抗HBsAg人IgG表达载体的构建及其在CHO细胞中的表达

目的 尝试将１株来源于人源性噬菌体抗体库的抗ＨＢｓＡｇ人Ｆａｂ,转换成完整的抗ＨＢｓＡｇ人ＩｇＧ。方法 采用重叠ＰＣＲ方法,在抗ＨＢｓＡｇ人Ｆａｂ和Ｖκ的ＶＨ基因的５’端接上人工合成的人κ链的前导序列,构建表达完整ＩｇＧ１的真核表达载体,转染ＧＨＯ（ＤＨＦＲ＾－）细胞,用ＥＬＩＳＡ、ＲＴ－ＰＣＲ和免疫印迹检测抗ＨＢｓＡｇ人ＩｇＧ的表达。通过ＤＨＦＲ／ＭＴＸ体系及金属离子诱导提高Ｉｇ表达。结果：获得     

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     

通过受体介导法将核酶特异导入肝细胞以阻断HBV蛋白表达的初步研究

应用半乳糖末端糖蛋白受体（ＡＳＧＰ－Ｒ）介导的内吞作用，将外源基因导入真核细胞，与脂质体介导的转染和细胞表面转铁蛋白受体（Ｔｆ－Ｒ）介导的内吞作用相比，虽然三种方式均能有效介导外源基因的转移，但ＡＳＧＰ－Ｒ法具有肝细胞特异性，而脂质体法和Ｔｆ－Ｒ法不具此特性。将克隆于真核表达载体的针对乙型肝炎病毒（ＨＢＶ）ｍＲＮＡＰｒｅＣ／Ｃ区的核酶质粒ｐＣＭＶ－Ｒｉｐｃ特异性导入肝细胞并发挥作用，通过酶联免疫吸附法（ＥＬＩＳＡ）检测细胞培养液中的乙型肝炎表面抗原（ＨＢｓＡｇ）和ｅ抗原（ＨＢｅＡｇ），评价核酶在细胞水平对ＨＢＶ抗原表达的阻断作用。结果表明当核酶质粒ｐＣＭＶ－Ｒｉｐｃ与ＨＢＶ抗原表达质粒ｐＵＣ－２ＨＢＶ共转染ＨｅｐＧ２细胞时，核酶对ＨＢｓＡｇ和ＨＢｅＡｇ表达的抑制率分别为５５．２９％和６８．７３％。     

HBV感染与IgA肾病肾小管间质病变的关系

目的探讨ＩｇＡ肾病ＨＢＶ感染与肾小管间质病变的关系。方法利用原位分子杂交（ＨＢＶＤＮＡ）、免疫组化（ＨＢＡｇ、ＣＤ３、ＣＤ８）以及ＨＢＶＤＮＡＨＢＡｇ和ＨＢＡｇＣＤ４３双标记技术，对９１例ＩｇＡ肾病肾穿刺标本进行研究。结果肾组织内ＨＢＡｇ阳性率为６９．２％。ＨＢＶＤＮＡ原位杂交阳性率为４２９％。ＨＢＶＤＮＡ阳性的病例，双重标记染色发现ＨＢＶＤＮＡ阳性的肾小管上皮细胞可表达ＨＢｃＡｇ或／和ＨＢｓＡｇ。ＨＢＶ感染标记（ＨＢＶＤＮＡ、ＨＢｃＡｇ、ＨＢｓＡｇ）阳性组ＣＤ３阳性细胞和ＣＤ８阳性细胞数明显高于阴性组（Ｐ＜００１），并可见数量不等的Ｔ淋巴细胞入侵ＨＢｃＡｇ及ＨＢｓＡｇ阳性肾小管管壁或围绕其周围。结论感染ＨＢＶ的肾组织细胞能够表达ＨＢＡｇ，并诱导ＣＤ３阳性细胞和ＣＤ８阳性细胞浸润，从而加重肾小管、间质损害。ＨＢＶ感染对ＩｇＡ肾病的发生发展可能起着重要作用     

抗人Clq轻链可变区基因在E．coli中的表达

应用基因工程技术，将抗人Ｃｌｑ轻链可变区基因（ＣｌｑＶ＿Ｌ）由重组质粒ｐＧＥＭ－ＣｌｑＶ＿Ｌ中分离，克隆进表达载体ｐＪＬＡ５０２。该表达载体含ＣＩＴｓ８５７抑制子基因，通过开温诱导法，重组表达子在宿主菌ＨＢ１０１中表达，获得抗人Ｃｌｑ轻链可变区片段，ＳＤＳ－ＰＡＧＥ表明其分子量范围为１２～１３ＫＤ。这为进一步抗人Ｃｌｑ小抗体的表达积累了经验，有利于用于抗原－抗体结构功能的研究。     

