抗甲硝唑单链抗体融合蛋白的表达及其ELISA检测方法建立

为实现甲硝唑单链抗体(single-chain variable fragment,scFv)在大肠杆菌中的可溶性表达,以甲硝唑抗体可变区基因(VH、VL)为模板,扩增甲硝唑的scFv基因,scFv双酶切后与PLIP6/GN载体重组,构建重组质粒PLIP6/GNscFv,转化大肠杆菌JM109,阳性克隆转大肠杆菌BL21经温度诱导表达重组单链抗体,并通过聚丙酰胺凝胶电泳(sodium dodecyl sulfate polyacrylamide gel electrophoresis,SDS-PAGE)对其鉴定。采用竞争酶联免疫(enzyme linked immunosorbent assay,ELISA)检测重组单链抗体的抗原结合活性。结果表明诱导表达的scFv分子量接近33 kDa,能够特异性识别兽药甲硝唑,半抑制率(IC50)为(0.72±0.04)ng/mL。选择虾样品进行添加回收试验,建立的方法检测其回收率在88.54%到113.27%之间。这说明成功构建重组质粒PLIP6/GN-scFv并实现可溶性表达并可用于实际样品检测。     

抗克伦特罗单链抗体在毕赤酵母GS115中的表达

构建表达抗克伦特罗单链抗体(CBL scFv)的毕赤酵母菌株,筛选高拷贝转化子并诱导CBL scFv在毕赤酵母中的分泌表达。将构建的含有CBL scFv基因的重组真核表达载体pPICZαA-CBL经BstX I酶切线性化后,利用电转化方法转化毕赤酵母菌GS115感受态细胞,通过Zeocin抗性平板筛选高拷贝转化子,以甲醇诱导蛋白表达,对表达产物进行SDS-PAGE、Western blotting鉴定及ELISA活性分析。结果显示,通过筛选出的重组毕赤酵母菌株分泌表达了1个分子量约为41kDa的蛋白,该蛋白可与鼠抗His标签单克隆抗体发生特异性结合,且可被游离的CBL竞争性抑制,IC50值为5.1ng/mL。经检测,重组抗体表达量为0.095g/L。这说明分泌表达CBL scFv的毕赤酵母菌株构建成功,为后期CBL scFv发酵与制备奠定了基础。     

拟除虫菊酯类农药单链可变区抗体的制备、 鉴定及其特异性分析

目的制备拟除虫菊酯类农药可溶性单链可变区(single-chain fragment variable,scFv)抗体,鉴定其免疫活性并对其特异性进行分析。方法构建表达载体pET28a(+)-scFv和pET42a(+)-scFv并转化BL21(DE3),重组菌经诱导后通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)鉴定目的蛋白的表达。可溶性融合蛋白scFv/GST经Glutathione Sepharose 4B纯化后用FactorXa切除GST标签获得scFv,然后采用Western blotting法对其进行鉴定。scFv对7种拟除虫菊酯类农药的特异性采用间接竞争酶联免疫法进行分析。结果经0.1mmol/LIPTG诱导后,重组菌BL21(DE3)-p ET28a(+)-scFv和BL21(DE3)-pET42a(+)-scFv表达的目标蛋白分别为包涵体抗体和部分可溶性抗体。经鉴定,可溶性scFv相对分子量约为30k Da,效价为1:25600。同源半抗原Hap1对单链抗体sc Fv的IC50为2.43μg/mL,检出限为0.045μg/mL;scFv抗体对氯菊酯、氯氰菊酯、溴氰菊酯均有较高特异性,IC50分别为3.29、4.72和5.55μg/mL。结论本研究成功获得了拟除虫菊酯类农药的可溶性单链可变区抗体,该抗体对3种拟除虫菊酯农药具有很强的特异性,该研究可为拟除虫菊酯类农药多残留免疫分析提供依据。     

