抗人黑色素瘤单链抗体基因的克隆和分泌型表达

应用ＲＴ－ＰＣＲ技术从分泌抗人黑色素瘤单克隆抗体的杂交瘤细胞ＨＢ８７６０中克隆了抗体轻、重链可变区基因,然后用（Ｇｌｙ４Ｓｅｒ）３连接肽基因将ＶＨ、ＶＬ连接成ＳｃＦｖ基因,并进行了序列测定．计算机分析表明ＶＨ,ＶＬ均符合小鼠抗体可变区的特征,为功能性重排的抗体可变区基因．ＶＨ、ＶＬ、ｌｉｎｋｅｒ拼接正确．ＳｃＦｖ基因全长７２９ｂｐ,其中ＶＨ基因长３６０ｂｐ,编码１２０个氨基酸,ＶＬ基因长３２４ｂｐ,编码１０８个氨基酸．在噬菌粒表达载体ｐＣＡＮＴＡＢ５Ｅ中表达了可溶性的ＳｃＦｖ蛋白,表达产物经流式细胞仪检测可特异地与黑色素瘤细胞结合,不与肝癌、胃癌及良性黑痣细胞结合     

抗结肠癌相关抗原单链抗基因的构建和表达

通过ＰＣＲ扩增和酶切分别得到抗结肠癌相关抗原抗体的重链可变区序列,轻链可变区序列及连接肽序列,将它们构建成为ＶＨ－ｌｉｎｋｅｒ－ＶＬ形式的单链抗体基因片段,并在大肠杆菌中进行了表达,ＳＤＳ－ＰＡＧＥ分析结果表明,以ｐＣｏｍｂ３为载体,在大肠杆菌ＪＭ８３中,ｓｃＦｖ未获得有效表达,而以ｐＥＴ－２２ｂ（＋）为载体的ｓｃＦｖ在大肠杆菌ＢＬ２１（ＤＥ３）中,３０℃诱导培养获得了高效表达,表达水平占全菌蛋白     

鼠源抗HFRSV衣壳蛋白McAbF3株单链抗体基因克隆构建及表达

为了获得原抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３＾１株轻链可变区基因,由连接肽体外连接获得单链抗体基因,在大肠杆菌中表达,从鼠源抗ＨＦＲＳＶ衣壳蛋白ＭｃＡｂＦ３株细胞中分离总ＲＮＡ,以ｏｌｉｇｏ（ｄＴ）１８为引物逆转录成ｃＤＮＡ,通过ＰＣＲ扩增出抗体的轻链（ＶＬ）和重链可变区（Ｖ１１）基因,由连接本外连接获得单链抗体（ＳｅＦｖ）基因。将此单链抗体（ＳｅＦｖ）基因插入原核表达载体ＰＥＴ２８ａ,经大肠杆菌（     

抗HBsAg二硫键稳定性Fv抗体（dsFv）基因的构建与表达

采用ＰＣＲ定点突变方法,成功地构建了抗ＨＢｓＡｇｄｓＦｖ抗体的轻、重链突变基因,ＮｄｅＩ和ＥｃｏＲＩ酶切后分别插入ｐＥＴ２０ｂ表达质粒,经测序证明在重链第４４位氨基酸和轻链第１００位氨基酸已突变形成半胱氨酸（Ｃｙｓ）。ＶＨ和ＶＬ重组质粒分别转化到大肠肝菌ＢＬ２１（ＤＥ３）中,ＩＰＴＧ诱导后,经ＳＤＳＰＡＧＥ电泳表明在１２ｋＤ处有包含体蛋白表达,表达蛋白含量分别为２８％和３５％。ＶＨ和ＶＬ包含体蛋白经ＧｕＨＣｌ变性后,等量混合在复性折叠液中结合,形成了一个约２４ｋＤ并有一定活性的ｄｓＦｖ蛋白。抗ＨＢｓＡｇｄｓＦｖ抗体表达及复性的成功,为今后基因工程抗体的研究及应用奠定了基础。     