抗人C1q轻链可变区基因在E.coli中的表达

应用基因工程技术，将抗人Ｃ１ｑ轻链可变区基因的重组质粒ｐＧＥＭ－Ｃ１ｑＶＬ中分离，克隆进表达载体ｐＪＬＡ５０２。该表达截体含ＣＩＴｓ８５７抑制子基因，通过开温诱导法，重组表达子在宿主菌ＨＢ１０１中表达，获得抗人Ｃ１ｑ轻链可变区片段，ＳＤＳ－ＰＡＧＥ表明其分子量范围为１２－１３ＫＤ。这为进一步抗人Ｃ１ｑ小抗体的表达积累了经验，有利于用于抗原－抗体结构功能的研究。     

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     

用微弹轰击法介导的转基因对小鼠进行乙肝病毒遗传免疫研究

用包裹ＤＮＡ的金微弹轰击耳朵，将表达乙型肝炎病毒表面抗原（ＨＢｓＡｇ）基因的重组质粒转入小鼠皮肤细胞，在小鼠血清中检测ＨＢｓＡｇ及抗－ＨＢｓ抗体。实验组８只小鼠中，有３只在基因转移３天后检测到ＨＢｓＡｇ。１４天再轰击１次。１６天后２只鼠中检测到抗－ＨＢｓ抗体。２８天后４只鼠中检测出抗ＨＢｓ抗体。对照组４只小鼠中ＨＢｓＡｇ及抗－ＨＢｓ抗体均未检测出。本实验为今后进一步分析乙肝病毒的遗传免疫奠定了实验基础，提供了实验依据。     

用微弹轰击法介导的转基因对小鼠进行乙肝病毒遗传免？…

用包裹ＤＮＡ的金微弹轻击耳朵，将表达乙型肝炎病毒表面抗原（ＨＢｓＡｇ）基因的重组质粒转入小鼠皮肤细胞，在小鼠血清中检测ＨＢｓＡｇ及抗－ＨＢｓ抗体。实验组８只小鼠中，有３只在基因转移３天后检测到ＨＢｓＡｇ。１４天再轰击１次。１６天后２只鼠中检测到抗－ＨＢｓ抗体。２８天４只鼠中检测出抗ＨＢｓ抗体。对照组４只小鼠中ＨＢｓＡｇ及抗－ＵＨＢｓ抗体均未检测出。本实验为今后进一步分析乙肝病毒的遗传免疫奠定了实验     

EB病毒膜蛋白在CHO细胞中的表达

目的研制ＥＢ（Ｅｐｓｔｅｉｎ－Ｂａｒ）病毒基因工程疫苗。方法构建了含有Ｅｐｓｔｅｉｎ－Ｂａｒ病毒（ＥＢＶ）膜蛋白（ＭＡ）基因的重组表达质粒ｐＣＭＶ／ＭＡ，转染ＣＨＯ细胞，研究表达产物的生物学性状，及培养液中不同血清含量、冻存、Ｇ４１８对ＣＨＯ细胞分泌ＭＡ的影响。结果获得两个稳定表达ＭＡ的细胞株。免疫印迹检测表达产物的分子量约为３４０ｋＤ和２２０ｋＤ，间接免疫荧光和免疫斑点确定表达产物能与抗ＭＡ的单克隆抗体特异性结合，用薄层扫描和Ｌｏｗｒｙ法计算ＭＡ的表达量为每天１９μｇ／ｍｌ。经纯化的ＭＡ免疫小鼠，在血清中检测到抗ＭＡ的特异性抗体。结论为开发有效的表达系统用于ＥＢ病毒基因工程疫苗的生产提供有利的实验基础。     

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。     

Rapid detection of hepatitis B virus surface antigen by an agglutination assay mediated by a bispecific diabody against both human erythrocytes and hepatitis B virus surface antigen

Bispecific antibodies have immense potential for use in clinical applications. In the present study, a bispecific diabody against human red blood cells (RBCs) and hepatitis B virus surface antigen (HBsAg) was used to detect HBsAg in blood specimens. The bispecific diabody was constructed by crossing over the variable region of the heavy chains and the light chains of anti-RBC and anti-HBsAg antibodies with a short linker, SRGGGS. In enzyme-linked immunosorbent assays, this bispecific diabody showed specific binding to both RBCs and HBsAg. When this bispecific diabody was mixed with human blood specimens in the presence of HBsAg, the dual binding sites of the diabody caused agglutination of human RBCs. This diabody-mediated agglutination assay was then used to test 712 clinical blood specimens and showed 97.7% sensitivity and 100% specificity when the results were compared with those of the conventional immunoassay, which was used as a reference. This autologous RBC agglutination assay provides a simple approach for rapid screening for HBsAg in blood specimens.     