人体中可溶性VEGFR-2酪氨酸激酶在大肠杆菌中的表达、纯化及活性测定

针对人体血管内皮生长因子受体2(Vascular endothelial growth factor receptor 2,VEG-FR-2)受体型酪氨酸激酶(Receptor tyrosine kinase,RTK)进行体外表达,并对表达和纯化条件进行优化。构建表达VEGFR-2酪氨酸激酶重组大肠杆菌,通过研究不同诱导温度、IPTG浓度、诱导时间和超声条件等对蛋白表达的影响,采用Ni-NTA亲和层析法、Western Blot法ELISA方法对重组蛋白进行纯化、鉴定和活性测定。成功地将1 000 bp左右的VEGFR-2酪氨酸激酶DNA片段插入载体pQE30中,在优化条件下重组蛋白较好地可溶性表达。SDS-PAGE表明重组蛋白相对分子质量约为36 000,与预期一致,Western Blot分析表明它能与抗Flk-1和抗His抗体特异反应,ELISA检测结果表明重组蛋白具有利用ATP催化底物磷酸化的激酶活性。通过重组DNA技术使人体VEGFR-2酪氨酸激酶在大肠杆菌得到可溶性表达,纯化重组蛋白具有较高的活性。     

抗大田软海绵酸单抗独特型抗体的制备及鉴定

目的:研制大田软海绵酸(okadaic acid,OA)单抗的独特型抗体并鉴定其模拟抗原特性,为建立无毒检测提供借鉴.方法:利用小鼠腹水法大量制备抗OA单抗,经辛酸饱和硫酸铵法纯化后用胃蛋白酶酶切制备F(ab')<,2>片段,免疫日本大耳白兔,取其血清,琼脂扩散法检测血清抗体与抗OA单抗反应活性,ELISA方法鉴定血清...     

抗克百威单链抗体的可溶性表达载体的构建及鉴定

为构建克百威(carbofuran,CBF)单链抗体(sc Fv)可溶性表达载体,实现其在大肠杆菌中的表达。以p CANTAB5E-sc Fv质粒为模板扩增CBF的重链(VH)及轻链(VL)片段,通过引物设计引入由15个氨基酸组成的连接肽(Gly4Ser)3,经重叠延伸拼接重链及轻链片段,并通过PCR扩增得到sc Fv基因,再与载体p LIP6/GN连接,转化大肠杆菌BL21,阳性克隆质粒经PCR鉴定并测序。重组菌经0.6μmol/L异丙基硫代半乳糖苷(IPTG)及低温诱导表达重组单链抗体,并通过SDS-PAGE和Western blotting对其鉴定。采用ELISA法检测重组单链抗体的抗原结合活性。结果表明重组质粒p LIP6/GN-sc Fv含有插入片段,sc Fv与碱性磷酸酶(AP)相连得到重组蛋白的分子量接近78 ku,可被游离的CBF竞争性抑制,IC50值为26.80 ng/m L。这说明成功构建了重组质粒p LIP6/GN-sc Fv并实现了其在大肠杆菌中的可溶性表达,为研究其在免疫分析方法中的应用奠定了基础。     

双抗夹心ELISA检测食品中大肠杆菌O157:H7方法研究

研究获得纯化抗大肠杆菌O157:H7IgY抗体,经检测10mg/ml纯化IgY抗体的效价为1:320;以大肠杆菌O157:H7免疫新西兰大耳白兔,获得兔抗大肠杆菌O157:H7IgG抗体,效价达1:25600。以兔抗大肠杆菌O157:H7IgG抗体稀释3200倍作为捕获抗体,抗大肠杆菌O157:H7IgY抗体为检测抗体建立双抗夹心ELISA方法检测大肠杆菌O157:H7,正交试验分析表明,捕获抗体于37℃包被2h、不封闭、抗原与捕获抗体于37℃结合2h、检测抗体浓度为0.25mg/ml、与抗原于37℃结合1h为最优反应条件。该方法对纯培养菌液检出限为105CFU/ml,具有良好的敏感性及特异性。染菌样品经在EC增菌液中选择性培养后进行双抗夹心ELISA检测,接种量为0.1～1CFU/g(ml)的样品在培养12h后可检出阳性反应,1～10CFU/g(ml)的样品在培养8h后可检出阳性反应。     

