人源抗乳腺癌单链抗体ScFv的基因克隆

乳腺癌的发病率在我国呈上升趋势。鼠源性单克隆抗体(mAb)抗不适用于人体,而人源化mAb则在乳腺癌的诊断、靶向治疗中具有较高的应用价值。为此,我们从乳腺癌细胞体外致敏乳腺癌患者外周血中分离的淋巴细胞,利用噬菌体抗体库技术克隆了人抗体基因,以期获得抗乳腺癌的人源化单链抗体。1材料和方法1.1 材料 质粒提取试剂盒(Wizard plus SV minipreps DNApurification)、DNA纯化试剂盒(Wizard DNA clean up)、SfiI和Not I内切酶、T4 DNA连接酶均购于Promega公司;RNA纯化     

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。     

抗人肺癌单链抗体噬菌体库的构建及特异性单链抗体的鉴定

目的 构建人源噬菌体抗体库,并从中筛选出抗肺癌人源单链抗体。方法 提取肺癌患者癌旁淋巴结组织,通过RT-PCR扩增出重链可变区基因（VH）和轻链可变区基因（VL）,再经剪切-重叠-延伸PCR(SOE-PCR)将VH 和VL连接得到单链抗体（ScFv）。将双酶切后的ScFv基因片段克隆入噬菌体表达载体pCANTAB5E,得到初级噬菌体抗体库。以肺腺癌细胞株A549为抗原对抗体库进行4轮筛选富集,鉴定抗体库性能。将得到的阳性克隆用IPTG诱导表达并进行检测。结果 成功构建噬菌体单链抗体库。经筛选富集,噬菌体收获率得到增加,第4轮是第1轮的115倍。随机选取10个克隆,通过ELISA法检测到其中7个与A549细胞呈阳性反应,阳性率为70%。SDS-PAGE及ELISA检测证实得到人源抗肺癌单链抗体。结论 成功构建人源单链抗体噬菌体库,从中获得具有较高特异性的抗人肺癌单链抗体。     

抗人纤维蛋白噬菌体单链抗体库的构建和鉴定

目的应用噬菌体展示技术构建抗人纤维蛋白单链抗体(scFv)文库,筛选高亲和力抗人纤维蛋白scFv并进行鉴定。方法利用人纤维蛋白免疫小鼠,分别扩增小鼠VH和VL基因,经重叠延伸聚合酶链反应(PCR)将VH和VL基因拼接成scFv基因,SfiⅠ/NotⅠ双酶切克隆入pCANTAB 5E噬菌粒载体,转化E.coli TG1构建成库,采用人纤维蛋白原对抗体库进行负筛选,人纤维蛋白进行正筛选,酶联免疫吸附分析(ELISA)检测阳性克隆的抗原特异性并进行测序分析。结果构建了库容为8.7×106的抗人纤维蛋白scFv库,ELISA测定显示scFv具有较高的抗原特异性;抗人纤维蛋白scFv基因序列长732 bp,编码244个氨基酸,VH和VL基因均有明确的3个互补决定区和4个骨架区。结论成功构建了抗人纤维蛋白scFv文库,并筛选到高亲和力的抗人纤维蛋白scFv,为新型血栓显像剂的开发奠定了实验基础。     

从噬菌体抗体库克隆抗人RBC单链抗体基因

目的用噬菌体抗体库技术克隆抗人红细胞（ＲＢＣ）的单链抗体（ＳｃＦｖ）基因。方法以人ＲＢＣ免疫小鼠，取脾细胞，ＲＴ－ＰＣＲ法扩增ＶＨ和Ｖκ基因，组装到ＳｃＦｖ－噬菌体抗体表达载体中，构建了噬菌体抗体库，以人ＲＢＣ为固相抗原，对该库进行了四轮“吸附－洗脱－扩增”筛选，获得了抗人ＲＢＣ的ＳｃＦｖ基因。结果ＤＮＡ序列分析表明其可变区基因分别属于小鼠ＶＨⅢ亚群和ＶκⅤ亚群。结论所表达的ＳｃＦｖ可与不同人ＲＢＣ结合，与ＡＢＯ血型抗原无关，可用于构建快速血凝诊断的工程抗体     

