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。     

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。     

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。     

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。     

抗内毒素噬菌体抗体库的构建及筛选

目的构建一个鼠源性的抗内毒素单链噬菌体抗体库,从中筛选出对内毒素具有较高亲和力的单链抗体。方法从小鼠脾细胞中提取总RNA,通过RT-PCR技术扩增出小鼠抗体重链、轻链可变区基因（VH,VL）,用Linker将VH,VL交联形成单链抗体可变区片段（ScFv）。经NotⅠ,SfiⅠ双酶切后与经同样双酶切的pCANTAB5E载体相连,转化入大肠杆菌TG1以构建鼠抗内毒素单链噬菌体抗体库。在援救噬菌体抗体库后,用内毒素淘筛特异性的ScFv,富集的噬菌体阳性克隆重新感染TG1。在96孔板分别援救单个含特异性ScFv的TG1菌落,最后随机挑选出190个菌落经ELISA检测抗内毒素ScFv。结果小鼠血清中抗内毒素的效价为1∶12800。提取的总RNA浓度为12.3813μg/ml,纯度较好。扩增出的VH长约340bp,VL约320bp,ScFv约800bp。转化入TG1后有约1.9×107个菌落。淘筛一轮过后即有3×104阳性菌落长出,190个菌落经ELISA检测有2个阳性克隆。结论成功地构建了一个库容量为1.9×107的鼠抗内毒素单链噬菌体抗体库,并从中筛选出了2株抗内毒素ScFv。     

全人源食管癌单链抗体scFv 基因文库的构建

 目的 制备全人源化食管癌单链抗体scFv基因文库。方法 取食管癌病人癌肿周围淋巴结作为B细胞的来源，提取总RNA，用RT-PCR的方法获得抗体可变区基因cDNA文库。首先分别网格筛选确定扩增VH和VL基因片段的引物对，以cDNA为模板扩增VH和VL基因片段，再以它们为模板分别扩增VH-linker与VL-linker，用SOE-PCR技术将它们拼接成SCFv，再引入酶切住点Sfi I和Not I，胶回收PCR产物获得SCFv。将SCFv基因克隆入噬菌粒载体pCANT心5E后电转入Ecoli TG1。PCR法鉴定抗体基因插入率，1．5％琼脂糖凝胶电泳鉴定阳性克隆酶切产物。结果 食管癌周围淋巴结的提取总RNA琼脂糖电泳结果中可见清晰的28S、18S条带；VH基因的大小约为450bp。VL基因为350bp，组装后的scFv基因约为850bp。PCR连接产物的转化效率为2×10^7efu／μg，SCFv的阳性插入率为91．7％（22／24）。结论 食管癌相关的人源单链抗体基因文库的构建为进一步筛选单链抗体库奠定了基础。     

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。     

大容量人乳腺癌单链抗体库的构建与初步鉴定

目的利用细胞内重组技术构建大容量人乳腺癌单链抗体(scFv)库。方法收集30例乳腺癌外周转移淋巴结,依次提取总RNA、mRNA,RT-PCR扩增全套可变区基因(VL、VH),各自克隆到T载体,构建T载体库。然后从T载体上酶切VL、VH分步克隆到噬粒载体pDAN5,经电转化TG1得到初级库。辅助性噬菌体感染初级库,使噬菌体抗体表面呈现。提纯噬菌体抗体,滴定浓度后感染大肠杆菌BS1365进行细胞内重组。噬菌体挽救后再次提纯滴定噬菌体,感染大肠杆菌DH5αF',得到次级库,并测序验证。结果VH、VLT载体库分别为3.5×108和1.7×108,初级库库容量2.5×109。次级库库容量为2.5×1011。结论所采用的引物能够高效扩增目的片段,T载体的应用能够提高抗体基因的克隆效率,细胞内重组将进一步扩大库容量及增加其多样性,这种基于RT-PCR、T载体过渡、细胞内重组的路线能够构建大容量抗体库。     

特异性抗肝癌噬菌体单链抗体的应用研究

目的体外观察特异性抗肝癌(HCC)噬菌体单链抗体(ScFv)对HCC细胞生长和凋亡的影响.方法通过构建的特异性抗HCC噬菌体ScFv库在大肠杆菌HB2151中表达,制备游离ScFv,应用细胞培养、DNA凝胶电泳和流式细胞仪(FCM)技术检测抗体对HCC细胞生长和凋亡的影响.结果特异性抗HCC噬菌体ScFv处理的HCC细胞的生长速度明显减慢,克隆形成和附壁能力减弱,细胞存活率降低,DNA凝胶电泳可见典型凋亡梯状带;FCM检测发现抗体处理后HCC细胞凋亡率达32.6%,明显高于对照组(P＜0.01).结论应用噬菌体表面呈现技术和基因克隆技术构建的特异性抗HCC噬菌体ScFv具有抑制HCC细胞生长、促进HCC细胞凋亡的功能.     

