抗血小板膜糖蛋白单克隆抗体SZ-2单链抗体的构建、表达及功能研究

目的：降低鼠源性抗体的免疫原性及分子量，为其进一步研究、应用奠定基础。方法：应用RT—PCR技术，克隆SZ-2重链、轻链可变基因。应用基因重组技术构建SZ-2单链抗体表达载体pET22-2scFv，导入大肠杆菌BL21(DE3)Plys，诱导表达。流式细胞术、ELISA、Western blot检测SZ-2 scFv与血小板结合能力，瑞斯托霉素、凝血酶和ADP诱导血小板聚集试验对表达产物功能进行研究。结果：克隆基因序列符合小鼠轻、重链可变区基因特征；pET22—2scFv表达质粒拼接正确；表达产物以包涵体形式为主，表达量占菌体总蛋白的25％；纯化、复性后证明表达产物具有与血小板结合的活性，并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。结论：成功表达了SZ—2单链抗体，该小分子抗体具有与血小板结合的活性，并可抑制瑞斯托霉素、凝血酶诱导的血小板聚集。     

CEA单链抗体基因在大肠杆菌中的克隆及表达

目的尝试将ＣＥＡ单链抗体在大肠杆菌中进行高效表达。方法以ＣＥＡ单链抗体基因为模板，设计４对引物，ＰＣＲ扩增，将４条ＤＮＡ扩增片段分别插入原核表达载体ｐＢＶ２２０中；重组质粒转染大肠杆菌ＴＧ１，４２℃热诱导外源蛋白的表达；包涵体经８ｍｏｌ／Ｌ脲溶解及谷胱甘肽系统复性；过离子交换柱进行纯化；ＥＬＩＳＡ夹心法测活性。结果得到了ＳＤ序列与起始密码ＡＴＧ分别相隔着５，６，７，８个核苷酸的重组质粒；ＳＤＳ－ＰＡＧＥ显示转染入重组质粒的ＴＧ１菌经热诱导后有分子量约为３×１０４的外源蛋白，表达量最高达９３０ｍｇ／Ｌ培养液，约占菌体总蛋白的５０％；ＥＬＩＳＡ活性测定结果表明复性后的ＳｃＦｖ有较高的抗原结合活性。结论ＣＥＡ单链抗体在大肠杆菌中获得了较高水平的表达，且经复性后有较高的抗原结合活性。     

抗乙肝病毒表面抗原单链抗体在大肠杆菌中的高效表达

在大肠杆菌中高效表达抗乙肝表面抗原的单链抗体。方法：通过ＰＣＲ将单链抗体基因重组到原核细胞表达载体ＰＴ１７中,构建单链抗体高效表达载体ＰＴ７ＳＣ。将ＰＴ７ＳＣ质粒转化大肠杆菌ＢＬ２１,ＴＰＴＧ诱导进行表达。     

HBsAg人源噬菌体单链抗体的筛选及其在临床诊断中的应用

目的 筛选乙型肝炎病毒(HBV)表面抗原(HBsAg)的人源噬菌体单链抗体，并探讨其在临床治疗和诊断中的应用价值。方法 以HBsAg阳性血清超速离心纯化的HBsAg为固相抗原，从噬菌体单链可变区半合成抗体库中经过5轮“吸附—洗脱—扩增”筛选过程，获得特异性较强的HBsAg人源单链可变区抗体(ScFv)；用该抗体对10例石蜡包埋的乙型肝炎患者肝组织进行免疫组化鉴定。结果 酶联免疫吸附法(ELISA)结果表明，制备的HBV人源单链抗体能与HBsAg抗原特异性结合；免疫组化结果表明，该抗体能够特异性识别乙型肝炎患者肝组织中的HBsAg抗原，与正常肝组织及丙型肝炎病毒(HCV)的抗原均无交叉反应。结论 此法制备的单链抗体亲和性好，特异性强，且制备方法简便，周期短，为HBV病原的检测提供了新的有效试剂，为今后HBsAg人源抗体的研究和应用奠定了基础。     