抗人RBC血型B抗原双价小分子抗体基因的构建与表达

目的： 分离抗人红细胞血型Ｂ抗原单克隆抗体（ｍＡｂ） ５Ｄ１２ 可变区基因, 构建并表达其双价小分子抗体（ｄｉａｂｏｄｙ） 融合蛋白, 为用原核细胞生产抗血型Ｂ抗原工程抗体进行基础研究。方法： 设计合成抗体重、轻链可变区引物, 通过加端ＰＣＲ技术, 在ｍＡｂ ５Ｄ１２ ＶＨ 和ＶＬ基因两端引入连结短肽和适当的限制性酶切位点,体外连接构建５Ｄ１２ ｄｉａｂｏｄｙ 基因, 并将其克隆入融合表达载体ｐＧＥＸ４Ｔ２ 中, 在Ｅ－ｃｏｌｉ ＴＧ１ 中表达。运用ＰＣＲ和双脱氧核苷酸链末端终止法分析其核苷酸序列。结果： ＤＮＡ 序列分析表明, ５Ｄ１２ ｄｉａｂｏｄｙ 基因全长为６９９ｂｐ;其中ＶＨ 长３５１ｂｐ , 编码１１７ 个氨基酸; ＶＬ 长３２４ｂｐ , 编码１０８ 个氨基酸, 两者以五肽连接头相连; 重组体表达产物经ＳＤＳＰＡＧＥ显示, ＧＳＴ５Ｄ１２ ｄｉａｂｏｄｙ 表达产物的相对分子质量（ Ｍｒ） 为５１ ０００ 左右, 与预期结果相符。光密度扫描结果表明, ＧＳＴ５Ｄ１２ ｄｉａｂｏｄｙ 融合蛋白占菌体总蛋白的２４－６％ , 表达产物主要以不溶性的包涵体形式存在。结论： 成功地构建并表达了５Ｄ１２ 双价小分子抗体基因, 在原核细胞中获得较高水平     

人工合成免疫刺激DNA联合HBsAg DNA疫苗在HBV转基因鼠的免疫应答研究

目的 :探讨免疫刺激DNA序列联合基因免疫在HBV转基因鼠的免疫应答。方法 :用人工合成硫代修饰的免疫刺激DNA寡核苷酸 (CpGODN)与HBVS区基因真核表达载体 (V HBs)联合免疫HBsAg转基因鼠 ,通过ELISA观察小鼠血清HBsAg及抗 HBs抗体水平 ,并用免疫组化 (SP法 )及病理HE染色观察小鼠肝组织HBsAg表达量的改变及肝组织炎症活动度。结果 :V HBs联合CpGODN组 6只免疫鼠中有 2只血清抗 HBs抗体阳性 ,其平均效价为 (5 6 2 1± 15 16 )mU ml,血清HBsAg浓度在免疫后第 8周时有 2只转阴 ,而单用V HBs组及V 10 12对照组小鼠血清抗 HBs抗体均阴性、HBsAg含量无明显降低。V HBs +CpGODN组肝组织HBsAg的表达量低于V HBs组及对照组 ,并可见大量炎细胞浸润 ,炎症组织活动度积分明显高于V HBs组及对照组。结论 :CpGODN联合V HBs可增强其免疫应答及抗病毒效应。     

Di-diabody: a novel tetravalent bispecific antibody molecule by design

The clinical development of bispecific antibodies (BsAb) as therapeutics has been hampered by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods have been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. These recombinant antibody molecules possess dual antigen-binding capability with, in most cases, monovalency to each of their target antigens. Here, we describe an efficient approach for the production of a novel tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a bispecific/divalent diabody to, via the hinge region, the N-terminus of the CH(3) domain of an IgG. The novel BsAb, which we termed "di-diabody", represents a tetravalent diabody dimer resulting from dimerization between the hinge region and the CH(3) domains. A di-diabody was constructed using two antibodies directed against the two tyrosine kinase receptors of vascular endothelial growth factor, expressed both in a single Escherichia coli host and in mammalian cells, and purified to homogeneity by a one-step affinity chromatography. Compared to the bispecific/divalent diabody, the tetravalent di-diabody binds more efficiently to both of its target antigens and is more efficacious in blocking ligand binding to the receptors. The di-diabody retained good antigen-binding activity after incubation at 37 degrees C in mouse serum for 72 h, demonstrating good product stability. Finally, expression of the di-diabody in mammalian cells yielded higher level of production and better antibody activity. This design and expression for BsAb fragments should be applicable to any pair of antigen specificities.     