抗克伦特罗单链抗体基因构建及蛋白结构模拟

目的：构建抗克伦特罗单链抗体(scFv)基因cbl,进行蛋白质结构模拟并对其理化特性进行分析。方法：采用重叠延伸PCR 方法,以能特异性分泌抗克伦特罗mAb 的杂交瘤细胞株5D1 为原料,构建抗克伦特罗scFv 基因cbl,进行测序,采用生物信息软件对氨基酸序列推导并进行结构预测,同时对理化性质进行分析。结果：抗克伦特罗scFv 基因cbl 全长720bp,对应225 个氨基酸,单链抗体分子量约为25826.6,等电点预测值8.78,为碱性蛋白质;二级结构显示抗克伦特罗单链抗体含α螺旋20 处,β折叠99 处,β转角28 处,随机卷曲93 处。cbl 三级结构建模显示重链(VH)和轻链(VL)形成一个疏水的" 口袋",Linker 游离于此结构之外,符合scFv 的结构特点,明显具有抗原结合位点的空间构象,理论上应该具有良好的抗原结合活性。结论：构建一个抗克伦特罗scFv 基因cbl,应用信息学技术所获得的预测和分析结果为抗克伦特罗单链抗体的进一步表达、纯化和活性研究提供信息。     

动物源性食品中抗司帕沙星单克隆抗体的研制与鉴定

为了制备高灵敏、高特异性的司帕沙星(SPFX)单克隆抗体,鉴定免疫学特性。采用蛋白质偶联技术DCC法制备免疫抗原SPFX-BSA和包被抗原SPFX-OVA,免疫BALB/c小鼠,用间接ELISA和阻断ELISA选出产生优质多克隆抗体的的小鼠进行细胞融合,经过多次亚克隆筛选出分泌高敏感的特异性抗SPFX单克隆抗体的杂交瘤细胞株,体内诱生腹水法制备抗SPFX的单克隆抗体并对其免疫学特性进行鉴定。结果表明,间接ELISA检测显示免疫的6只小鼠效价均达1.28:10~4以上,融合后筛选出四株杂交瘤细胞株4H4-C8,4H4-C4,4H4-B1和4H4-E9;其细胞上清效价分别为1:1.4×10~3,1:8.0×10~2,1:5.0×10~2,1:6.7×10~2;4H4-C8株腹水效价为1:2.6×10~6,抗SPFX的单克隆抗体对SPFX的IC_(50)为31μg/L,且具有较好的特异性。本研究成功合成SPFX人工抗原,制备出优质的单克隆抗体,为SPFX免疫学快速检测方法的建立奠定坚实的基础。     

黑大蒜和鲜大蒜中可溶性蛋白成分的比较研究

为探究黑大蒜和鲜大蒜中可溶性蛋白成分的差异,采用研磨浸提、硫酸铵沉淀、凝胶柱层析的方法,获得黑大蒜和鲜大蒜可溶性蛋白;采用阿尔玛蓝（Alamar Blue）法检测其体外抗肿瘤活性,采用牛津杯法检测其抗菌活性,并利用十二烷基硫酸钠-聚丙烯酰胺凝胶电泳法分析其可溶性蛋白成分的差异。结果表明：从黑大蒜和鲜大蒜中提取的可溶性蛋白均具有抗人肝癌细胞株HepG2的活性,鲜大蒜中可溶性蛋白的抗肿瘤活性高于黑大蒜中可溶性蛋白的抗肿瘤活性,且具有耐热性;鲜大蒜中可溶性蛋白还具有抗大肠杆菌和抗金黄色葡萄球菌的活性,其分子质量在10～55 kD之间;黑大蒜中的可溶性蛋白不具有抗菌活性,其分子质量在10 kD左右。黑大蒜与鲜大蒜的可溶性蛋白在种类上差异较大,在抗人肝癌细胞株HepG2、抗大肠杆菌和抗金黄色葡萄球菌的功能上差异显著。     

