抗胃癌鼠单抗3G9 Fab段基因的克隆及其在大肠杆菌…

本文报道用聚合酶链延伸反应（ＰＣＲ）从分泌抗胃癌鼠单抗的杂交瘤细胞系３Ｇ９中克隆出了重链Ｆｄ段和ｋ链的基因。ＤＮＡ序列测定表明３Ｇ９的ＶＨ、Ｄ、ＪＨ分别属于ＶＨ７１８３、ＤＦＬ１６．１和ＪＨ３；Ｖｋ和Ｊｋ分别属于ＶｋⅡ和Ｊｋ２。将３Ｇ９Ｆｄ段和ｋ链ｃＤＮＡ克隆到表达载体ｐＣｏｍｂ３中，在大肠杆菌中获得了表达，用还原和非还原的ＳＤＳ－ＰＡＧＥ电泳作免疫印迹实验，证明了Ｆａｂ段的表达。并用酶联免过滤实     

抗人TNF-α单抗基因的克隆及鉴定

目的克隆抗人ＴＮＦ－α鼠单抗得可变区基因以构建人－鼠嵌合抗体表达载体。方法采用ＲＴ－ＰＣＲ技术,以前导肽序列的引物从１个分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆抗体轻链、重链可变区基因（Ｖκ,ＶＨ）,在大肠杆菌中表达Ｆａｂ段核实其功能活性。结果分别得到了２个Ｖκ和２个ＶＨ基因。ＤＮＡ序列测定表明,其中１个轻链可变区基因为骨髓瘤细胞系中固有的无功能基因。１个重链可变区基因经原核系统表达测活表明无抗体活性。另一个轻链和重链可变区基因的成熟蛋白编码部分与从第一骨架区引物所克隆的、可在大肠杆菌表达出抗体活性的Ｖκ、ＶＨ序列相符。将该轻链、重链基因分别克隆到了人－鼠嵌合轻链、重链表达载体中。结论通过原核表达系统核实,获得了抗ＴＮＦ－α单抗的可变区基因     

抗胃癌鼠单抗3H11V区基因的克隆及人—鼠嵌合轻链的表达

利用前导肽序列设计引物，采用ＲＴ－ＰＣＲ技术从分泌抗人胃癌的单抗杂交瘤细胞系３Ｈ１１分离克隆了抗体的轻重链可变区基因序列。经ＤＮＡ序列分析表明所获基因含有全部前导序列，其成熟蛋白编码部分与从第一骨架区引物所克隆的序列相符。将轻链基因组建到人－鼠嵌合轻链表达载体中，转染至小鼠骨髓瘤细胞Ｓｐ２／０中，可获得人鼠嵌合轻链的表达，证明所克隆的基因具有表达活性，为人－鼠嵌合抗体的构建奠定了基础。     

抗p185单抗5E12Fab段基因的克隆及表达

目的：克隆抗p185单抗5E12的Fab段基因并在原核细胞进行表达。方法：用逆转录．聚合酶链反应技术（RT-PER），以可变区第一骨架区的通用引物从分泌抗p185单抗的杂交瘤细胞系5E12中克隆Fd段和K链的基因，重组到Fab表达载体中，在大肠杆菌中表达噬菌体抗体和可溶性Fab；根据前导肽序列设计引物，通过PER介导的定点突变将V区基因氨基端序列恢复为5E12的原始序列；以NIH3T3／erbB-2细胞ELISA法、免疫组化法等进行特异性鉴定。结果：以第一骨架区的通用引物从小鼠杂交瘤细胞5E12中克隆到Fd段和k链的基因，在大肠杆菌中表达出Fab段抗体但无特异性抗原结合活性，分别将Fd段和k链V区基因的氨基端序列矫正为亲本单抗的原始序列后，恢复了Fab段的抗原结合活性。单独恢复Fd段可变区氨基端序列可恢复抗原结合活性。但同时恢复k链后活性却有所下降。结论：成功构建了抗p185小分子抗体Fab并进行功能性表达，为进一步构建抗p185鼠单抗其他小分子抗体及其人源化改造打下基础；进一步证实抗体氨基端序列对抗体活性的重要性，为今后以PER方法构建小分子抗体的工作提供了有益的借鉴。     