从噬菌体抗体库中筛选抗角蛋白单链抗体

目的　从半合成噬菌体抗体库中筛选人源性抗角蛋白单链抗体 (scFv)并进行鉴定。方法　以人表皮角蛋白为抗原 ,通过“吸附 洗脱 扩增”过程从噬菌体抗体库中筛选特异性抗角蛋白单链抗体 ,对其抗原结合活性、识别角蛋白的相对分子质量 (Mr)和序列进行分析鉴定。结果　经过筛选 ,获得 6株能与角蛋白特异性结合的阳性克隆 ,所识别角蛋白的Mr 相同 ,均在 5 6 0 0 0～ 5 70 0 0之间 ;序列分析显示 ,所获抗体克隆的可变区基因分别属于免疫球蛋白基因家族的不同亚群。结论　利用噬菌体抗体库技术可以不经免疫制备出高特异性的人源性抗角蛋白单链抗体     

人源性胃癌单链抗体库的构建

目的: 构建人源天然库容量大、多样性好的核糖体展示单链抗体(scFv)库.方法: 采集人新鲜外周血分离淋巴细胞,提取总RNA,用RT-PCR扩增人抗体VH和VL基因,同时扩增作为间隔区的人抗体CK基因;采用重叠延伸PCR(简称SOE PCR)技术连接VH-VL-CK基因,同时引入T7启动子和核糖体结合位点序列,体外构建核糖体展示scFv库模板,连接T-Vector转化E.coli DH5α大肠杆菌,蓝白筛选,挑取阳性克隆测序以鉴定scFv组装.结果: 成功构建了库容量达3.1×1013的人源性胃癌核糖体展示scFv库.结论: 构建的大容量人源性胃癌核糖体展示抗体库可以成为进一步筛选多种特异性人源抗体的实验平台,为开发治疗性人源抗体奠定了很好的实验基础.     

大容量人源噬菌体抗体库的构建

目的:构建大容量易于改造成Diabody的噬菌体单链抗体库,筛选人源性抗体。方法:从正常成人外周血和新生儿脐血中分离淋巴细胞提取RNA,经RT-PCR扩增轻重链可变区基因(VL和VH),通过重叠PCR(over-lapPCR)将VL和VH拼接成单链抗体(ScFv)基因(其中VH两侧是两个非同源的loxp基因),并克隆入噬菌体表达载体PDF,得到初级噬菌体抗体库。将初级抗体库以高MOI超感染Cre+菌株BS1365,通过loxp-cre定位重组系统,介导轻重链在菌内重组配对,然后低感染XL1-Blue菌,得到大容量的次级抗体库。以多种不同抗原对库进行筛选,得到多样性较好的特异性抗体。结果:经超感染重组,得到1·2×1010的大容量抗体库。经三种蛋白抗原筛选,均得到多株特异性较好的噬菌体抗体,并成功构建为活性较好的Diabody。结论:经Loxp-Cre定位重组系统在单细胞内重组,能够高效地构建大容量噬菌体单链抗体库。     

从半合成噬菌体抗体库中克隆抗地高辛人单链抗体

目的：从半合成噬菌体抗体库中克隆抗地高辛（Dig）人单链抗体（ADA ScFv）,为Dig中毒的诊断和治疗提供人源性抗体。方法：①以改良法制备Dig-BSA偶联物②以固相化的Dig-BSA偶联物对人噬菌体ScFv抗体库进行了4轮筛选,通过ELISA筛选出能结合Dig的抗体克隆并鉴定其活性,通过ELIS竞争抑制实验分析其特异性,对阳性克隆的DNA进行测序分析。结果：①在对抗体库的筛选过程中可见有明显的富集;②获得一株可结合Dig及其类似物的阳性克隆;③阳性克隆的可变区分别属于VH5和Vλ1亚群。结论：利用噬菌体抗体库技术获得了能与Dig及其它洋地黄类药物结合的人ADA ScFv,经过一步分析及抗体工程改造后,有可能为临床上诊断及治疗洋地黄类药物中毒提供具有实用价值的人源性抗体。     

