人红细胞单链抗体基因的克隆及其序列分析

目的：获得抗人红细胞的单链抗体基因。方法：以分泌抗人红细胞单抗（ＨＲＣ）的杂交瘤细胞总ＲＮＡ为模板，分别扩增出轻链可变区（ＶＬ）和重链可变区（ＶＨ）基因，用连接肽将其连接成具ＶＬ－ｌｉｎｋｅｒ－ＶＨ结构的单链抗体基因。结果：ＤＮＡ序列分析证实，获得的单链抗体基因的轻，重链分别属于鼠Ｉｇｋａｐｐａ轻链第Ⅲ亚组和Ｉｇ重链第Ⅱｃ亚组，结论：构建了抗人红细胞单链抗体的载体，提供了与其它基因连接，为建立病人     

鼠抗人纤维蛋白抗体单链Fv片段的三维结构模建

用Ｂｉｓｙｍ公司开发的计算机辅助分子设计系统模建了鼠人纤维蛋白抗体Ｆｖ片段的三维结构。Ｆｖ是由重链可变区和轻链可变区两个结构域组成的具有抗原结合能力的最小抗体片段。先分别模建了Ｖｈ和Ｖ１两个结构域，然后搭建出Ｆｖ片段的整体三维结构，并对模建的结构进行了分子力学和动力学优化。     

抗结肠癌抗体重链可变区和κ轻链基因的克隆

目的：从一株抗结肠癌细胞系ＬＳ１７４Ｔ的单抗细胞株ＣＬ－４中克隆出抗结肠癌抗体重链可变区和κ轻链基因。方法：用一步法提取总ＲＮＡ，逆转录形成ｃＤＮＡ，设计并合成一套扩增小鼠抗体重链可变区和完整κ轻链的通用引物，通过ＰＣＲ扩增基因，分别克隆入ｐＵＣ１９，作核苷酸序列分析。结果：用重链引物扩增获得长约３６０ｂｐ的片段；用轻链引物扩增获得约６４０ｂｐ的片段，序列测定获得它们的ＤＮＡ序列。结论：克隆的重链可变区基因长度为３６０ｂｐ，属于小鼠重链可变区Ｉ（Ｂ）亚族；κ轻链长度为６４２ｂｐ，其中可变区为３２１ｂｐ，属于小鼠κ轻链Ⅴ亚族。     

纳米抗体在肿瘤分子显像中的应用

纳米抗体是一种由骆驼源的重链可变区组成的单域抗体,具有相对分子质量小、亲和力高和化学稳定性好等特性,非常适于进行肿瘤以及其他疾病的PET、SPECT显像和辅助治疗研究。纳米抗体在分子影像诊断方面的优势很可能使其成为新一代核医学显像剂。     

人CD34单链抗体基因的构建及序列测定

目的：获得抗人ＣＤ３４单链抗体基因。方法;选用一株分泌抗ＣＤ３４抗原单克隆抗体的杂交瘤细胞株,提取总ＲＮＡ,经ＲＴ－ＰＣＲ扩增出此抗本的重链可变区和轻链可变区基因,与Ｋａｂａｔ抗体库比国得到其框架区和互补决定区的氨基酸序列,重新合成两引引物去掉保留的分泌信号前导序列并加入适当的限制性内切酶酶切位点,再经ＰＣＲ得到所需要的重链可变区和轻链可变区基因,经一弹性连接肽连接构建成抗人ＣＤ３４的单链抗体（ｓ     