甜菜坏死黄脉病毒内蒙分离物RNA3序列分析及编码25kD蛋白基因在大肠杆菌中的表达

以甜菜坏死黄脉病毒（ＢＮＹＶＶ）内蒙分离物的总ＲＮＡ为模板,通过反转录－ＰＣＲ扩增获得ＢＮＹＶＶＲＮＡ３全长ｃＤＮＡ。将其克隆到ｐＧＥＭ－７Ｚｆ（＋）上,得到重组质粒ｐＧＢＹ５６。序列分析结果表明,内蒙分离物ＲＮＡ３基因组全长为１７７５ｎｔ,其中包含３个开放阅读框架,分别编码２５ｋＤ蛋白、４．６ｋＤ蛋白和一种由５９个氨基酸组成的Ｎ蛋白。与法国Ｆ２分离物、德国Ｇ１分离物和日本Ｓ分离物相比,其核苷酸序列的同源性分别为９６．４％、９６．８％和９７．３％。将２５ｋＤ蛋白编码基因克隆到ｐＪＷ２上,构建了该基因的原核表达载体。ＳＤＳ－ＰＡＧＥ和Ｗｅｓｔｅｒｎｂｌｏｔｉｎｇ分析结果表明,２５ｋＤ蛋白基因在Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３）中经温度（４２℃）诱导后,可特异地表达２５ｋＤ蛋白     

乙肝表面抗原专一的单链嵌合抗体在大肠杆菌中的表达

利用重组ＰＣＲ技术将乙肝表面抗原专一单抗的Ｖｋ与人源的Ｃ基因拼接成轻链嵌合抗体Ｖ－Ｃ,再通过编码柔性肽段（Ｇｌｙ４Ｓｅｒ）的Ｌｉｎｋｅｒ与Ｖ连接成单链嵌合抗体ＳｃＦＶ－Ｃ,并分别在大肠杆菌的热敏与分泌型表达系统中进行表达。表达产物的Ｗｅｓｔｅｒｎ－ｂｌｏｔ与间接ＥＬＩＳＡ检测结果表明,ＳＣＦｖ－Ｃ产两种系统中的表达产物均具有抗原结合活性,并已在分泌肽的指导下ＳｃＦｖ－Ｃｋ能被Ｅ．ｃｏｌｉ分泌出胞外。     

重组人融合基因Nm23—H1／HbFGF的构建及其在大肠杆菌中表达？…

根据Ｎｍ２３－Ｈ１与ＨｂＦＧＦｃＤＮＡ序列,人工合成一段中间核酸序列,将它分别与Ｎｍ２３－Ｈ１ｃＤＮＡ的上游引物及ＨｂＦＧＦｃＤＮＡ的下游引物组成两对引物,通过ＰＣＲ（聚合酶链式反应）构建出融合基因Ｎｍ２３－Ｈ１／ＨｂＦＧＦ,将其定向克隆于质粒载体ｐＢＶ２２０上,经诱导,ＳＤＳ－ＰＡＧＥ分析,表达产物分子量为３４ｋＤ,表达量占菌体总蛋白的１４％,表达产物以包涵体形式存在,ＥＬＩＳＡ和Ｗｅｓｔｅｒｎ     

抗肝癌单链抗体的活性检测

将抗肝癌单克隆抗体 ＨＡｂ２５的轻、重链可变区基因通过Ｌｉｎｋｅｒ连接起来,构建成抗肝癌单链抗体ｓｃＦｖ２５,然后亚克隆入含 Ｅ－ｔａｇ检测标签的 ｐＥＴ１５ｂ表达载体进行融合表达。表达产物（ｓｃＦｖ２５－Ｅ－ｔａｇ）采用 ＳＡＢＣ法对肝癌细胞涂片进行染色,利用鼠抗Ｅ－ｔａｇ抗体间接检测初步纯化的ｓｃＦｖ２５的活性,同时采用竞争结合实验检测ｓｃＦｖ２５的亲和活性。ｓｃＦｖ２５能够与靶细胞特异性结合,阳性部位主要定位于细胞膜上;ｓｃＦｖ２５可封闭５３％的亲本抗体的亲和活性。ｓｃＦｖ２５较好地保持了亲本抗体的特异性和亲和活性。     

文摘

000042抗结肠癌相关抗原单链抗体基因的构建和表达[中]/朱林霞…//生物工程学报.-2000,16(1).-82～85通过ＰＣＲ扩增和酶切分别得到抗结肠癌相关抗原抗体的重链可变区序列、轻链可变区序列及连接肽序列,将它们构建成为ＶＨ-ｌｉｎｋｅｒ-ＶＬ形式的单链抗体基因片段,并在大肠杆菌中进行表达。ＳＤＳ-ＰＡＧＥ分析结果表明,以ｐＣｏｍｂ3为载体,在大肠肝菌ＪＭ83中,ｓｃＦｖ未获得有效表达,而以ｐＥＴ-22ｂ( )为载体的ｓｃＦｖ在大肠杆菌ＢＬ21(ＤＥ3)中,30℃诱导培养获得了高效表达,表达水平占全菌蛋白的35.5%。(杨淑培)000043大肠杆菌原核…     