Development of a Single Chain Variable Fragment Antibody and Application as Amatoxin Recognition Molecule in Surface Plasmon Resonance Sensors

A phage display library of single chain variable fragment (scFv) antibodies was constructed to screen specific scFv antibodies against amatoxins. One recombinant scFv antibody with high specificity for amatoxins, termed scFv-A4, was isolated from the library by biopanning. SDS-PAGE analysis showed that the soluble scFv-A4 protein was expressed successfully, and the protein posed relatively good specificity with an IC₅₀ of 77.48 ng/mL and IC₁₅ of 1.91 ng/mL using indirect competitive ELISA (ic-ELISA). On the basis of the expressed scFv-A4 protein, a rapid and simple new immunoassay using surface plasmon resonance (SPR) sensor for the detection of amatoxins was developed. The IC₅₀ and IC₁₅ of new immunoassay were 7.66 and 0.17 ng/mL. The sensitivity of immunoassay using SPR sensor was about 10 times that of ic-ELISA. These results showed the potential usefulness and advantages of new immunoassay using SPR sensor for the detection of toxins.     

Immunochemical Properties of Native and Heat Denatured Ovalbumin

Affinity antibodies purified against native ovalbumin were found more reactive (higher avidity) against heat-denatured ovalbumin than against the native molecule by three different immunochemical methods. Quantitative immunoprecipitation in soluble phase revealed that more antigen-antibody complexes were insoluble with native ovalbumin than with heat-denatured ovalbumin; 25% of antibodies were still present in supernatant at equivalence as measured by ELISA. At the same conditions with heat-denatured ovalbumin, very small amounts of antibodies were precipitated while almost no activity was found in the supernatant. Classical ELISA or competitive ELISA test allowed detection down to 100 ng/mL of native ovalbumin and 10 ng/mL of denatured ovalbumin.     

Effects of cytoplasmic and periplasmic chaperones on secretory production of single-chain Fv antibody in Escherichia coli

The effects of cytoplasmic and periplasmic chaperones on the secretory production of an anti-bovine ribonuclease A single-chain variable fragment (scFv) 3A21 in Escherichia coli were investigated. Co-expression of a cytoplasmic chaperone, GroEL/ES, DnaK/DnaJ/GrpE, trigger factor, or SecB with 3A21 scFv affected the proportions of antigen-binding activity in the cytoplasmic soluble fraction, the periplasmic fraction, and the extracellular medium, but there was no significant difference in the total activity compared to the control without chaperone co-expression. On the other hand, co-expression of a periplasmic chaperone, Skp or FkpA, with the exception of DsbC, greatly increased the binding activity in all the soluble fractions. Co-expression of both Skp and FkpA had no synergistic effect. Combinations of cytoplasmic and periplasmic chaperones decreased the productivity. In shake-flask cultures of cells co-expressing Skp or FkpA, considerable amounts of 3A21 scFv were detected in the extracellular medium by enzyme-linked immunosorbent assay (ELISA) and Western blot, and the extracellular production level of 3A21 scFv was calculated to be around 40mg/l. The binding activity of 3A21 scFv co-expressed with Skp was slightly higher than that with FkpA. These results indicate that the co-expression of periplasmic chaperones Skp and FkpA is extremely useful for the secretory production of scFvs in a culture medium using E. coli, but cytoplasmic chaperones and multiple-chaperone combinations may not be effective.     

