１可变区基因以ＰＣＲ错配技术进行随机突变,构建次级突变抗体库,仍未筛出阳性克隆。最后用ＰＣＲ定点突变技术,将ＳｃＦｖ可变区基因氨基端序列恢复成３Ｈ１１的原始序列,终于得到可与胃癌细胞结合的ＳｃＦｖ克隆。结论说明ＰＣＲ引物引入的可变区氨基端序列的改变有时可对ＳｃＦｖ的活性有很大影响     

噬菌体随机12肽库筛选肝癌患者血清肿瘤标记物的初步研究

目的：从噬菌体随机多肽文库中,筛选出能与肝癌患者血清特异性结合的短肽分子.方法：采用肝癌患者血清作为配基,筛选以融合蛋白形式在丝状噬菌体M13外壳蛋白Ⅲ表达的随机12肽文库.按吸附一洗脱一扩增的淘筛过程,经3轮淘筛后,随机挑取噬菌体克隆用ELISA检测其特异性,评价分析其诊断肝癌的价值.结果：经3轮淘筛后,特异性结合的噬菌体富集增加近100倍.用.ELISA检测第3轮筛选后随机挑取的单个噬菌体克隆,其中特异性最好的3个克隆具有诊断肝癌的潜在价值.结论：噬菌体展示肽库技术,可以有效进行肝癌相关抗原肽的筛选研究,为获得特异性诊断试剂进而为肝癌的诊断提供依据.     

胃癌细胞特异性结合肽的筛选及鉴定

目的 筛选与胃癌细胞特异性结合的多肽。方法 以正常细胞为吸附细胞,胃癌细胞为筛选靶细胞对噬菌体随机12肽库进行消减筛,用细胞酶联免疫吸附试验（ELISA）、免疫细胞和组织化学法及裸鼠正常组织结合实验鉴定阳性克隆并进行DNA测序。结果 经三轮筛选,利用ELISA从随机挑选的24个噬菌体克隆中得到8个与胃癌细胞具有高结合力的噬菌体阳性克隆,经免疫细胞化学及裸鼠正常组织结合试验鉴定,发现第20、24两个克隆能与胃癌细胞特异性结合,而不与正常细胞和裸鼠组织结合,噬菌体阳性克隆氨基酸序列无同源性。结论 得到两个序列不同的特异性结合胃癌细胞的噬菌体克隆,这可为进一步的研究提供实验依据。