抗丙型肝炎病毒非结构蛋白NS3单链抗体的制备及免疫组织化学研究

目的 研制抗丙型肝炎病毒（HCV）非结构蛋白NS3的人源噬菌体单链抗体,并探讨其在临床诊断中的应用价值。方法 以重组的丙型肝炎病毒非结构蛋白NS3为固相抗原,利用亲和筛选的原理,从噬菌体抗体库中经过5轮“吸附－洗脱－扩增”的筛选过程及酶联免疫吸附试验（ELISA）、斑点免疫杂交试验和DNA序列分析,获得HCV NS3的人源单链抗体;用该抗体与不同来源的HCV NS3抗原进行反应;对10例石蜡包埋的丙型肝炎患者肝组织进行免疫组化鉴定。结果 ELISA结果表明,所制备的HCV NS3人源单链抗体能与不同来源的HCV NS3抗原特异性结合（吸光度A值为1.38）;免疫组化结果表明,该抗体能够特异性识别丙型肝炎患者肝组织HCV NS3抗原,与正常肝组织及乙型肝炎病毒（HBV)的表面抗原均无交叉反应。结论 此法制备的单链抗体亲和性好,特异性强,且制备方法简便,周期短,可用于HCV NS3病原的检测。     

抗AIF单链抗体在大肠杆菌中的可溶性表达和鉴定

目的:获得具有生物活性的抗酸性同功铁蛋白(AIF)单链抗体(scFv)。方法:将AIF4c9scFv基因亚克隆于原核表达载体pPOW3,构建重组表达质粒pPOW4c9,电转化大肠杆菌DH5α,以温度诱导重组抗AIFscFv表达,经镍螯合层析柱纯化后,用ELISA和Westernblot检测表达蛋白与AIF的结合特性。结果:抗AIFscFv抗体在大肠杆菌中获得可溶性表达,亲和力常数KD为3.18×10-8mol/L;纯化后的可溶性AIFscFv含量约为1.6mg/L。重组蛋白能特异识别AIF,并且具有良好的抗体生物学活性。结论:在大肠杆菌中表达并纯化了特异性可溶性抗AIFscFv抗体。     

抗CD3 scFv基因的真核表达及其生物学活性鉴定

目的：真核表达抗CD3单链抗体(scFv)，并研究其生物学活性。方法：将编码抗CD3 scFv的DNA片段插入真核表达载体pDisplay中。对所构建重组表达载体进行测序确认后，以电穿孔法将重组质粒导入Hela细胞，以原位杂交法检测抗CD3 scFv的表达，以3H TdR掺入法检测其在体外对T细胞的活化作用，以MTT比色法观察转染的。Hela细胞与T细胞混合培养后，诱发的细胞毒性T细胞的杀伤作用。结果：成功地构建了抗CD3 scFv的真核表达载体，并在Hela细胞中获得表达。所分泌的抗CD3 scFv在抗CD28mAb存在的条件下，能够刺激T细胞活化。将转染的Hela细胞与T细胞混合培养能够诱发CTL的杀伤作用。结论：真核表达的抗CD3 scFv具有刺激T细胞活化的活性，可用于构建对肿瘤进行免疫治疗的双功能分子。     

抗人Lipocalin型前列腺素D合酶单链抗体基因的构建与表达

利用RT-PCR技术,从稳定分泌抗人Lipocalin型前列腺素D合酶(Lipocalin-typeprostaglandinDsynthase,L-PGDS)单克隆抗体的鼠杂交瘤细胞中获得抗体可变区基因;通过一柔性连接短肽[(Gly)4Ser]3,SOEPCR法将此重、轻链可变区拼接成完整的抗人L-PGDS单链抗体基因,并装入表达载体pET-28a(+),在BL21宿主菌中进行表达。经SDS-PAGE检测显示,在低剂量0.02mmol/LIPTG诱导和较低温度25℃或28℃条件下培养,重组菌表达了一定量的可溶性单链抗体。后经Ni-NTA亲和层析柱纯化,得率为2mg/100ml,纯度达92%;双夹心ELISA和免疫荧光竞争抑制实验证实,纯化后的单链抗体具有较好的亲和力和特异性。     