抗伏马菌素B_1单链抗体的原核表达及其抗体活性和稳定性分析

研究了对抗伏马菌素B1单链抗体在不同原核表达条件下表达情况及其抗体稳定性,以促进其在免疫学分析中的应用。通过构建表达载体pET22b-1D11和pMAL-1D11,分析了诱导温度、诱导剂浓度、宿主菌和高溶解性标签蛋白对单链抗体表达形式的影响;采用酶联免疫吸附实验法(ELISA)分析了单链抗体的热稳定性和有机溶剂耐受性。结果表明:条件优化后,单链抗体及其融合蛋白主要以包涵体形式表达;热处理会降低单链抗体活性甚至使其完全失活,甲醇会降低基于单链抗体的ELISA体系的灵敏度。     

抗呋喃唑酮代谢物单链抗体基因构建及蛋白结构分析和活性鉴定

从单克隆杂交瘤细胞出发,构建抗3-氨基-2-噁唑烷酮（3-amino-2-oxazolidinone,AOZ）衍生物单链抗体基因,并对其蛋白质进行理化性质分析及结构预测和活性鉴定。首先以呋喃唑酮代谢物AOZ衍生物单克隆抗体杂交瘤细胞1D2为原料,提取总RNA后克隆重链（VH）、轻链（VL）可变区基因,然后使用重叠延伸聚合酶链式反应技术用连接肽(G4S)3将VH、VL基因连接构建抗AOZ衍生物单链抗体基因,经测序后采用生物信息软件对抗体编码的氨基酸序列进行推导并展开生物信息学分析,再将此基因与Plip6/GN表达载体连接后进行表达并采用直接竞争酶联免疫分析法（direct competitive enzyme-linked immunosorbent assay,dcELISA）鉴定其活性。结果表明：抗AOZ衍生物单链抗体基因全长750?bp,编码250?个氨基酸,相对分子质量为27?012.20,理论等电点为9.13,属于碱性氨基酸;其二级结构预测结果显示抗体蛋白含20?处α-螺旋、25?处β-转角、114?处无规卷曲、91?处延伸带结构;三级结构预测的正面俯视图显示重、轻链由连接肽相连形成一个“环桶状”结构,侧面显示为“口袋状”,这与常规单链抗体的结构特征一致,dcELISA结果也显示该抗体具有良好的抗原结合活性。本研究为后续AOZ残留检测免疫分析方法的建立及抗AOZ衍生物单链抗体的改造奠定一定基础。     

Production of single-chain Fv antibodies against melamine from a rabbit phage display library for the development of a melamine immunoassay

A rabbit phage display library of single chain Fv (scFv) antibodies was constructed to screen specific scFv antibodies against melamine. Rabbits were immunized with melamine immunogens prepared via glutaraldehyde method. Two recombinant scFv antibodies with high specificity for melamine, termed A9 and B4, were isolated via library panning. SDS-PAGE analysis showed that the soluble A9-scFv and B4-scFv antibodies were expressed successfully and the scFvs showed good sensitivity with an IC₅₀ of 12.64 and 10.32 μM to melamine, respectively. The cross-reactivity of 2 scFv antibodies with cyanuric acid and chloromycetin were below 1%. The high similarity of 2 scFv nucleotide sequences indicated that they may originate from 1 B cell clone that had undergone somatic diversification. As a consequence, the conjugation synthesis was successful and the selected scFvs could be used for sensitive detection of melamine in foods and feeds by immunoassay.     

抗-Cry2Aa单链抗体分子互作及荧光免疫检测研究

Cry2Aa毒素是一种新型生物农药,分析该毒素与特异性单链抗体分子互作,并建立快速检测毒素残留的方法对保障食品和生态安全有重要意义。本研究以同源蛋白为模板对单链抗体进行建模,并进行了Cry2Aa毒素与单链抗体的分子对接模拟,确定关键结合位点,以此为基础将单链抗体作为检测抗体,建立了间接竞争时间分辨荧光免疫分析方法,对大米样品中Cry2Aa毒素进行了检测。利用生物信息学工具模拟获得单链抗体三维结构模型,分子对接结果显示重链可变区81NY82和121SGNY124区域及轻链可变区的245YSSN248氨基酸残基在与毒素结构Ⅱ区识别过程中起关键作用,为建立高效检测方法奠定基础,进一步基于该单链抗体建立的时间分辨检测方法灵敏度(IC10)为0.08 ng/mL,中抑制浓度(IC50)为7.99 ng/mL,线性检测范围(IC20~IC80)为0.24~263.77 ng/mL,且与常规双抗夹心ELISA法检测呈良好线性关系。     