TNF-α单抗Fab段基因的克隆及其在大肠杆菌的表达

目的进行ＴＮＦ－α单抗Ｆａｂ段基因的克隆并在大肠杆菌中表达。方法用逆转录－聚合酶链反应技术（ＲＴ－ＰＣＲ）从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆重链Ｆｄ段和κ链基因；并用ＥＬＩＳＡ和免疫印迹分析证明表达的Ｆａｂ段特异结合ＴＮＦ－α。结果从分泌抗人ＴＮＦ－α的鼠单抗杂交瘤细胞系中克隆出了重链Ｆｄ段和κ基因。经ＤＮＡ序列测定表明，ＶＨ、Ｄ、ＪＨ分别属于ＶＨ３Ｄ、ＤＳＰ２和ＪＨ４；Ｖκ和Ｊκ分别属于Ｖκ１和Ｊκ１。将该Ｆｄ和κ链ｃＤＮＡ克隆到表达载体ｐＣｏｍｂ３Ｈ中，在大肠杆菌中获得了表达。结论ＥＬＩＳＡ和免疫印迹分析表明，表达的Ｆａｂ段可特异地和ＴＮＦ－α结合。     

抗HBs人VH基因中异常序列对抗体活性影响的研究

目的 从噬菌体抗体库中克隆到１株ＶＨ第３骨架区中含有异常序列的抗ＨＢｓ人Ｆａｂ基因,探讨该ＶＨ基因中异常序列对抗体活性的影响。方法 采用重叠ＰＣＲ方法去除了这段异常序列,构建表达载体,转化大肠杆菌表达出抗ＨＢｓＡｇ噬菌体抗体和可溶性Ｆａｂ,测定了它们与抗原的结合活性。结果 与原抗体比较,改造后的抗体与抗原的结合活性明显下降,分子模建提示这段异常序列对ＶＨ ＣＤＲ１和ＣＤＲ２的部分氨基酸残基的位置有     

嵌合抗CD20抗体Fab片段在大肠杆菌中的表达及活性鉴定

目的：构建抗ＣＤ２０嵌合抗体Ｆａｂ片段表达载体,并在大肠杆菌中进行高效可溶性分泌表达。结果：利用ＰＣＲ方法从抗ＣＤ２０单链抗体（ＳｃＦｖ）表达载体上扩增抗ＣＤ２０抗体轻链可变区基因（ＶＬ）、重链可变区基因（ＶＨ）,然后将ＶＨ、ＶＬ基因重组到Ｆａｂ表达载体ｐＹＺＦ中,构建抗ＣＤ２０Ｆａｂ表达载体ｐＹＺＦ１ｃｄ２０,并在２７Ｃ７菌中高效表达。结果：经Ｆａｂ表达载体转化的２７Ｃ７菌株,进行表达培养,经分     

从人源性噬菌体抗体库分离出1株含有异常序列的抗HBsAg Fab克隆

曾从人源性噬菌体抗体库中筛选出１株抗人乙肝表面抗原的Ｆａｂ我隆，为了筛选出新的抗ＨＢｓＡｇＦａｂ段，采用抗原屏蔽法，用已得到的Ｆａｂ段封闭相应的抗原决定基，对该抗体库进行了再次筛选，得到了１株新的人抗ＨＢｓＡｇＦａｂ段克隆经序列分析发现其轻链可变区基因来源于Ｖｋ１亚群和Ｊｋ４基因，重链可变区基因来源于ＶＨ１亚群和ＪＨ４基因，但在ＶＨ第７７位和第７８位氨基酸基之间出现了７个多余的氨基酸残基，经对基因     

