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     

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。     

改进的锚定反转录PCR法克隆中国人庚型肝炎病毒3′末端基因

应用改进的锚定反转录ＰＣＲ（ａｎｃｈｏｒｅｄＲＴ－ＰＣＲ）法，在总ＲＮＡ的３′端“加尾”，Ｏｌｉｇｏｔｅｘ提纯ｐｏｌｙ（Ａ）＋ｍＲＮＡ，Ｏｌｉｇｏｄ（Ｔ）－ａｎｃｈｏｒ引物合成第１链ｃＤＮＡ，ａｎｃｈｏｒ引物及特异引物进行“半巢式”ＰＣＲ，从献血员血清中扩增出ＨＧＶ３′末端基因片段。序列分析结果表明：此中国株ＨＧＶ３′末端基因序列与美国株存在较大差异，但阅读框架相似。用此方法克隆单链ＲＮＡ病毒基因组的３′末端。由于许多动物病毒和９０％以上的植物病毒均为单链ＲＮＡ基因组，故该技术也可应用于其它病毒基因组未知末端的基因克隆。     

PCR介导的大鼠再生肝cDNA文库构建

简介了ＰＣＲ技术介导的ｃＤＮＡ文库构建方法，其基本原理是在第一链ｃＤＮＡ３’端连接多聚Ｇ，进而以ｏｌｉｇｏ（ｄＴ）和ｏｌｉｇｏ（ｄＣ）为引物，对第一链ｃＤＮＡ进行扩增，这一技术可能在分离亚细胞群特异基因中有重要作用。     

登革病毒NS1基因的平端PCR产物的快速克隆

用ＰＣＲ产物进行克隆已广泛用于分子生物学的各个领域。ＰＣＲ产物的克隆主要通过两个途径：其一，在引物的３’末端加上限制性内切酶的识别序列，使扩增片段产生粘性末端，再连接到有相应末端的载体中去。但是，由于许多限制酶对处在ＤＮＡ片段末端的识别序列的作用效率远较处于内部的序列为低。因此，该方法在实际应用中常常不能获得满意的结果。其二，用ＰＣＲ产物进行平端连接，这种方法无需酶切，可适用于任何扩增产物，故应用较广。但由于ＴａｑＤＮＡ聚合酶具有不依赖模板的末端转移酶活性而导致扩增片段３’端带有腺苷酸残基，而使…     

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     

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     

中国大陆庚型肝炎病毒结构区与非结构区部分cDNA基因的序列分析

作者在建立检测庚型肝炎病毒酶免疫和ＰＣＲ技术的基础上，将抗－ＨＣＶ上阳性病人血清经逆转录－巢式ＰＣＲ分离部分ＨＧＶｃＤＮＡ片段，用与ＨＧＶ基因互补的特异寡核苷酸引物进行双脱氧核苷酸链末端终止法－ＰＣＲ直接测序。结果表明，中国大陆ＨＧ－３０２Ⅰ株部分ｃＤＮＡ序列与Ｌｉｎｎｅｎ等报道的ＨＧＢ美国株ｃＤＮＡ序列有极高的同源性。     

用多重及套式PCR同时检测血清中的HBV和HCV方法的建立

首次应用多重及套式ＰＣＲ技术同时检测人血清中的ＨＢＶ和ＨＣＶ。将抽提的ＨＢＶＤＮＡ和（或）ＨＣＶＲＮＡ在含ＡＭＶ逆转录酶、ＴａｑＤＮＡ多聚酶以及ＨＢＶ和ＨＣＶ外套引物的ＰＣＲ缓冲引物的ＰＣＲ缓冲液中进行逆转录后连续进行ＰＣＲ扩增，以第一轮扩增产物为模板，在含ＨＢＶ和ＨＣＶ内套引物的ＰＣＲ反应体系中进行第二轮扩增，产物经电泳后，以ＤＮＡ分子量标志物或已知片段做参考，出现５２３ｂｐ和（或）２６０ｂｐ产     