噬菌体随机肽库淘选桔霉素模拟表位的研究

目的:从噬菌体随机肽库中淘选模拟桔霉素表位的噬菌体粒子。方法:以抗桔霉素的单克隆抗体为配基,分别免疫亲和淘选以融合蛋白形式表达在丝状噬菌体M13外壳蛋白Ⅲ上的随机7肽库和12肽库,以ELISA方法鉴定阳性克隆,同时进行DNA测序以分析插入的7肽和12肽的氨基酸序列。结果:经过3轮淘选,在7肽库中淘选到20株能与该抗体特异性结合的阳性克隆,在12肽库中淘选到33株能与该抗体特异性结合的阳性克隆,且该结合均能被桔霉素阻断,模拟表位的共有序列为X-组氨酸-赖氨酸-X-X-X-X,X为任意氨基酸。以7肽库中亲和力最强的克隆(P10)建立了竞争ELISA检测方法,线性范围为10～325ng/ml,检测下限为10ng/ml;以12肽库中亲和力最强的克隆(P1)建立了竞争ELISA检测方法,线性范围为10～439ng/ml,检测下限为10ng/ml。结论:噬菌体展示技术可成功淘选到桔霉素模拟表位,高度保守的His和Lys的存在,提示His和Lys在CIT与其配体的结合中可能起重要作用。     

基于ELISA克罗诺杆菌单链抗体制备与鉴定

利用基因工程技术制备克罗诺杆菌特异性单链抗体(sc Fv)。从克罗诺杆菌单克隆抗体的杂交瘤细胞中提取总RNA,利用RT-PCR反转录合成第一链c DNA后再扩增出抗体的重链可变区(VH)和轻链变区(VL)基因片段,采用重叠延伸PCR的方法,用柔性多肽Linker接头(Gly4Ser)3将VH基因和VL基因拼接成sc Fv基因片段,XhoⅠ﹑Eco RⅠ限制性内切酶双酶切sc Fv后克隆到原核表达载体p ET-26b中构建重组质粒sc Fv-pet-26b,挑取阳性克隆提取重组质粒后转入大肠杆菌BL21中进行诱导表达,通过His柱进行亲和层析,最后利用ELISA检测单链抗体的活性。成功构建了表达克罗诺杆菌单链抗体的基因工程菌株,通过SDS-PAGE和ELISA试验结果表明,诱导表达的罗诺杆菌单链抗体分子量约为30 k Da,其能与罗诺杆菌特异性结合,可作为免疫检测罗诺杆菌的候选抗体分子。     

脱氧雪腐镰刀菌烯醇ELISA定量检测试剂盒的研制

研制了脱氧雪腐镰刀菌烯醇ELISA试剂盒,用于检测谷物粮食中的脱氧雪腐镰刀菌烯醇(Deoxynivalenol,DON)。在抗DON单克隆抗体的基础上,用间接竞争酶联免疫吸附检测试剂盒各项指标。结果为:试剂盒最低检测浓度为20ng/mL,检测线性范围为50～400ng/mL,IC50103ng/mL。平均加标回收率>80%,批内变异系数小于10%,批间变异系数<15%。用试剂盒测定盲样,其结果与德国拜发试剂盒及免疫亲和柱-高效液相色谱检测结果无显著性差异。说明试剂盒指标符合相关技术要求,具有快速、灵敏、准确、方便等特点。