将抗体库所获抗-HBs Fab转换为全长人IgG

目的 将大肠杆菌表达的抗－乙肝表面抗原（ＨＢｓ）人Ｆａｂ转换为真核表达的全长人ＩｇＧ１。方法 用重叠ＰＣＲ法将人工合成的人Ｖｋ前导序列拼到抗－ＨＢｓ的ＶＨ和ＶＫ上,构建人ＩｇＧ１真核表达载体。转染真核细胞,通过ＥＬＩＳＡ、ＲＴ－ＰＣＲ、免疫印迹检测抗－ＨＢｓ人ＩｇＧ的表达。结果 用轻链和重链同时表达的单一载体在ＣＨＯ细胞中获得了抗－ＨＢｓ人ＩｇＧ的表达。结论 通过噬菌体抗体库所获Ｆａｂ段可经拼接人     

抗菌肽MagaininⅡ单链抗体基因的构建及其表达

目的 将克隆的抗体轻重链可变区基因拼接成单链抗体基因并将其在大肠杆菌中表达。方法 通过ＲＴ－ＰＣＲ从两株抗ＭａｇａｉｎｉｎⅡ杂交瘤细胞株（２Ｄ１,３Ｆ８）中克隆出ＶＨ和ＶＬ基因,然后利用重组ＰＣＲ技术,将ＶＨ和ＶＬ基因通过柔性肽段（ＧＬＹ４Ｓｅｒ）３的Ｌｉｎｋｅｒ拼接成单链抗体基因（ＳｃＦｖ）,将ＳｃＦｖ克隆到表达载体ｐＣＡＮＴＡＢ５Ｅ上并将其分别在大肠杆菌Ｅ．ｃｏｋｉ ＨＢ２１５１及ＴＧ１中进行     

基因工程抗体分泌影响因素研究进展

基因工程抗体（gene engineering antibody，GEAb）是继多克隆血清和单克隆抗体之后的第三代抗体，在许多医学领域都极具应用潜力。众所周知，预防和治疗感染性疾病常用的药物是疫苗和抗生素，但对于如SARS、获得性免疫缺陷综合征（AIDS）等难以获得相应疫苗或疫苗治疗效果不理想的病毒感染，目前仍缺乏有效的治疗方法，     

鼻咽癌抗独特型单克隆抗体的制备和鉴定

用针对鼻咽癌癌细胞相关抗原的单克隆抗体Ｆｃ１（Ａｂ１）作免疫原，采用常规免疫和杂交瘤制备方法，获得了一株抗Ｆｃ１Ｖ区独特型的杂交瘤：２Ａ９。双夹心ＥＬＩＳＡ显示２Ａ９所分泌的抗体（Ａｂ２）对Ｆｃ１有效强的亲和力。ＥＬＩＳＡ结合抑制试验结果显示，Ａｂ２能抑制Ｆｃ１对鼻咽癌细胞ＣＮＥ１的反应。这就证明Ａｂ２作用于Ｆｃ１抗体的Ｖ区。用Ａｂ２与钥孔戚血蓝素（ＫＬＨ）交联物免疫小鼠产生的抗－抗独特型抗体（Ａｂ３）的血清，能与Ｆｃ１竞争ＣＮＥ１细胞株上的原始靶抗原。免疫组化证实，Ａｂ３与Ｆｃ１对鼻咽癌细胞有相同的反应，均为细胞膜染色。可以看出Ａｂ３与Ａｂ１（Ｆｃ１）具有相同的配位。以上结果表明：２Ａ９杂交瘤细胞所分泌的抗独特型抗体Ａｂ２是带有鼻咽癌相关抗原内影像的单克隆抗体。     

Epstein-Barr-Virus- und lymphocytotoxische Serumantikörper in Sarkoidose-Patienten