抗血板GPⅢa单克隆抗体SZ—21单链抗体片断的表达和特性

将能与血小板膜糖蛋白Ⅲa特异结合的单克隆抗体SZ－21构建成单链抗体。应用RT－PCR技术,从分泌SZ－21单克隆抗体的杂交瘤细胞中克隆出VH和VL可变区基因,并构建成SZ－21单链抗体（SZ－21ScFv)。将该单链抗体基因亚克隆到高效表达质粒pET20b,得到pET20b-21ScFv。通过IPTG诱导在大肠杆菌E coli BL21(DE3)PlysS中表达了功能分子。表达产物主要以包涵体形式存在,表达量占菌体蛋白的21％。经过包涵体的溶解、Ni-NTA树脂亲和吸附纯化和蛋白质的体外重折叠等一系列的变性和复性过程,获得具有生物活性的高纯度单链抗体片段。ELISA和Western blot证实该融合蛋白保留了与亲本抗体同样的血小板结合特性。SZ－21单链抗体体外能够抑制ADP诱导的血小板聚集,其抑制能力呈剂量依赖性,在浓度为20mg/L时达到最大抑制率。流式细胞仪检测证实该单链抗体能与内皮细胞反应。该单链抗体有望用于血栓性疾病的治疗。     

Production of polyclonal antibodies against Pelargonium zonate spot virus coat protein expressed in Escherichia coli and application for immunodiagnosis

Pelargonium zonate spot virus (PZSV) is identified recently in tomato plants in the United States. To develop serological diagnostic tools for the detection of this virus, the production of good quality antibodies is a necessity. The coat protein (CP) gene of a California isolate of PZSV was cloned into a bacterial expression vector (pTriEX-4 Ek/LIC). The plasmid pTriEX-4-PZSV-CP was transformed into Escherichia coli Rosetta 2(DE3)pLacI and the recombinant PZSV-CP was expressed as a fusion protein containing N-terminal hexa-histidine and S tags. Expressed PZSV-CP was purified under denaturing conditions by affinity chromatography yielding 3 mg refolded protein per 200 mL of bacterial culture, and used as an antigen for raising PZSV-CP antiserum in rabbits. Specificity of the antiserum to PZSV was shown by Western blot and ELISA. When used in Western blot analysis, the antiserum was able to detect the recombinant protein, the PZSV coat protein and PZSV infected plant samples. The antiserum was successfully used in indirect-ELISA at dilutions of up to 1:16,000 to detect PZSV in infected leaf samples. Direct ELISA was successful only with denatured antigens. This is the first report on production of polyclonal antiserum against recombinant coat protein of PZSV and its use for detection and diagnosis of virus using serological methods.     

溶藻弧菌抗独特型抗体scFv的原核表达及免疫原性鉴定

目的 构建溶藻弧菌抗独特型单链抗体(scFv)的原核表达载体,并对其表达的蛋白进行免疫原性鉴定.方法从分泌溶藻弧菌抗独特型单克隆抗体的杂交瘤细胞株(2F4)中已获得的该抗体重链可变区基因(VH)和轻链可变区基因(VL),通过基因重组构建scFv及原核表达载体pET32a-AL,转化大肠杆菌BL21后用IPTG诱导表达.表达后的重组蛋白利用动物免疫试验进行免疫原性鉴定.结果 scFv基因序列全长747碱基对,编码249个氨基酸,符合小鼠免疫球蛋白可变区基因特征,含有框架区(FRs)、抗原互补决定区(CDRs)及抗体特征性的半胱氨酸残基.ELISA测定和动物免疫试验显示抗独特性抗体的scFv具有藻弧菌一样的免疫原性.结论 成功构建了抗溶藻弧菌独特型抗体scFv原核表达载体并表达于包涵体中,表达的融合蛋白具有与溶藻弧菌一样的免疫原性,为溶藻弧菌抗独特型单链抗体scFv成为鱼用基因工程疫苗奠定了初步基础.     