抗HBsAg/RBC双特异噬菌体抗体的构建

目的 通过不同外壳蛋白的展示，构建抗HBsAag/RBC双特异噬菌体抗体。方法通过DNA重组技术，将抗HBsAg Fab与噬菌体基因8融合，抗RBC ScFv与噬菌体基因3融合，这2种融合基因以不同的启动子驱动，并克隆于同一噬菌体表达载体上，得到双特异噬菌体抗体表达载体，转化大肠杆菌后获取噬菌体抗体上清，用ELISA和红细胞凝集试验检测其双特异活性。结果ELISA和RBC凝集实验证明，本方法可形成双特异噬菌体抗体，可使含有HBsAg的RBC悬液产生凝集。用展示性能得到提高的蛋白8变种取代野生型蛋白8可提高双特异噬菌体抗体的活性。结论 HBsAb和RBC两种抗体分子能够通过不同噬菌体外壳蛋白同时展示于同一噬菌体表面，形成双特异噬菌体抗体。可用于人外周血HBsAg的快速血凝法检测。     

抗人红细胞抗体和HIV抗原融合蛋白的构建及活性鉴定

目的构建及表达抗红细胞(RBC)单链抗体(ScFv)和HIV抗原的融合蛋白,并做活性测试。方法将鼠抗人红细胞单链抗体可变区重链、轻链基因与HIV抗原基因相连,重组于高效表达载体中,并在大肠埃希菌中表达。结果经ELISA检测和红细胞凝集检测,证明表达的融合蛋白具有HIV抗原和抗人红细胞的双重活性,可使含有HIV抗体的红细胞悬液凝集。结论融合蛋白构建成功,可用于HIV的快速检测。     

Unexpected recombinations in single chain bispecific anti-CD3-anti-CD33 antibodies can be avoided by a novel linker module

CD33 is an attractive immunotarget on the surface of tumor cells from patients with acute myeloid leukemia (AML). In a first attempt for immunotargeting of AML blasts we constructed two bispecific antibodies in the single chain bispecific diabody (scBsDb) format by fusing the variable domains of monoclonal antibodies directed against CD3 and CD33. Unfortunately, protein expression of both scBsDbs resulted in varying mixtures of fragmented and full length proteins. As the non-functional fragments competed with the functional full length antibodies we tried to understand the reason for the fragmentation. We found that the anti-CD3 and anti-CD33 antibody genes show striking sequence homologies: during B cell development the same V_h J558 heavy and V_l kk4 light chain genes were selected. Moreover, the closely related D genes DSP2 (9 and 11) were combined with the same JH4 gene. And finally, during VJ recombination of the light chain the same JK5 element was selected. These homologies between the two monoclonal antibodies were the reason for recombinations in the cell lines generated for expression of the scBsDbs.Finally, we solved this problem by (i) rearranging the order of the heavy and light chains of the anti-CD3 and anti-CD33 domains, and (ii) a replacement of one of the commonly used glycine serine linkers with a novel linker domain. The resulting bispecific antibody in a single chain bispecific tandem format (scBsTaFv) was stable and capable of redirecting T cells to CD33-positive tumor cells including AML blasts of patients.     

乙型肝炎核酸疫苗NV—HB／s肌注小鼠后HBsAg的表达和抗 …

目的 研究乙型肝炎核酸疫苗肌注小鼠后在体内的表达及小鼠体液免疫应答的规律,同时观察接种局部组织的病理变化。方法 以乙型肝炎核酸疫苗ＮＶ－ＨＢ／ｓ肌注小鼠,然后定期分批处死,采集肌注局部组织检测ＨＢｓＡｇ（ＡＢＣ免疫组化法）,采集外周血标本检测ＨＢｓＡｇ和抗－ＨＢｓ（ＥＬＩＳＡ法０,并常规病理检查。     

人源性抗地高辛单链抗体(scFv)及diabody的获取

目的:从大容量噬菌体抗体库中筛选人源性抗地高辛单链抗体(scFv),并构建双价抗体(diabody)。方法:以固相化的Dig对构建的大容量噬菌体抗体库进行筛选,4轮后挑取集落,用ELISA法鉴定其特异性,并对抗Dig阳性抗体基因进行DNA指纹分析及测序分析。选取活性好的抗体基因进行改造,构建diabody。结果:在抗体库的筛选过程中可见到明显的富集现象,获得4株可与Dig特异性结合的人源单链抗体,经DNA指纹分析及测序分析证明为不同抗体基因,分泌抗Dig抗体的阳性克隆的可变区基因轻链均属于λ第1亚群,重链基因分别属于第3和第4亚群。选取4号克隆进行基因改造,构建diabody的表达载体,获得活性较好的diabody。结论:利用噬菌体抗体技术获得了人源性抗Dig的抗体,并改造成应用前景较好的diabody,为临床诊断及治疗洋地黄类药物的中毒提供了具有实用价值的人源抗体。