抗人SARS病毒单链抗体库的构建

目的:利用细胞内重组的方法,构建大库容量的人源性抗SARS病毒单链抗体(scFv)库,为筛选SARS病毒抗原的人源性抗体奠定基础。方法:取6例SARS康复患者的80mL外周血,分离淋巴细胞后提取总RNA。分离纯化mRNA并反转录成cDNA后,扩增抗体重链、轻链可变区基因片段。然后经重叠延伸PCR装配成scFv基因并克隆入噬粒载体pDAN5中,电转化大肠杆菌TG1构建初级抗体库。制备初级库噬菌体后,感染大肠杆菌BS1365进行细胞内重组制备次级抗体库。结果:初级库库容量为3×109,在大肠杆菌BS1365中重组后得到3×1011的次级抗体库。结论:细胞内重组方法的应用可使大库容量抗体库的构建变得简单易行。构建的抗SARS病毒单链抗体次库不仅接近人类天然抗体库的水平,而且多样性好,为筛选抗SARS病毒抗原的抗体提供了保证。     

两种血清Her-2/neu蛋白检测试剂的比较研究

目的探讨两种血清Her-2/neu蛋白检测试剂检测结果的一致性,为合理选择检测试剂提供依据。方法应用西门子公司化学发光法(chemiluminescence immunoassay,CLIA)和DRG公司ELISA法两种试剂对乳腺癌组195份、健康对照组60份和其他肿瘤患者组100份样本进行Her-2/neu蛋白检测,比较两种试剂检测结果的一致性。结果 355份样本中有351份测定结果一致,总符合率为98.87%(351/355);两组检测结果的相关系数r值为0.9919,P=0.000。健康对照组和其他肿瘤组患者的Her-2/neu蛋白检测值均低于临界值。结论两种血清Her-2/neu蛋白检测试剂可比性强,化学发光法试剂是一种良好的检测方法,能够满足临床需求。     

Significance of TFF3 protein and Her-2/neu status in patients with gastric adenocarcinoma

Increased knowledge of molecular alterations involved in gastric carcinogenesis has provided useful information for diagnosis and prediction. In this study, we investigated the clinicopathological significance of TFF3 expression and Her-2/neu status in gastric adenocarcinoma and explored the correlation between TFF3 expression and Her-2/neu status. A hundred and twenty-six (126) patients having undergone curative gastrectomy were enrolled. Immunohistochemistry for TFF3 was performed on tumor tissues and non-neoplastic resection margins. Her-2/neu status was assessed by immunohistochemical staining and fluorescence in situ hybridization (FISH) on tumor tissues. As a result, TFF3 expression and Her-2/neu positivity were detected in 46.8% and 11.9% of the cases, respectively. Patients with negative TFF3 exhibited longer survival than the positive group (P = 0.0142), while patients with positive Her-2/neu only showed a tendency toward longer overall survival (P > 0.05). However, Her-2/neu was significantly associated with improved disease-free survival (P = 0.0234). Multivariate analysis demonstrated Her-2/neu and TFF3 to be independent prognostic indicators of recurrence, and a significantly poor prognosis for expression of TFF3 in patients with Her-2/neu negative tumors. TFF3 is an independent indicator for overall survival in gastric cancer, while Her-2/neu, as a marker of long time survival prediction, seems to be limited.     

Her-2/neu、DPC4和P16在胰腺癌中的表达及其意义

目的观察Her-2/neu、P16和DPC4基因在胰腺癌中的表达,并探讨其与胰腺癌发生、发展的关系。方法应用免疫组织化学法检测Her-2/neu、P16和DPC4基因在34例胰腺癌和12例胰腺良性病变和正常组织中的表达水平,将结果与患者的临床病理学资料进行统计。结果胰腺癌Her-2/neu表达水平和P16表达缺失水平显著高于胰腺良性病变和正常组织(P<0.05);胰腺癌DPC4表达水平低于胰腺良性病变和正常组织。高、中度分化胰腺癌DPC4表达水平显著高于低分化胰腺癌(P<0.05);有淋巴结转移的胰腺癌Her-2/neu和P16表达水平显著低于无淋巴结转移的胰腺癌(P<0.05);随TNM分期增高,Her-2/neu表达阳性率显著降低(Ⅰ期85.7%;Ⅱ期54.5%,Ⅲ期36.4%,Ⅳ期0.0%)(P<0.05);P16表达阳性率亦显著降低(Ⅰ期100.0%;Ⅱ期72.7%,Ⅲ期54.5%,Ⅳ期20.0%)(P<0.05)。Her-2/neu与P16和DPC4与Her-2/neu的表达密切相关(P<0.05);P16与DPC4的表达极密切相关(P<0.01)。结论Her-2/neu、P16和DPC4的表达对估计胰腺癌的生物学行为和判断预后有较大帮助。P16和DPC4的表达对胰腺癌的进展可能有协同作用。     