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。     

抗γ-精浆蛋白人-鼠杂合Fab噬菌体抗体库的构建及导向筛选

目的:建立人-鼠杂合Fab噬菌体抗体库,筛选抗γ-精浆蛋白(γ-sm)人源性抗体轻链。方法:PCR扩增前列腺癌患者外周血淋巴细胞人抗体全套轻链基因,克隆人含鼠源抗γ-sm抗体Fd基因的pComb3X噬粒中,建立人-鼠杂合Fab噬菌体抗体库。通过稀释滴定、限制性酶切等分别对库容、基因重组率和多样性进行鉴定。以M13K07辅助噬菌体超感染,利用纯化的γ-sm对杂合库进行筛选,对筛出的阳性克隆进行功能检测。结果:构建了库容量为1．2×107CFU、重组率为90％、多样性好的人-鼠杂合噬菌体抗体库。ELISA检测出2个强阳性克隆,Western blot确证为抗γ--sm的人-鼠杂合Fab,竞争ELISA显示其表观亲和力约为亲本鼠抗体亲和力的71．8％。DNA测序显示筛到的人源轻链可变区基因属IGKV4-1*01胚系家族。结论:成功得到人源性抗γ-sm抗体轻链,为进一步获得全人源化抗体奠定了基础。     

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.     

抗人肺腺癌单链抗体的构建及在大肠杆菌中的表达

目的:构建抗肺腺癌的单链抗体(scFv),研究其表达条件,为将这一小分子抗体应用于临床奠定基础.方法:将抗人肺腺癌单克隆抗体的重链及轻链可变区基因插入表达载体pFUW80、pTH、pTF,分别在大肠杆菌XL1-Blue、Top10、GI698/GI724中诱导表达,得到噬菌体抗体或包涵体.用SDS-PAGE和ELISA鉴定检测活性.结果:SDS-PAGE的结果表明,在pTH和pTF的表达产物中,有一条43kD的特异条带,其分子量与预期相符,单链抗体以包涵体形式出现,其表达量达细菌总蛋白的18.6%.ELISA的结果表明,噬菌体抗体和经复性处理的包涵体具有与亲本单抗相同的特异性.结论:成功地构建和在大肠杆菌中表达了抗肺腺癌单链抗体.     

Selection of high affinity human neutralizing antibodies to VEGFR2 from a large antibody phage display library for antiangiogenesis therapy.

Compelling evidence suggests that vascular endothelial growth factor (VEGF) and its receptors play an important role in angiogenesis associated with tumor growth and metastasis. VEGF exerts its biologic activities through 2 transmembrane tyrosine kinase receptors: the fms-like tyrosine kinase receptor (Flt-1, or VEGFR1) and kinase insert domain-containing receptor (KDR or VEGFR2). We have previously produced a panel of antibodies directed against KDR from mice immunized with the recombinant form receptor. These antibodies efficiently neutralized VEGF-induced KDR activation and mitogenesis of human umbilical vascular endothelial cells (HUVEC). Murine antibodies, however, may not be suitable candidates for human therapy because of their propensity to elicit human anti-mouse antibody response. Here we isolated several high-affinity human Fab antibody fragments directed against KDR from an antibody phage display library constructed from the pooled B lymphocytes of nonimmunized healthy human donors. These human Fab fragments bind specifically to KDR with nanomolar affinity and block KDR/VEGF interaction with IC(50) of approximately 2-20 nM. Further, they effectively inhibit VEGF-stimulated mitogenesis of HUVEC and migration of human leukemia cells. Epitope mapping studies demonstrated that all neutralizing human antibodies bound the epitope(s) located within the first 3 N-terminal immunoglobulin-like domains of KDR, the same region that encompasses the binding site of VEGF. Our results suggest that these human anti-KDR antibodies may have potential application in the treatment of cancer and other diseases in which pathologic angiogenesis occurs.     