Adhesion of Escherichia coli on to a series of poly(methacrylates) differing in charge and hydrophobicity

The adhesion of three Escherichia coli strains on to six poly(methacrylates) differing in hydrophobicity and surface charge was measured as a function of time under laminar flow conditions. Polymers used were poly(methy) methacrylate) (PMMA), poly(hydroxyethy) methacrylate) (PHEMA) and copolymers of MMA or HEMA with either 15% methacrylic acid (MAA) or 15% trimethylaminoethyl methacrylate-HCl salt (TMAEMA-CI). Bacterial and polymer surfaces were characterized by means of water contact angles and zeta potentials. Both the sessile drop contact angles and the zeta potentials of the bacterial surfaces were significantly different. No significant differences in the sessile drop contact angles of the polymer surfaces were observed. Using the Wilhelmy plate technique large contact angle hysteresis was observed for the different polymer surfaces. Surfaces of copolymers with MAA had more negative zeta potentials than those of the corresponding homopolymers. Surfaces of copolymers with TMAEMA-CI had positive zeta potentials. The highest numbers of adherent bacteria were found on materials with positive zeta potentials, irrespective of the bacterial strain used. Bacterial adhesion on to copolymers with MAA was less than on to the corresponding homopolymers. Bacterial equilibrium adhesion values correlate with the zeta potentials of the polymer surfaces (r > 0.85). On substrates with less negative zeta potentials high numbers of adhered bacteria were observed. Additionally, the equilibrium bacterial adhesion values could be related with receding contact angles of polymer surfaces with negative zeta potentials (r > 0.86). High equilibrium adhesion values were obtained for polymers with high contact angles. No correlation between the zeta potentials and contact angles of the bacteria with the adhesion values was found.     

Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli

The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli . A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.     

抗人卵巢癌×抗人CD3×抗CD28VH单链三特异抗体的胞内可溶表达、纯化及体外活性测定

目的　研究抗人卵巢癌 (ovariancarcinoma ,oc)×抗人CD3×抗CD2 8VH 单链三特异抗体(singlechaintrispecificantibody,scTsAb)在大肠杆菌中的可溶表达与纯化及纯化后产物的活性测定 ,从而为其应用于卵巢癌治疗的临床研究打下基础。方法　将已构建的scTsAb表达载体转化大肠杆菌BL2 1(DE3)Star菌株 ,采用低温 (30℃ )、低剂量IPTG(0 .2mmol L)诱导 ,进行胞内可溶表达。根据抗卵巢癌三特异抗体 (ocTsAb)等电点较高 (pI9.0 ) ,而菌体蛋白大多为酸性蛋白的特点 ,利用DEAE弱阴离子交换层析(pH8.0 )进行一步纯化 ,并利用ELISA及FACS的方法检测纯化后抗卵巢癌三特异抗体的活性。结果　(1)SDS PAGE鉴定低温诱导时可溶比例达到 5 6 %。 (2 )绝大多数菌体蛋白被DEAE层析柱吸附 ,而抗卵巢癌三特异抗体在穿透液中流出 ,SDS PAGE检测纯度达到 90 %。 (3)ELISA结果显示纯化后的抗卵巢癌三特异抗体与重组CD2 8纯抗原 ,Jurkat(CD3 )细胞膜提取抗原 ,SKOV3细胞膜提取抗原均有特异性结合。 (4 )FACS结果证明纯化后的抗卵巢癌三特异抗体与Jurkat(CD3 )活细胞、SKOV3活细胞有特异性结合。结论　低温诱导胞内可溶表达的抗人卵巢癌×抗人CD3×抗CD2 8VH 单链三特异抗体经弱阴离子交换层析一步纯化后仍保持原有免疫学活性 ,这     