人源宋内痢疾杆菌脂多糖抗独特型抗体的筛选和基因结构特点分析

将纯化的宋内痢疾杆菌脂多糖的鼠单克隆抗体（５Ｂ１２）固相化，然后对混合噬菌体抗体库（ＨＩＶ、ＨＡＶ、ＨＢＶ）进行５轮“吸附－洗脱－扩增”的淘筛，使特异性噬菌体抗体得到了富集，阳性率达５３．１３％。竞争抑制试验证明，其中６株与宋内痢疾杆菌脂多糖的竞争抑制率大于５０％。酶切电泳图谱显示，淘筛的阳性克隆均无轻链基因，重链基因约４００ｂｐ。对分离的两株阳性克隆进行序列分析研究，发现这两个克隆ＤＮＡ序列相同，均为人免疫球蛋白重链可变区序列，属人ＩｇＧ基因Ⅲ家族，长度为３８４ｂｐ，与期望的６６０ｂｐ相差２７６ｂｐ。但这种重链部分缺失和轻链完全缺失的抗体片段却有明确的与５Ｂ１２的结合活性。     

Mouse-specific antibody responses to a monoclonal antibody during repeated immunoscintigraphy investigations: Comparison of antibody titres and imaging studies in a rat model

As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 gmg/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 g, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both¹³¹I-labelled antibody and with antibody labelled with¹¹¹In by diethylenetriamine-penta-acetic acid (DTPA) chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both¹³¹I- and¹¹¹In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given¹¹¹In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable.     

Mouse-specific antibody responses to a monoclonal antibody during repeated immunoscintigraphy investigations: comparison of antibody titres and imaging studies in a rat model

As a model for human mouse-specific antibody responses in patients undergoing immunoscintigraphy, we have investigated in rats the production of mouse-specific antibodies (MA) to the mouse monoclonal antibody 791T/36. At intervals of between 5 and 16 weeks the rats were given repeated cycles of intravenous (IV) injections of antibody with or without a simultaneous intradermal (ID) injection. The IV dose was 60 micrograms/kg, a dose similar to that used in many clinical immunoscintigraphy studies. The ID injection was 2 micrograms, which mimicks the skin test dose often given in clinical imaging protocols. The study was carried out with both 131I-labelled antibody and with antibody labelled with 111In by diethylenetriamine-penta-acetic acid (DTPA) chelation. MA was measured with a passive haemagglutination assay using sheep red blood cells coated with the monoclonal antibody. Of rats given ID injections of unlabelled antibody at the same time as the IV imaging doses, 9/20 produced MA during 4 cycles of injections. In contrast, only 2/16 rats given only the IV dose produced MA. Both 131I- and 111In-labelled antibody appeared equally immunogenic with 5/18 and 6/18 overall responders, respectively. The production of MA was associated with a significant perturbation in the biodistribution of the IV dose of labelled antibody as seen by gamma-camera imaging of the rats given 111In-labelled antibody. There was clearance of immune complexes to the liver, this organ accumulating up to 90% of the whole body count rate of radiolabel. MA titres of between 1/100 and 1/78,000 caused equal perturbation of biodistribution, although below 1/100 the effect was more variable.     

抗原化抗体及其应用

抗原化抗体是指在抗体分子可变区的互补决定区中插入外源蛋白寡肽的人工构建抗体，本文综述了抗原化抗体的性质，构建方法，应用及展望。     

Monoclonal antibody hapten radiopharmaceutical delivery

One hundred micrograms of monoclonal antibody (MoAb) CHA255 with a binding constant Kb of 4 X 10(9) was complexed with indium-111 labelled BLEDTA II, BLEDTA IV, benzyl EDTA, and an EDTA conjugate of Fab. The 24-h tumour and organ distribution of BALB/c mice bearing KHJJ tumours was studied for each compound alone, the antibody complex, and 3 h following a chelate chase of the antibody complex. Whole body biological half-life was measured for 7 days with and without a chelate chase for each antibody complex. The 24-h whole body counts dropped 20 to 60% and blood concentration fell over 89% within 3 h of administering the chelate chase. Theoretical equivalent human organ doses were calculated from the 24-h organ concentrations, effective half-life, and MIRD 11 S values (absorbed dose per cumulated activity). Liver and spleen were the target organs, with the dose ranging from 0.50 to 3.91 rads mCi-1. The reduction in organ radiation dose varied up to 95% following the chelate chase. Rapid selective renal clearance of chelate labelled radiopharmaceuticals by competitive inhibition (chelate chase) of their reversible binding to monoclonal antibodies enhances tumour imaging and improves the radiation dosimetry.     