Recombinant single‐chain variable fragment antibodies against extracellular epitopes of human multidrug resistance protein MRP3 for targeting malignant gliomas

Multidrug resistance protein 3 (MRP3), a multidrug resistance protein identified by serial analysis of gene expression as a glioblastoma multiforme (GBM)‐associated molecule, is highly expressed in GBM, but not in normal brain cells. Thus, MRP3 is a candidate for GBM immunotargeting, but to date, no monoclonal antibody has been isolated that can target an extracellular MRP3 epitope. By phage display, we have isolated 3 recombinant, fully human, single‐chain Fv (scFv) antibodies, M25, M58 and M89, which specifically react with the extracellular N‐terminus of human MRP3. In ELISA, these scFvs reacted only with the peptide used for screening and not with other MRP3‐derived peptides. Flow cytometric analysis revealed that these scFv fragments bind specifically to viable human GBM cells displaying different MRP3 expression levels, but not to MRP3‐null cells. Furthermore, these scFv antibodies failed to react with tumor cells overexpressing other MRP proteins, including MRP1, MRP2, MRP4 and MRP5. M25 and M58 also bound to viable neurospheres. Iodogen‐labeled scFvs demonstrated a yield of 56–76%. The immunoreactive fractions of the radiolabeled M25, M58 and M89 scFvs were 32, 52 and 69%, respectively. M25 exhibited 20% internalization into D2159MG neurospheres, M58, 33% into D54MG cells and M89, 26% into D247MG. Immunohistochemical evaluation of human gliomas to determine the localization of MRP3 antigen using scFvs M25 and M58 showed a dense cytoplasmic and membranous staining pattern. These Fv‐based recombinant antibodies, which possess superior tumor penetration capabilities and selectively target tumor cells that express MRP3, may potentially be used in immunotherapy and diagnosis for brain tumors and other cancers.     

非小细胞肺癌T7噬菌体展示文库的构建

Objective: Currently, only a limited numbers of tumor markers for non small lung cancer (NSCLC) diagnosis, new biomarker, such as serum autoantibodies may improve the early detection of lung cancer. Our objective is construction human lung squamous carcinoma and adenocercinoma T7 phage display cDNA library from the tissues of NSCLC patients. Methods: mRNA was isolated from a pool of total RNA extract from NSCLC tissues obtained from 5 adenocarcinomas and 5 squamous carcinomas, and then mRNA was reverse transcribed into double stranded cDNA. After digestion, the cDNA was inserted into T7Select 10-3 vector. The phage display cDNA library was constructed by package reaction in vitro and plate proliferation. Plaque assay and PCR were used to evaluate the library. Results: Two T7 phage display cDNA library were established. Plaque assay show the titer of lung squamas carcinoma library was 1.8×106 pfu, and the adenocarcinoma library was 5×106pfu. The phage titer of the amplified library were 3.2×1010 pfu/mL and 2.5 x 1010 pfu/mL. PCR amplifica-tion of random plaque show insert ratio were 100% (24/24) in adenocarcinorna library and 95.8% in human lung squamas carcinoma library (23/24). Insert range from 300 bp to 1 500 bp. Conclusion: Two phage display cDNA library from NSCLC were constructed.     

小鼠结肠癌RNA转染mIL12修饰的树突状细胞诱发抗肿瘤活性

目的:构建肿瘤组织特异的噬菌体呈现型单链抗体库,用噬菌体体内筛选与肿瘤血管特异性结合的单链抗体,为癌症的诊断和治疗奠定基础。方法:取食道癌、胃癌、脑癌、肺癌、脊髓瘤患者肿瘤组织,提取各肿瘤组织膜蛋白混合后免疫BALB/c小鼠,取鼠脾脏淋巴细胞提取总RNA,用RT-PCR分别扩增抗体重、轻链可变区基因(VH和VL),经Linker连接形成ScFv基因片段,将ScFv基因片段与噬菌粒载体pCANTAB5E的连接产物转化大肠杆菌TG1,在辅助噬菌体M13KO7的作用下,获得重组噬菌体单链抗体库。以人宫颈癌HeLa细胞致瘤裸鼠为实验模型,用所构建的单链抗体库进行了4轮特异抗体的体内筛选。挑取了24个PCR鉴定为阳性的单链抗体做免疫组化分析,将在HeLa致瘤的组织切片上呈阳性染色、而在对照的肾组织切片上没有阳性染色的单链抗体进行序列测定。结果:选用7株不同的肿瘤细胞系建立裸鼠致瘤模型,以HeLa细胞致瘤裸鼠成瘤效果最好。用所构建的库容量为1.6×106的单链抗体库对肿瘤血管特异结合抗体进行体内筛选,得到1株单链抗体ScFv(VH-linker-VL),命名为ScFvH1。结论:成功构建肿瘤特异单链抗体库,并用此抗体库筛选到与肿瘤血管结合特异性较好的单链抗体,为肿瘤的早期诊断和治疗提供了一条新的技术路线。