大容量噬菌体抗体库的构建及鉴定

目的　构建大容量噬菌体单链抗体库 ,从中筛选人源单链抗体 (ScFv)。方法　从正常成人外周血和新生儿脐血分离淋巴细胞 ,用RT PCR扩增轻链可变区基因 (VL)和重链可变区基因(VH) ,通过重叠PCR法将VH 和VL 拼接形成ScFv基因 ,并克隆入噬菌体表达载体PDF ,得到ScFv初级噬菌体抗体库。以高MOI超感染cre 菌株BS136 5 ,通过loxp cre定位重组系统 ,介导轻重链的组合配对 ,得到大容量抗体库 ,用多种抗原对抗体库进行生物淘筛 ,鉴定抗体库的性能。结果　获得了 6×10 10 的大容量单链噬菌体抗体库。分别用卵清蛋白、胃蛋白酶、铁蛋白、人角蛋白、人TNF α、地高辛等6种抗原进行筛选 ,均得到多样性的特异性噬菌体抗体。结论　经loxp cre定位重组系统在单细胞内重组成功地构建了大容量单链噬菌体抗体库 ,初步尝试对 6种抗原进行筛选均获成功 ,提示该抗体库可用于制备具有应用前景的人源抗体     

Detection of Her-2/neu oncogene in breast carcinoma by chromogenic in situ hybridization in cytologic specimens

Determination of Her-2/neu oncogene amplification is important in the current treatment of breast carcinoma. In addition to fluorescence in situ hybridization (FISH) and immunohistochemical stain (HercepTest), chromogenic in situ hybridization (CISH) has been shown to be a sensitive and specific method to determine the Her-2/neu status of surgical specimens. The effectiveness of CISH in detecting the Her-2/neu oncogene in cytologic specimens has not been well documented. Twenty-five cases of fine needle aspirate smears and touch imprints from infiltrating ductal carcinomas were examined. Both CISH and FISH were performed on each case using a digoxigenin-labeled Her-2 DNA probe for CISH (Zymed) and both Her-2 and chromosome 17 probes for FISH (Vysis). Sixty tumor cells were evaluated in each case. The scoring system and interpretation of CISH were as follows: (1) no amplification (<5 brown dots/nucleus), (2) amplification (>10 brown dots/nucleus), and (3) low-level amplification (5-9 brown dots/nucleus). Of the 25 cases analyzed, 23 (3 amplified and 20 nonamplified) showed similar results for both methods. Two cases were discordant. In these cases, low-level amplification was suggested by CISH but nonamplification by FISH. One of the cases can be explained by polysomy for chromosome 17 by FISH. In conclusion, our preliminary data suggest that CISH is a useful technique to determine Her-2/neu oncogene status in cytologic specimens. In a case of low-level amplification, a CISH chromosome 17 probe should be used, or FISH is recommended for confirmation.     

Comprehensive immunohistochemical analysis of Her-2/neu oncoprotein overexpression in breast cancer: HercepTest™ (Dako) for manual testing and Her-2/neuTest 4B5 (Ventana) for Ventana BenchMark automatic staining system with correlation to results of fluorescence in situ hybridization (FISH)

Overexpression of Her-2/neu-oncoprotein is used as marker for Herceptin® therapy. To investigate the sensitivity and specificity of automatic immunohistochemistry (Benchmark, Ventana), we compared the results to the manual testing (Dako) in 130 breast carcinomas and validated the results by fluorescence in situ hybridization (FISH). Manual and automatic immunohistochemistry of Her-2/neu-oncoprotein using two different antibodies (HercepTest?, Her-2/neuTest 4B5) was analyzed. FISH was performed in all cases with uncertain or strong overexpression in either immunohistochemical stainings or with different immunohistochemical results. Same immunohistochemical results were seen in 73.8%. Two cases with overexpression, detected with Her-2/neuTest 4B5 and confirmed by FISH, showed no overexpression using HercepTest?. From 21 cases with 2+ by Her-2/neuTest 4B5, 15 cases had no gene amplification (two of them with 3+ HercepTest?); three cases showed a gene amplification (one of them with failing overexpression by HercepTest?); two other cases were polysomic; one could not be analyzed. Ventana immunohistochemistry seems to be of same reliability like Dako with a little better concordance to FISH in our study.     