Summary Sera from 47 patients with sarcoidosis were examined for antibodies to Epstein-Barr virus, 63.8% of sarcoidosis patients were positive, against 74% of the clinical and 36% of the normal controls. The incidence of moderately elevated EBV-antibody titers in sarcoidosis patients was significantly (p<0.05) different from that in the controls. No significance, however, was found between sarcoidosis patients and clinical controls. It is concluded that the EB-virus does not play a major etiologic role in sarcoidosis. The slightly elevated EBV-antibody titers in sarcoidosis may rather be due to an increased humoral activity to certain antigens as described previously.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft und das Landesamt für Forschung, Nordrhein-Westfalen.     

Effect of Plasmodium yoelii YM Infection on Vaccination with 19 kDa Carboxylterminus of the Merozoite Surface Protein 1 （MSP1 19）

The 19 kDa carboxylterminal fragment of merozoite surfaceprotein 1 (MSP119) is a leading malaria vaccine candidate[1]. Immunization of monkeys [ 2 , 3 ] or mice [ 4 , 5 ]with recombinant MSP119 confers protection against chal-lenge infection. Studies in m…     

Anti-5-hydroxytryptamine antibodies: studies on their cross-reactivity in vitro and their immunohistochemical specificity

Summary Antibodies to 5-hydroxytryptamine (5-HT) were obtained from 4 rabbits after injections of 5-HT coupled to bovine serum albumin by means of paraformaldehyde (PF). Two methods were used to monitor the developement of antibodies (AB): the one based on the in vitro competitive binding properties of the antibodies with³(H)5-HT, the other, on their in situ binding properties to endogenous 5-HT, using the peroxidase-anti-peroxidase immunohistochemical technique, applied to paraffin embedded sections of cat brainstem. No pharmacological processing, detergents or proteolytic enzymes were used. The specificity of the antiserum was tested by competitive procedures with 20 analogs using the in vitro and in situ techniques. In vitro studies were performed with 5-HT free analogs and with analogs previously coupled with PF to lysine. Radioimmunological tests showed that the antibodies recognize mainly the ethylamine (CH₂-CH₂-NH₂)-cham of the free analogs and that the best specificity was obtained with the 5-HT conjugate (5-HT-lysine-PF). The results suggest that the hapten is coupled through the phenolic positions C4 or C5. The in situ immunohistochemical extinction assays also revealed a distinct specificity for 5-HT. Possible optical and ultrastructural applications are illustrated in the raphé nuclei of the cat. These results confirm the reliability of radioimmunological tests for studying the specificity of AB directed against haptens, provided that haptens and analogs tested were first chemically transformed to resemble the immunogen (herewith lysine-PF coupling) with regard to its antigenic structure.     

抗红细胞双价小分子抗体的构建及表达

单链抗体（ＳｃＦｖ）是一个重链可变区（ＶＨ），一个轻链可变区（ＶＬ）由连接肽（Ｌｉｎｋｅｒ）连在一起构成的单价小分子抗体。为使其能组装成双价，对一个抗人红细胞的单链抗体进行了改造，将其连接肽由１７个氨基酸〔ＳＲ（ＧＧＧＧＳ）３〕缩短为６个氨基酸（ＳＲＧＧＧＳ），强迫不同分子间的ＶＨ和ＶＬ组合成Ｆｖ，从而形成双价小分子抗体（Ｄｉａｂｏｄｙ）。在大肠杆菌中分泌型表达后，显示具有血球凝集活性，证明其为双价。进一步用凝胶过滤分析，显示改建后抗体分子的分子量约为原单链抗体的两倍。     

Tyrosine Phosphatase-like Protein (IA-2) and Glutamic Acid Decarboxylase (GAD65) Autoantibodies: A Study of Chinese Patients with Diabetes Mellitus