牛疱疹病毒I型前早期基因BICPO的表达对牛免疫缺陷病毒LTR的反式激活…

由含有ＢＨＶ－１（Ｂｏｖｉｎｅ Ｈｅｒｐｅｓ Ｖｉｒｕｓ－１）前早期基因的基因组片段亚克隆ＩＣＰＯ（ＢＨＶ－１Ｉｎｆｅｃｔｅｄ Ｃｅｌｌ Ｐｒｏｔｅｉｎ Ｏ）的ＤＮＡ序列至表达载体ｐＳＶＤ３,构建质粒ｐＳＶ２．９。将该质粒与ＰＢＬＴＲ－Ｌｕｃ共转染小牛肺细胞,检测转染细胞裂解物的荧光素酶活性,ＢＩＣＰＯ的表达产物可以显著地激活ＢＩＶ ＬＴＲ启动子控制下的荧光素酶基因的表达。根据ＰＳＶ２．９与含有Ｂ     

Gene cloning, bacterial expression, in vitro refolding, and characterization of a single-chain Fv antibody against PreS1(21-47) fragment of HBsAg

The murine monoclonal antibody 125E11 is an IgG which recognizes PreS1(21-47) fragment of large hepatitis B surface antigen. It has been successfully used for clinical detection of HBV virion in serum of hepatitis B patients. In present study, the genes of variable region in heavy chain (VH) and light chain (VL) of 125E11 have been cloned. Sequence analysis of cloned VH gene and VL gene showed that they had general characterization of immunoglobin variable region genes. According to Kabat classification, VH gene and VL gene belong to VH10 family, subgroup IIID and Vkappa family subgroup I, respectively. An expression vector of 125E11 single-chain Fv antibody fusion protein, in which VH and VL peptide were connected by a flexible linker (Gly(4)Ser)(3), was constructed. The scFv fusion protein was highly expressed in Escherichia coli mainly in inclusion body form. Using urea and pH gradient gel filtration method, the refolding of scFv was efficiently achieved. The refolding efficiency reached about 11% and 2.7 mg refolded scFv was obtained from 1L of culture. The binding activity and specificity of 125E11 scFv against PreS1(21-47)-containing antigen were also analyzed.     

Expression of single-chain antibody fragments (scFv) specific for beet necrotic yellow vein virus coat protein or 25 kDa protein in Escherichia coli and Nicotiana benthamiana

The coding sequences for the variable regions of heavy and light chains of monoclonal antibodies (mAbs) to beet necrotic yellow vein virus (BNYVV) coat protein (cp) or the 25 kDa nonstructural protein (P25) were cloned into the pCOCK vector and expressed as single-chain antibody fragments (scFv) in Escherichia coli. For expression in higher plants the scFv were targeted either to the secretory pathway by including the sequences encoding the pectate lyase B (PelB) or the phytohemagglutinin (PHA) signal peptides in the vector constructs or they were targeted to the cytoplasm by omitting a signal peptide-encoding sequence from the constructs. The scFv were detected mainly in plants in which the PHA signal peptide had been used for targeting demonstrating for the first time the usefulness of this peptide for enabling scFv expression in plants. The scFv were not secreted into the culture fluids of suspension cultures, but were retained in the cells. The amount of expression of scFv in the best expressing plants was at least as high as in bacterial culture supernatants. In a dot blot immunoassay, 0.4 ng BNYVV cp or 0.8 ng P25 were detected by the respective scFv either from E. coli or from plants. The majority of the 21 plants expressing cp-specific scFv had near-normal growth whereas the three plants expressing P25-specific scFv grew poorly and did not form roots.     

肝癌细胞特异性结合单链抗体三维结构的同源模建

用MSI公司开发的计算机辅助分子设计系统模建肝癌细胞表面抗原特异性单链抗体三维结构。先分别模建VH(variable region of the heave chain)和VL(variable region of the light chain)两个结构域,然后搭建出scFv(single chain variable fragment)片段的整体三维结构,并对模建的结构进行分子力学和动力学优化;对结构的合理性验证显示模建结构是合理的。文章可为预测该特异性单链抗体的生物活性以及研制高亲和力、高特异性的双价抗体提供结构信息。     

一种核糖体展示单链抗体文库的构建与鉴定

用柑桔溃疡病致病菌Xanthomonas axonopodis pv. citri（Xac）全菌免疫小鼠,提取小鼠脾细胞mRNA,RT-PCR扩增小鼠抗体重链可变区（VH）和轻链可变区（VL）,采用linker (Gly4Ser)3连接VH和VL,构建用于核糖体展示方法筛选阳性单链抗体（scFvs）的文库。通过将scFv文库DNA转化大肠杆菌JM109,随机挑取克隆子测序以分析单链抗体文库的多样性。结果显示,用柑桔溃疡病菌免疫后的小鼠抗血清效价为2500倍左右,扩增的VH大小为350bp左右,VL的大小为650bp左右,linker连接后的单链抗体文库DNA大小为1200bp左右。测序结果显示,单链抗体文库多样性好。以Xac为靶,筛选到了抗Xac的特异性抗体,说明该抗体库可用于柑桔溃疡病菌单链抗体的筛选。     

