抗牛精子sp18蛋白单链抗体基因的克隆及表达

应用重组噬菌体抗体技术,从小鼠抗牛精子sp18蛋白的A5杂交瘤细胞系中分离总RNA,构建重组噬菌体抗体库。以牛精子作为包被抗原,经过三轮“亲和吸附-洗脱-扩增”淘选后,用phage-ELISA筛选出特异性阳性克隆。取其中特异结合牛精子细胞的重组噬菌体抗体pCSA1克隆进行序列测定。将其ScFv(single chain fragment variable)因克隆到pET-30a( )上,用IPTG进行诱导表达,成功得到了抗sp18单链抗体基因蛋白。     

抗乙肝病毒表面抗原PreS1(20-47)的单链抗体在转基因烟草根分泌物中的初步表达

为探索利用植物根分泌表达重组蛋白的可行性，构建了含有抗乙肝病毒表面抗原PreS1(20—47)单链抗体(ScFv)基因的表达载体。该ScFv基因转化烟草后在烟草根部细胞的细胞质和内质网中获得表达。实验结果表明，5’端融合ER导向信号肽的重组ScFv可通过根分泌表达。     

抗人卵巢癌×抗人CD3双特异性单链抗体的构建、表达及复性研究

构建了抗人卵巢癌×抗人CD3双特异性单链抗体 (scBsAb) ,在大肠杆菌中得到表达。用稀释复性 ,缓慢透析复性和凝胶过滤层析复性三种方法对scBsAb表达形成的包涵体进行复性研究表明 ,凝胶过滤层析复性能有效抑制蛋白的聚集。ELISA检测显示 ,复性后的scBsAb具有较高活性 ,能与相应抗原特异结合。     

铜绿微囊藻生物钟蛋白的表达纯化与抗体制备

为了获得融合表达的铜绿微囊藻(Microcystic aeruginosa)生物钟蛋白KaiA、KaiB、KaiC并制备其相应的多克隆抗体,将kaiA、kaiB、kaiC基因分别克隆到原核表达质粒pET-His中.重组质粒pET-His-KaiA,pET-His-KaiB和pET-His-KaiC经酶切和测序鉴定后,分别转化E.coli BL21(DE3)进行融合表达.经SDS-PAGE分析可知,融合表达的KaiA、KaiB和KaiC蛋白表达量可分别达到菌体总蛋白的25%、40%和20%.经亲和层析后融合蛋白KaiA和KaiB的纯度分别达95%和92%,而KaiC经胶回收纯化后纯度也可达93%.将纯化后的三种Kai蛋白作为抗原分别免疫小鼠制备多克隆抗体,经ELISA检测抗体滴度表明,制备的抗KaiA、抗KaiB和抗KaiC的多克隆抗体效价高,分别可达到1:50000、1:60000和1:100000.Western blotting结果表明:获得的多克隆抗体具有较高的效价,抗体能识别相应的Kai蛋白,具有较高的特异性,能用于铜绿微囊藻生物钟蛋白KaiA、KaiB和KaiC的表达节律检测.     

从噬菌体抗体库中筛选出抗原结合活性相近、表达水平不同的单链抗体

从抗人膀胱癌改形噬菌体抗体库中筛选出一组相对亲和力常数和细胞结合活性相近的单链抗体，而它们的结合活性均明显高于起始的鼠源单链抗体。在亲和力淘选的过程中，这一组抗体的表达量存在显著差异，这在一定程度上影响到筛选过程中ELISA鉴定的结果，使整个筛选过程并不仅仅是对高亲和力抗体的筛选，也同时得到了高表达量的抗体。     

抗HIV-1 gp120单链抗体导向SEAD227A融合蛋白原核温控型表达载体构建及表达

人工合成了能广泛识别HIV—1 gp120的单链抗体(SeFv120)基因和金黄色葡萄菌外毒素A(SEA)基因,将SEA基因第227位编码Asp(D)的密码子GAT转换成编码Ala(A)的密码子GCT,并对两基因进行密码子优化。构建原核温控型表达质粒pBV—120和pBV—SL120,经条件优化,pBV—120和pBV—SL120分别在E．coli BL21(DE3)plys中44℃诱导6h、42℃诱导7h获得最佳表达,表达量分别占菌体总蛋白的27．673％和32．519％。目的蛋白经包涵体洗脱、分子筛层析折叠复性后可与HIV—1抗原条发生良好的结合反应。     