新型抗EGFR人源化抗体基因的构建和表达

目的：构建并表达进一步人源化且效果显著增强的抗EGFR抗体。方法：通过计算机辅助设计的结果,合成新型抗EGFR抗体的轻链和重链可变区序列,拼接成完整的轻链和重链基因并克隆入pIRES双表达载体。瞬时转染293T细胞,用rProteinA亲和层析柱从细胞培养上清中纯化抗体,并进行SDS-PAGE和免疫印迹鉴定。采用Biacore3000技术检测抗体结合抗原的能力;细胞侵袭实验初步检测抗体的功能。结果：成功构建3种不同突变体表达载体C2、C3和C5,纯化的抗体在还原SDS-PAGE中表现为相对分子质量约为25×103和50×103两条带;免疫印迹分析表明,该人源化抗体可与羊抗人IgG特异性结合。Biacore3000实验结果表明,C3抗体与抗原具有良好亲和活性（亲和力为6.13×10-10mol/L）;细胞侵袭实验结果表明,C2和C3抗体对肿瘤细胞生长迁移均具有一定的抑制作用。结论：成功构建、表达了3种抗EGFR人源化抗体C2、C3和C5,其中C3具有良好的抗原结合能力和抑制肿瘤细胞生长迁移能力。     

抗体可变区稳定性评价及其初步应用

目的：通过研究抗体轻、重链间的结合能与其构象特征、理化性质间的内在关系,建立相应的数学模型,合理评价抗体分子的热稳定性,为抗体分子的设计、合理改造及亲和力成熟奠定基础。方法采用生物信息学和计算生物学相关方法对已有晶体结构的抗体结构信息进行分析,通过距离几何学、计算机图形学技术对抗体可变区的构象特征进行研究;基于分子间氢键形成理论、反应自由能理论,从热力学、动力学对抗体分子轻、重链可变区相互作用的动态结构及能量特征进行探讨;借助统计学原理、非线性拟合、回归分析等分析手段对抗体轻、重链作用能与其构象特征、理化性质建立相关性分析。结果通过分子模拟与统计学分析,抗体的轻、重链可变区结合能与其轻、重链间的氢键形成数目、范德华作用均存在线性相关性;与抗体理化性质存在基于多项式的非线性相关性。基于关键位置氨基酸的使用频率以及建立的分析模型,针对无法获得稳定工程细胞株的抗蓖麻毒素人源化功能抗体进行合理的评价及优化,借助理论指导,获得抗蓖麻毒素人源化抗体稳定的工程细胞株。结论抗体可变区的自身结构（构象、理化性质）对其稳定性有很大影响,可以通过抗体结构优化来提高抗体的稳定性。     

5441名应征入伍青年抗HIV检测结果分析

艾滋病感染者在我国逐年增加,其感染对象主要为青壮年。军队是一个担负特殊任务的主体。为保证兵员质量及所有官兵具有健康的体魄,对每年进入部队的新成员进行抗—HIV的普查十分必要。笔者仅以本年度入伍新兵抗—HIV普查情况报告如下。1 材料与方法1.1 凋查对象 均系本年度应征入伍青年。兵员分别来自四川、贵州、云南、广东、江苏、辽宁以及山东等地区;年龄为18～22岁,男性5347例,女性94例。血样标本均为当日空腹抽血,并及时送检。     

Analysis of monoclonal antibody uptake in tumours

Power (y = axb) and linear (y = a + bx) equations were derived, relating percentage uptake (y) of radionuclide tagged specific (SP) and non-specific (NSP) monoclonal antibody (MoAb) and blood flow (BF) to tumour weight (x) in animal models in the literature. Power equations were more significant than linear equations, that is, MoAb uptake and blood flow did not increase linearly with tumour weight. The power equations were similar to the equation relating surface area (SA) to the volume (V) of a sphere (SA = 4.84 V0.67), but with some categorical differences, namely aSP greater than aSA greater than aNSP, aBF and bSA greater than bNSP bBF.bSP was variable. These results can be correlated with tumour physiology. a and b for non-specific MoAb uptake were similar to those for blood flow, suggesting antibody uptake is blood-flow related. Tumours are vascularized from their surface, explaining their relationship to surface area. Autoradiographs show that the high aSP values are due to preferential uptake of specific MoAb by tumours.