抗人CD40功能性嵌合抗体的制备及其高效表达

目的构建抗CD40嵌合抗体的真核表达载体,实现在真核细胞中的高效表达。方法采用RT-PCR法,从分泌鼠源抗人CD40抗体的杂交瘤细胞系5C11中钓取其轻、重链基因,进行序列测定。将5C11轻重链可变区基因分别克隆入表达载体pCMV-VH和pCMV-VL,构建成嵌合抗体（c5C11）的轻链真核表达载体5C11L-pCMV及重链真核表达载体5C11H-pCMV。将嵌合抗体的轻重链真核表达载体共转染入293T细胞,利用夹心ELISA法测定上清中c5C11的浓度,通过流式细胞术检测c5C11与膜抗原的特异性结合。通过生物信息学相关方法对c5C11的氨基酸序列进行合理改造,尝试在保持生物学功能的基础上提高c5C11的表达量。结果成功钓取鼠源单抗5C11的轻重链可变区基因并构建了真核表达载体,实现了c5C11的分泌表达;嵌合抗体较好地保留了鼠源母本抗体的抗原识别能力。利用计算机辅助设计,一定程度上提高了c5C11的表达量。结论为后续研究嵌合抗CD40抗体对B淋巴瘤等肿瘤的治疗提供了一定的依据。     

抗结肠癌抗体重链可变区和k轻链基因的克隆

目的：从一株抗结肠癌细胞系ＬＳ１７４Ｔ的单抗细胞株ＣＬ－４中克隆出抗结肠癌抗体重链可变区和ｋ轻链基因，方法：用一步法提取总ＲＮＡ，逆转录形成ｃＤＮＡ，设计并合成一套扩增小鼠抗体重链可变区和完整ｋ轻链的通用引物，通过ＰＣＲ扩增基因，分别克隆入ｐＵＣ１９，作核苷酸序列分析，结果：用重链引物扩增获得长的３６０ｂｐ的片段，用轻链引物扩增获约６４０ｂｐ的片段，序列测定获得它们的ＤＮＡ序列，结论：克隆的重链可     

人源抗-HAV全分子抗体在CHO细胞中的表达

目的 :构建人源抗_HAV全分子抗体的CHO细胞表达体系。方法 :将抗_HAV抗体轻链全分子 (信号肽与VL_CL)基因克隆至表达载体pCI_neo中 ;将重链信号肽、重链 (γ)VH_CH1 和Fc基因进行重组形成重链全分子基因 ,再将其克隆到真核表达载体pCdhfr1中。将轻、重链全分子表达载体共转染CHO dhfr_细胞 ,用G_4 18和甲氨蝶呤(MTX)筛选细胞克隆 ,ELISA法检测细胞培养上清中的抗_HAV活性 ,增加MTX浓度进行加压筛选。用蛋白L亲和层析柱从培养上清中纯化重组抗体。结果 :构建的真核表达载体可实现抗_HAV全分子抗体在CHO细胞中的分泌表达 ,纯化的重组抗体在还原SDS_PAGE中表现为相对分子质量约 2 5 0 0 0、5 0 0 0 0两条带 ;Western印迹表明 ,重组抗体全分子和重链分子均可和羊抗人Fc抗体发生特异性免疫反应。结论 :抗_HAV全分子抗体在CHO细胞中表达 ,为基因工程生产具有完整功能的全分子抗体奠定了基础     

抗人BlyS嵌合抗体的研制及功能鉴定

目的:在具有中和功能的母本抗体基础上,进行人源化改造,获得具有相同功能的嵌合抗体.方法:本研究以一株具有中和活性的抗人BlyS鼠单抗(B20)为母本,将抗人BlyS鼠单抗B20的轻、重链可变区基因分别插入到含有人κ链和IgG1重链恒定区基因的真核表达载体中,构建了人-鼠嵌合抗体表达载体,转染真核细胞,通过ELISA、RT-PCR、免疫印迹、体外中和实验分别对嵌合抗体(CB20)进行检测.结果:RT-PCR结果显示,转染后细胞系有人-鼠嵌合抗体mRNA的转录;ELISA、免疫印迹实验鉴定表明,该嵌合抗体与人BlyS特异性结合;体外中和实验表明,此抗体能中和BlyS对B淋巴细胞促增殖效应.结论:成功地构建人-鼠嵌合抗体CB20.     