HIV-1核心蛋白的表达与纯化

将编码人I型免疫缺陷病毒(HIV1)核心蛋白p24gag的基因序列克隆到原核表达载体pET28(b)中,高效表达了N,C端融合His·Tagp24蛋白,所表达的重组p24蛋白占菌体总蛋白的46%。在变性条件下,使用NiNTA亲和层析法纯化了p24蛋白,纯度为94%。菌体中及纯化、复性后的目的蛋白均能与抗HIV1p24单克隆抗体发生特异性反应。用纯化的p24蛋白免疫小鼠,4周时小鼠血清抗p24抗体效价达1∶400。实验结果表明:大肠杆菌表达的HIV1p24蛋白纯化后可用作HIV1检测试剂的原料。     

抗CD3基因工程嵌合抗体片段Fab′的构建、表达和活性测定

PCR扩增抗CD3单抗轻链可变区(VL)和重链可变(VH)片段基因,将其重组到Fab ′表达载体中,构建成抗CD3嵌合抗体Fab′表达载体,转化大肠杆菌16C9进行可溶性表达 . 产物经蛋白G亲和层析柱纯化.免疫荧光竞争结合实验和3H掺入实验证实能与小鼠抗CD3 IgG HIT3a竞争性结合表达CD3的T淋巴细胞,并促进细胞增殖.     

应用全合成噬菌体抗体展示库技术实现抗膀胱癌单链抗体的定向进化

单链抗体在肿瘤治疗的临床应用中常会遇到两方面的问题，即低亲和力和鼠源性导致的免疫原性。为此，我们对鼠源抗膀胱癌单链抗体进行人源化改造，并从噬菌体抗体库中筛选具有高亲和力的人源化的单链抗体分子。运用噬菌体展示技术，成功构建了库容为10^6人源化的噬菌体抗体库，用膀胱癌细胞系膜抗原进行三轮筛选，得到了2个结合抗原活性高于起始的鼠源单链抗体的人源化抗膀胱癌单链抗体。     

八肋游仆虫一种富含谷氨酸的蛋白GARP的表达与纯化

为研究游仆虫中含有三核苷酸重复序列的GARP基因编码的GARP蛋白在细胞中的功能,利用定点突变技术,将GARP基因中的TGA突变为通用半胱氨酸密码子TGT.突变后的GARP基因构建于原核表达载体pRSETc中,得到重组质粒pRSETc-GARP,将pRSETc-GARP质粒转化至大肠杆菌BL21(DE3)中,经IPTG诱导,带有纯化标签的重组蛋白His6-GARP获得可溶表达,表达产物与抗His抗体在约26 kDa处有很强的交叉反应.His6-GARP蛋白在不同pH条件下通过两次阴离子交换层析和一次凝胶层析三步纯化达到电泳纯.     

Noncompetitive immunoassay of small analytes at the femtomolar level by affinity probe capillary electrophoresis: direct analysis of digoxin using a uniform-labeled scFv immunoreagent

A general method for noncompetitive immunoassay of small analytes using affinity probe capillary electrophoresis (APCE) is demonstrated using digoxin as a model analyte. A uniform immunoreagent was prepared from a single-chain antibody (scFv) gene specific for digoxin. Site-directed mutagenesis introduced a unique cysteine residue for uniform labeling with a thiol-reactive fluorochrome. After expression in E. coli, the scFv was purified by immobilized metal affinity chromatography (IMAC) using an added C-terminal 6-histidine sequence. The protein was renatured and labeled while immobilized on the IMAC resin. After 0.02-microm filtration to remove microaggregates, the resulting reagent was highly uniform and stable at -12 degrees C for at least 1 year. Three formats of APCE using the scFv reagent were explored. A "mix-and-inject" assay optimized for low detection limits demonstrated analysis of 10 pM digoxin in aqueous standard solutions in 10 min. A rapid mix-and-inject format in a short capillary allowed detection of 1 nM digoxin in 1 min. Digoxin samples in serum and urine were injected directly after 10-fold dilution. In combination with solid-phase extraction, 400 fM digoxin was detected in 1 mL of serum. Including solid-phase extraction, reproducibility was within 2.5%, and the linear range was 3 orders of magnitude. The strategy adopted in this paper should be of general use in the low-level analysis of small analytes.     