Recombinant single-chain Fv antibodies that recognize the p25 protein of the Maedi-Visna virus

Single chain Fv (scFv) antibodies (generated by phage display technology, molecules representing new and efficient tools in the research and diagnostics of infectious diseases) against the capsid protein (p25) of Maedi-Visna virus were selected. Several clones of p25 specific scFv antibodies were identified; one of them was expressed as a soluble scFv molecule, purified by immobilized metal-affinity chromatography and further characterized by sequencing and determination of the kinetic equilibrium association constant. Sequence analysis showed that the rearranged VL and VH domains of the analyzed scFv clone used sequences from the VL3 family (germline DPL16/VL3.1) and VH1 family (germline VH20), respectively. The kinetic equilibrium association constant was determined as KA = 1.12 +/- 0.52 L/mumol.     

Immunolabeling of CD3-positive lymphocytes with a recombinant single-chain antibody/alkaline phosphatase conjugate

G3(3) is a novel murine monoclonal antibody directed against the CD3 antigen of human T lymphocytes which could be used to analyze lymphoid malignancies. We have produced and characterized a recombinant colorimetric immunoconjugate with the antigen-binding specificity of antibody G3(3). A gene encoding a single-chain antibody variable fragment (scFv) was assembled using the original hybridoma cells as a source of antibody variable heavy (VH) and variable light (VL) chain genes. The chimeric gene was introduced into a prokaryotic expression vector in order to produce a soluble scFv fused to bacterial alkaline phosphatase. DNA sequencing and Western blotting analyses demonstrated the integrity of the soluble immunoconjugate recovered from induced recombinant bacteria. The scFv/AP protein was bifunctional and similar in immunoreactivity to the parent G3(3) antibody. Flow cytometry and immunostaining experiments confirmed that the activity of the scFv/AP protein compares favourably with that of the parent antibody. The scFv/AP conjugate was bound to CD3 antigen at the surface of T cells and was directly detected by its enzymatic activity. Thus this novel fusion protein has potential applications as an immunodiagnostic reagent.     

天然兔源噬菌体单链抗体库的构建与鉴定

目的:构建天然兔源噬菌体单链抗体库。方法:采用RT-PCR法从未免疫的兔子脾脏中克隆得到抗体重链可变区(VH)与轻链可变区(VL)基因,重叠PCR将VH和VL拼接成scFv片段,将scFv连接到噬菌粒pComb3XSS上,电转入XL1-Blue菌中,得到单链抗体库,并用此抗体库筛选抗肌酸激酶抗体。结果:构建了容量为4×108,基因重组率95%的单链抗体库,DNA指纹图谱显示抗体库多样性良好。以肌酸激酶为抗原,从该库中筛到3株抗肌酸激酶的抗体。结论:分析表明构建的天然兔源单链抗体库质量良好,可用于快速筛选、制备多种单链抗体。     

基于核糖体展示技术进行抗狂犬病毒人源抗体的制备

构建了核糖体展示人源抗狂犬病毒单链抗体(scFv)库,筛选制备特异抗狂犬病毒糖蛋白(RVGp)的稳定性人源抗体.应用核糖体抗体库技术,从经狂犬病毒Vero疫苗免疫的志愿者外周血淋巴细胞中分离、构建核糖体展示scFv基因库.体外转录翻译后,以RVGp重组蛋白作筛选抗原,采用亲和富集法淘选RVGp特异性scFv抗体基因.在原核系统pET22b(+)/BL21(DE3)中实现scFv抗体片段的可溶性表达,ELISA鉴定阳性克隆.然后对筛选的scFv进行稳定性改构,构建VH-Lc-VK稳定性抗体,并对其生物学活性进行初步研究.成功构建了库容量约为6.2×1012的核糖体展示scFv抗体基因库.在180个筛选克隆中,克隆RB24、RB71、RB109和RB156显示出较高的ELISA值,其基因序列分析结果显示,它们是全新的人源抗RVGp抗体.改构后的抗RVGp VH-Lc-VK抗体的稳定性明显改进,可特异识别RVGp并有效中和狂犬病毒,抑制狂犬病毒对靶细胞的感染.以上结果表明,人源抗RVGp特异性抗体的获得,为狂犬病的有效预防、诊断和治疗提供了新的途径,而且将为其他人源抗体的制备提供理论依据和技术基础.