人源抗HIV—1整合酶单链抗体基因的构建及表达

获得人源抗ＨＩＶ－１整合酶单链抗体基因并在大肠杆菌及细胞中进行表达。方法：从人的抗ＨＩＶ－１整合酶的Ｆａｂ抗体基因片段中通过ＰＣＲ扩出此抗体的重链可变区和轻链可变区基因,经一弹性连接肽连接构建成人源抗ＨＩＶ－１整合酶的单链抗体基因。     

生物样品中蓖麻毒素的双夹心酶联免疫定量检测方法及应用

目的建立定量检测生物样品中蓖麻毒素的双夹心ELISA方法并研究毒素在大鼠体内的分布。方法利用抗蓖麻毒素的单克隆抗体（MAb）3D74和辣根过氧化物酶（HRP）标记的小鼠抗蓖麻毒素单克隆抗体4C13,建立了定量检测血清和组织样品中蓖麻毒素的双夹心ELISA方法,并对方法学进行了必要的验证。结果建立的双抗夹心ELlSA方法检测纽织中蓖麻毒素的浓度范围为1．25～320-g／ml,最低定量限为2．5ngy／ml,日内和日间精密度分另Ij小于3．5％和9％。将该方法应用于蓖麻毒素组织分布的定量测定,结果显示,大鼠静脉染毒后毒素可以在心、肝、脾、肺、肾、肠和脂肪等组织分布,以脾脏的分布为最高,其次为肝脏,染毒后0．5和2h脾与血清的比值分别为3．9和7．2。在脑和肌肉中未检出毒素。结论建立的双夹心ELISA方法简便、灵敏,可以用于定量检测血清和组织等生物样品中的毒素含量,为蓖麻毒素的毒物代谢动力学研究及毒素中毒的快速检测提供了技术支撑。     

抗基孔肯亚病毒人-鼠嵌合抗体真核表达载体的构建和表达

目的：表达具有中和活性的抗基孔肯亚病毒人．鼠嵌舍抗体。方法：用RT-PCR法克隆具有中和活性的抗基孔肯亚病毒鼠源单克隆抗体CHIK-IF1的轻重链可变区基因，克隆人IgG1恒定区基因组基因，构建了轻重链嵌舍基因和表达质粒pCdhfrlL，pcDNA3、1H，共转染二氢叶酸还原酶缺陷型CHO细胞，用ELISA检测培养上清中嵌舍抗体的表达，MTX加压筛选表达量高的克隆，扩大培养收集上清并纯化，纯化的嵌合抗体进行SDS-PAGE、Western印迹检测和抗原结合活性的检测。结果与结论：在CHO细胞中成功表达了抗基孔肯亚病毒的嵌舍抗体，为制备抗基孔肯亚病的被动免疫制剂奠定了良好的基础。     

抗人红细胞单链抗体基因的克隆与表达

目的：获得抗人红细胞单链抗体（single-chain antibody,ScFv)基因,并在大肠杆菌中表达具有能与人红细胞特异性结合的ScFv。方法：以从分泌抗人红细胞单克隆抗体细胞株中提取细胞的总RNA为模板,利用扩增鼠源性抗体可变区基因的通用引物,通用RT－PCR分别获得轻链（VL）、重链（VH）可变区基因,并通过15个氨基酸的加接肽（Gly4Ser)3按VL－接头－VH的顺序连接起来,组建成ScFv基因。结果：核苷酸序列分析证明,获得的单链抗体基因VH基因属于小鼠抗体重链可变区基因家族的V（A）组,VL基因属于小鼠抗体k轻链可变区基因家族的IV组。与PGEX－5X－3表达载体上的GST融合蛋白表达后,在相对分子质量54000有一明显的空载体对照没有的蛋白带（ScFv28000,GST26000）。间接免疫荧光证明该表达产物能与人红细胞特异性结合。结论：获得抗人红细胞ScFv基因和具有抗体活性的抗人红细胞ScFv,为建立患者自身红细胞凝集试验奠定了基础。     

Designer genes: recombinant antibody fragments for biological imaging.