Single-chain variable fragment (scFv) antibodies optimized for environmental analysis of uranium

Recombinant single-chain variable fragment antibodies (scFv) were specifically generated and selected for the measurement of environmental uranium with an antibody-based sensor. These sFvs, which recognized UO(2)(2+) complexed to 2,9-dicarboxyl-1,10-phenanthroline-acid (DCP), were produced using genetic material obtained from the spleen cells of rabbits immunized with UO(2)(2+)-DCP conjugated to keyhole limpet hemocyanin. Immunoglobulin light chain and heavy chain genes were amplified and cloned into the phagemid pSD3 for generation of a recombinant antibody library and phage-displayed antibodies. The screening process was designed to isolate antibodies that bound to a "loaded" noncovalent complex with high affinity, while selecting against binding to an "unloaded" complex. After five rounds of panning, individual positive scFv clones were used to infect E. coli TG1 and soluble scFv antibodies were purified and characterized. Binding studies showed that the best scFv bound tightly to the UO(2)(2+)-DCP complex (K(d), 19.6 nM). However, because of the depletion experiments performed on this library during the panning process, this scFv bound 1200-fold less tightly (K(d), 23.5 μM) to metal-free DCP. This scFv (clone 3A) was subsequently used to accurately determine the UO(2)(2+) concentrations in environmental water samples using a sensor based on kinetic exclusion analysis. The present studies demonstrate that recombinant scFvs with properties engineered for specific applications (i.e., biosensor-based measurement of metals in groundwater) can be prepared if the correct genetic material and techniques are employed. The phage display system permitted the generation of proteins with very specific binding properties (in this case, high affinity for a metal-chelate complex and low affinity for metal-free chelator). The recombinant scFvs isolated in these studies will be the basis for rapid and affordable assays for the detection of residual uranium in environmental water samples.     

Immunoenzymometric assay for a small molecule,11-deoxycortisol, with attomole-range sensitivity employing an scFv-enzyme fusion protein and anti-idiotype antibodies

To overcome the sensitivity limit in immunoassays for small molecules (haptens), we established a noncompetitive immunoenzymometric assay (IEMA) format that can detect attomole-range hapten molecules. We selected 11-deoxycortisol (11-DC; Mr 346.5), a corticosteroid serving a diagnostic index for pituitary-adrenal function, as a model target hapten. A fusion of a single-chain Fv fragment (scFv) specific for 11-DC and alkaline phosphatase (ALP) was generated for use as an enzyme-labeled antibody, instead of the conventional chemically linked enzyme-antibody conjugates. After binding reaction of 11-DC and fixed amounts of the fusion protein (scFv-ALP), the unbound fusion protein was removed by incubation with a mouse beta-type anti-idiotype antibody recognizing the scFv paratope. These complexes were captured by magnetic separation using anti-mouse IgG antibody-coated magnetic beads. Following magnetic sedimentation of the beads, immune complexes of scFv-ALP and 11-DC remained in the supernatant were further purified by capture on microtiter plates with immobilized alpha-type anti-idiotype antibody. As measured fluorometrically, ALP activity from bound immune complexes on the plates increased with increasing 11-DC, which is characteristic of a noncompetitive relationship. This IEMA afforded an extremely low detection limit (20 amol/assay), a very wide measurable range, and practical specificity. The plasma 11-DC levels determined for healthy subjects were validated as reliable.     

Engineering peptide linkers for scFv immunosensors

Using A10B single-chain fragment variable (scFv) as a model system, we demonstrated that the flexibility of scFv linker engineering can be combined with the inherent quick and adaptable characters of surface coupling chemistry (e.g., electrostatic, hydrogen bonding, or covalent attachment) to attach scFv to preformed functionalized self-assembled monolayers (SAMs). Six arginines, which were separated by glycine or serine as spacer, were incorporated in the peptide linker to form a 15-mer peptide linker (RGRGRGRGRSRGGGS). The polycationic arginine peptide was engineered into the A10B scFv-RG3 to favor its adsorption at anionic charged template surface (11-mercaptoundecanoic acid (MUA) and poly(sodium 4-styrenesulfonate (PSS))). This new approach was compared with the other engineered scFv constructs. Our results demonstrated that the anionic charged SAM template facilitated the oriented immobilization of scFvs on the SAM template surface as well as reduced the possibility of protein denaturation when directly immobilized on the solid surface. A 42-fold improvement of detection limits using MUA/A10B scFv-RG3 (less than 0.2 nM experimentally determined) was achieved compared to A10B Fab antibody and a 5-fold improvement was observed compared to A10B scFv that was engineered with a cysteine in the linker sequence. Using protein A-coated gold nanoparticles, a picomolar experimental detection limit was achieved. With 20 amino acids to choose from, engineered recombinant scFv in combination with SAM technology and nanoparticle mass amplification provide an emerging strategy for the development of highly sensitive and specific scFv immunosensors.     