Vesicular stomatitis virus expressing a chimeric Sindbis glycoprotein containing an Fc antibody binding domain targets to Her2/neu overexpressing breast cancer cells

Vesicular stomatitis virus (VSV) is a candidate for development for cancer therapy. It is an oncolytic virus that is safe in humans. Recombinant virus can be made directly from plasmid components. We attempted to create a virus that targeted specifically to breast cancer cells. Nonreplicating and replicating pseudotype VSV were created whose only surface glycoprotein (gp) was a Sindbis gp, called Sindbis-ZZ, modified to severely reduce its native binding function and to contain the Fc-binding domain of Staphylococcus aureus protein A. When titered on Her2/neu overexpressing SKBR3 human breast cancer cells, pseudotype VSV coated with Sindbis-ZZ had <1% the titer of pseudotype VSV coated with wild-type Sindbis gp. Titer was increased 50-fold when the Sindbis-ZZ pseudotype was conjugated with 4D5, a mouse monoclonal antibody directed against the Her2/neu receptor. Titers of antibody-conjugated virus were increased 36-fold on a second human breast cancer cell line, MCF7/H2, which expressed lower concentrations of Her2/neu receptor on the cell surface. At multiple concentrations of antibody, titers on SKBR3 cells were significantly greater when the virus was incubated with Herceptin, an antibody with a human Fc, than with 4D5, a mouse antibody, reflecting the known higher affinity of the protein A Fc-binding domain for human Fc. Analysis of the protein composition of the pseudotype VSV found low expression of the modified Sindbis gp on the virus accounting, in part, for a viral titer that did not exceed 1.2 x 10(5)/ml. This work demonstrates the ability to easily create, directly from plasmid components, an oncolytic replicating VSV with a restricted host cell range.     

Overexpression of HER2/neu in solid tumours: an immunohistochemical survey

AIMS: Using a standardized immunohistochemical assay we have evaluated 575 primary neoplasms of different histogenesis to determine the incidence of HER2 overexpression in some of the most common categories of human solid neoplasms. This study addresses the variable incidence of HER2 overexpression previously published for some tumour types. METHODS AND RESULTS: The immunohistochemical staining was performed on paraffin sections of surgical specimens and a well-defined scoring system based upon numbers of HER2 receptors expressed on the cell surface was applied. Overexpression of HER2 as defined as a HER2 score of equal or greater than 2 was seen in breast cancer (22%), pulmonary adenocarcinoma (28%), colorectal adenocarcinomas (17%), pulmonary squamous (11%) and gastric adenocarcinomas (11%). As expected, the proportion of cases with a HER2 score of 3 was highest in breast cancer. Contrary to published results prostate and pancreas adenocarcinomas showed a very low incidence of HER2 overexpression. CONCLUSIONS: Overexpression of HER2 is detected immunohistochemically in a proportion of epithelial neoplasms of diverse histogenesis in addition to ductal breast cancer. The standardized format of the assay will allow comparative analyses of studies performed at different institutions.     

eglA P-Her2/neu-IL-12融合基因穿梭载体的构建

目的:构建重组厌氧芽胞梭菌saccharobutylicum内源性β-1,4葡聚糖酶启动子(eglA p人)-表皮生长因子受体2胞外区(hHer2/neu ECD人)-白细胞介素12(rhIL-12)融合基因穿梭表达载体(pIMP1 eglA p-hHer2/neu ECD-rhIL-12),为进一步制备eglAp-hHer2/neu-rhIL-12融合基因修饰的厌氧芽胞梭菌,并探讨其对Her2/neu+实体肿瘤的基因治疗作用奠定基础。方法:以含有全长hHer2/neu序列的真核表达质粒(pcDNA3.1 hHer2/neu)为模板,采用PCR方法,扩增hHer2/neu ECD基因片段;将其插入重组rhIL-12真核表达质粒pcD-NA6 rhIL-12(p1)中的rhIL-12基因上游,获得pcDNA6 hHer2/neu ECD-rhIL-12(p2)真核表达质粒;设计合成eglAp基因中一段55 bp序列作为模板,通过连续PCR的方法,扩增eglAp片段(335 bp),并构建T-eglA p(p3)亚克隆质粒;通过酶切连接的方法,将eglA p片段插入p2质粒中的hHer2/neu ECD基因上游,...