Type 1 diabetes in most Asian populations may not have a salient autoimmune basis when assessed with single determinations of the major markers, islet cell antibodies (ICAs) and glutamic acid decarboxylase antibodies (GAD65ab). With the inclusion of antibodies to tyrosine phosphatase-like protein IA-2 (IA-2ab) as an additional major marker, we re-examined autoimmune diabetes in a group of Chinese patients. We studied 272 subjects at various stages of disease with blood samples procured for biochemical analysis. ICAs were measured by immunofluorescence, GAD65ab and IA-2ab by radioimmunoassay. Sixty-seven patients fulfilled clinical diagnosis of type 1 diabetes and the remaining 205 patients were type 2. Prevalence of single autoantibody type in recent-onset type 1 diabetes (<1 year duration; n =47 ) showed 10.6% with ICAs, 44.7% GAD65ab and 36.2% IA-2ab. GAD65ab account for more than two-thirds of the markers found in type 1 diabetes. Combined analysis further showed that 51.1% had at least one antibody type, 31.9% with two or more antibodies and 8.5% with all three antibodies. Islet autoimmunity presence in childhood-onset type 1 diabetes improved with the addition of IA-2ab, though less impact was seen in the adult-onset. Similarly, combined analysis for type 2 patients with recent diabetes showed a modest increase to 13% with islet autoimmunity compared to 8% when assessed by GAD65ab alone. Combining IA-2ab and GAD65ab assays results detected slightly more immune-mediated diabetes, compared to using a single GAD65ab determination. Non-autoimmune causes need to be considered in the pathogenesis of type 1 diabetes in Chinese, particularly in adults.     

习惯性流产与几种自身抗体的关系

本文对３１６例不明原因的习惯性流产者，用酶联免疫分析进行抗心磷脂抗体（ＡＣＡ）、抗血小板抗体（ＰＡ）及抗脱氧核糖核酸抗体（抗ＤＮＡ）测定，并与正常人比较。结果流产组ＡＣＡ、ＰＡ、抗ＤＮＡ阳性率分别为２３．７％、１６．７％、１３．６％，均显著高于正常对照组（Ｐ分别为〈０．０１，〈０．０１，〈０．０５），其中以ＡＣＡ－ＩｇＧ、ＡＣＡ－ＩｇＭ、ＰＡ－ＩｇＧ、ＰＡ－ＩｇＭ及ｓｓ－ＤＮＡ为主，它们与习惯性流     

Production and characterization of a human monoclonal anti-idiotype to anti-ribosomal P antibodies

Autoantibodies to ribosomal P protein (anti-P) are a specific hallmark of systemic lupus erythematous (SLE). Several authors found significant associations of anti-P antibodies with neuropsychiatric, hepatic, and renal disease. We now report the isolation by phage display of human anti-idiotype (Id) monoclonal antibody fragments as single-chain Fv fragment (scFv) against anti-P antibodies. The V gene repertoires were derived from the RNA obtained from the B cells of a SLE patient. Affinity-purified anti-P antibodies were used for the selection of bacterial clones producing anti-P-specific scFv antibody fragments and little reactivity with normal IgG and other IgG antibodies. The anti-Id antibody recognizes a public idiotope broadly cross-reactive with polyclonal anti-P antibodies and inhibited binding of anti-P to ribosomal P antigen in immunoassays and on Jurkat cells. The anti-Id scFv antibody fragment may have therapeutic implications in SLE. They may also be used as probes in the study of the structure of the idiotype.     

白芍总甙对免疫系统的影响

白芍总甙(TGP)对羊红细胞免疫的小鼠脾淋巴细胞体外诱生溶血素(IgM)抗体和刀豆素A诱导小鼠脾淋巴细胞体外增殖反应均呈现低浓度(<0.4μg/ml)促进和高浓度(>0.4μg/ml)抑制的双向调节作用。TGP(5mg/kg,ip)可明显促进辐照加输注受训胸腺细胞体内诱导抗原特异性Th细胞。以上结果提示,TGP对免疫系统的影响有明显的浓度与机能依赖性特征。