Nonregeneration protocol for surface plasmon resonance: study of high-affinity interaction with high-density biosensors

Surface plasmon resonance (SPR) has been used in determining kinetics and thermodynamics of biological interaction in the past decades. One difficulty encountered in this technology is the need for a proper regeneration, which means the removal of analytes from the bound complexes to regenerate the activity of the ligands. Regeneration is not always practical since the harsh regeneration reagents may destroy the bioactivity of the ligands. It is even more difficult for complexes with high affinity constants. In this paper, we report a nonregeneration protocol for SPR techniques in which subsequent ligand/analyte interactions can be measured without regeneration; thus ligand biological activity could be retained. Kinetics, binding models, and mathematics of this protocol are discussed in detail using rabbit IgG as the analyte and engineered recombinant antibody A10B single-chain fragment variables (scFv) as the ligand. The affinity constant of rabbit IgG binding with A10B scFv measured by using a nonregeneration protocol was (2.5 +/- 0.2) x 10(7) M(-1), which was comparable with the value determined with a conventional regeneration SPR method ((2.2 +/- 1.5) x 10(7) M(-1)) and quartz crystal microbalance (1.9 x 10(7) M(-1)). A paradigm of streptavidin-biotin binding was analyzed to validate this protocol. The affinity constant for each binding subunit of streptavidin to the immobilized biotin was determined to be (7.3 +/- 0.2) x 10(6) M(-1), which was comparable with the solution-based value of 2 x 10(7) M(-1). The nonregeneration protocol requires a relatively high ligand density on the biosensor surface so that more data points can be obtained before surface saturation. The small size of scFv enables them to be constructed in the biosensors for such purpose.     

Biologically produced bifunctional recombinant protein nanoparticles for immunoassays

Nanoparticles are increasingly used as labels for analytical purposes. In general, nanoparticles need to be functionalized with binding molecules (mostly antibodies or fragments thereof) and label substances using a multistep process that requires several manufacturing and purification steps. Here, we present a biological method of producing functionalized nanoparticles for effective use as label agents in a bioaffinity assay. The particles are based on the globular protein shell of human ferritin. A single chain Fv fragment (scFv) of an antibody is used as the binding moiety and Eu3+ ions as the label substance. Conventional chemical conjugation of the particle and antibody fragment is replaced with genetic fusion between the ferritin subunit and scFv genes. The material, for example, the fusion construct is produced in a single bacterial culture as insoluble forms that are easily purified by centrifugations. The subunits are solubilized and self-assembled, and label ions are introduced by shifting the pH. The functionality of these particles is demonstrated with a bioaffinity assay. This method of producing nanoparticles with inherent antigen binding activity presents several possibilities for the simple production of specific, functional nanoparticles. Production is fast, economical, and environmentally sustainable, making the system advantageous, particularly in applications requiring large quantities of specific nanoparticles.     

Engineered recombinant single-chain fragment variable antibody for immunosensors

A recombinant single-chain fragment variable (scFv) antibody (designated A10B) was engineered to contain two histidines within the linker peptide used to join the scFv heavy and light chains. A piezoimmunosensor using the scFv was successfully developed. A10B scFv bound to the gold piezoimmunosensor surface were correctly oriented, retained antigen-binding activity, and coupled at high surface concentration. These results, and results obtained from an earlier study using an scFv containing a linker cysteine, suggest that the location on the linker sequence in which the amino acids were incorporated was well tolerated by the scFv and did not interfere with scFv antigen-binding activity. The scFv-modified QCM sensor was thoroughly characterized and used to specifically detect antigen in crude serum sample and had a sensitivity of 2.3 +/- 0.15 nM (n = 4) with a linear range over 2.3 x 10(-9) - 3.3 x 10(-(8) M. The piezoimmunosensor was also used to study the kinetics and thermodynamics of antigen/scFv antibody binding.     

中国明对虾蜕皮抑制激素在大肠杆菌中的表达与分离纯化

采用大肠杆菌表达系统对中国明对虾蜕皮抑制激素(MIH)进行了体外重组表达研究.重组蛋白以包涵体的形式存在于菌体中.尿素-SDS-PAGE分析表明,经1mmol/L的IPTG诱导4h后,重组蛋白获得了大量表达,在分子量为13 kD处有一条与预测大小一致的特异性蛋白条带;经金属螯合柱纯化后,得到了电泳纯的融合蛋白.Western blotting检测结果表明:重组表达的融合蛋白与兔抗刀额新对虾MIH的多克隆抗体特异结合,证实该融合蛋白为中国明对虾MIH.通过胶内酶切与LC-ESI-MS分析进一步证实融合蛋白的部分肽段与日本对虾MIH一致.MIH融合蛋白的成功表达为进一步深入研究其在中国明对虾蜕皮过程分子作用机制奠定了基础.