Monoclonal antibodies (MAbs), with high specificy and high affinity for their target antigens, can be utilized for delivery of agents such as radionuclides, enzymes, drugs, or toxins in vivo. However, the implementation of radiolabeled antibodies as "magic bullets" for detection and treatment of diseases such as cancer has required addressing several shortcomings of murine MAbs. These include their immunogenicity, sub-optimal targeting and pharmacokinetic properties, and practical issues of production and radiolabeling. Genetic engineering provides a powerful approach for redesigning antibodies for use in oncologic applications in vivo. Recombinant fragments have been produced that retain high affinity for target antigens, and display a combination of rapid, high-level tumor targeting with concomitant clearance from normal tissues and the circulation in animal models. An important first step was cloning and engineering of antibody heavy and light chain variable domains into single-chain Fvs (molecular weight, 25-27 kDa), in which the variable regions are joined via a synthetic linker peptide sequence. Although scFvs themselves showed limited tumor uptake in preclinical and clinical studies, they provide a useful building block for intermediate-sized recombinant fragments. Covalently linked dimers or non-covalent dimers of scFvs (also known as diabodies) show improved targeting and clearance properties due to their higher molecular weight (55 kDa) and increased avidity. Further gains can be made by generation of larger recombinant fragments, such as the minibody, an scFv-CH3 fusion protein that self-assembles into a bivalent dimer of 80 kDa. A systematic evaluation of scFv, diabody, minibody, and intact antibody (based on comparison of tumor uptakes, tumor:blood activity ratios, and calculation of an Imaging Figure of Merit) can form the basis for selection of combinations of recombinant fragments and radionuclides for imaging applications. Ease of engineering and expression, combined with novel specificities that will arise from advances in genomic and combinatorial approaches to target discovery, will usher in a new era of recombinant antibodies for biological imaging.     

Humanized versus murine anti-human epidermal growth factor receptor monoclonal antibodies for immunoscintigraphic studies

The anti-human epidermal growth factor receptor (EGF-R) humanized antibody h-R3 (IgG(1)), which binds to an extracellular domain of EGF-R, was used to evaluate the biodistribution on nude mice xenografted with A431 epidermoid carcinoma cell line. Results are compared with its murine version ior egf/r3 monoclonal antibody (mAb). Twenty-one athymic female 4NMRI nu/nu mice were injected intravenously with 10 microg/100 microCi of (99m)Tc-labeled mAbs. The mAb ior C5 that recognizes an antigen expressed preferentially on the surface of malignant and cytoplasm of normal colorectal cells was used as negative control. Immunoreactivity of (99m)Tc-labeled mAbs was measured by enzyme linked immunosorbent assay on A431 cell line and the immunoreactive fractions determined by Lindmo method. Among all organs significative accumulation was found in tumor (6.14 +/- 2.50 %ID/g, 5.06 +/- 2.61 %ID/g for murine and humanized mAbs, respectively) 4 h after injection. The immunoreactive fractions were found to be 0.88 and 0.81 for murine and humanized mAb, respectively. Thus, we expect better results using the humanized mAb h-R3 for diagnostic immunoscintigraphy.     

Radiobromination of anti-HER2/neu/ErbB-2 monoclonal antibody using the p-isothiocyanatobenzene derivative of the [76Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1-) ion

The monoclonal humanized anti-HER2 antibody trastuzumab was radiolabeled with the positron emitter (76)Br (T(1/2) =16.2 h). Indirect labeling was performed using the p-isothiocyanatobenzene derivative of the [(76)Br]undecahydro-bromo-7,8-dicarba-nido-undecaborate(1-) ((76)Br-NBI) as a precursor molecule. (76)Br-NBI was prepared by bromination of the 7-(p-isothiocyanato-phenyl)dodecahydro-7,8-dicarba-nido-undecaborate(1-) ion (NBI) with a yield of 93-95% using Chloramine-T (CAT) as an oxidant. Coupling of radiobrominated NBI to antibody was performed without intermediate purification, in an "one pot" reaction. An overall labeling yield of 55.7 +/- 4.8% (mean +/- maximum error) was achieved when 300 microg of antibody was labeled. The label was stable in vitro in physiological and denaturing conditions. In a cell binding test, trastuzumab remained immunoreactive after labeling.