A convenient enzyme-linked immunosorbent assay for testing whether monoclonal antibodies recognize the same antigenic site. Application to hybridomas specific for the β2-subunit of Escherichia coli tryptophan synthase

Seven hybridoma clones, producing antibodies directed against the β₂-subunit of Escherichia coli tryptophan synthase, have been obtained from mouse cells. To test whether the corresponding monoclonal antibodies recognize different epitopes on β₂, an ELISA double antibody binding system has been developed and is reported here. The antigen is first coated onto a microtitration plate. Two monoclonal antibodies are then added either separately or simultaneously, and the amount of bound antibody is quantitatively measured by use of immunoglobulin (rabbit anti-mouse IgG) linked to β-galactosidase. Additivity of the bound enzymatic activity is observed when the monoclonal antibodies bind to distinct epitopes.Using this test, it is shown that, of the 7 anti-β₂ monoclonal antibodies obtained, 5 recognize the same antigenic site and the 2 others recognize a second site.     

Detection of pap, sfa and afa adhesin-encoding operons in uropathogenic Escherichia coli strains: Relationship with expression of adhesins and production of toxins

A total of 243 Escherichia coli strains isolated from patients with urinary tract infections (UTI) were investigated for the presence of pap, sfa and afa adhesinencoding operons by using the polymerase chain reaction. It was found that 54%, 53% and 2% of the strains exhibited the pap, sfa and afa genotypes, respectively. Pap⁺ and/or sfa⁺ strains were more frequent in cases of acute pyelonephritis (94%) than in cases of cystitis (67%) (P < 0.001) and asymptomatic bacteriuria (57%) (P < 0.001). The pap and/or sfa operons were found in 90% of strains expressing mannose-resistant haemagglutination (MRHA) versus 37% of MRHA-negative strains (P < 0.001). The presence of pap and sfa operons was especially significant in strains belonging to MRHA types III (100%) (without P adhesins) and IVa (97%) (expressing the specific Gal-Gal binding typical of P adhesins). Both pap and sfa operons were closely associated with toxigenic E. coli producing a-haemolysin (Hly⁺) and/or the cytotoxic necrotizing factor type 1. There was an apparent correlation between the pap and sfa operons and the O serogroups of the strains. Thus, 93% of strains belonging to O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 and O83 possessed pap and/or sfa operons, versus only 32% of strains belonging to other serogroups (P < 0.001). The results obtained in this study confirm the usefulness of our MRHA typing system for presumptive identification of pathogenic E. coli exhibiting different virulence factors. Thus, 85% of strains that possessed both pap and sfa adhesinencoding operons showed MRHA types III or IVa previously associated with virulence of E. coli strains that cause UTI and bacteraemia.La PCR a permis de détecter les opérons pap, sfa et afa chez 243 souches de Escherichia coli isolées d'infections de l'arbre urinaire. On observe que respectivement 54, 53 et 2% des souches sont du génotype pap, sfa et afa. Les souches pap⁺ et/ou sfa⁺ sont plus fréquentes dans les pyelonephritis aiguës (94%) que dans les cystites (67%) (P < 0,001) et dans les bactériuries asymptomatiques (57%) (P < 0,001). Les opérons pap et/ou sfa sont décelés chez 90% des souches MRHA⁺ (hémagglutination mannoserésistante) et 37% des souches MRHA⁻ (P < 0,001). La présence des opérons pap et sfa est spécialement significative chez les souches appartenant au type III de MRHA (100%) sans adhésines P, et au type IVa (97 %) exprimant la liaison Gai-Gal typique des adhésines P. Les opérons pap et sfa sont tous deux étroitement associés aux souches toxigéniques produisant l'α-hémolysine (Hly⁺) et le facteur cytotoxique nécrotique de type 1. Une corrélation apparaît entre les opérons pap et sfa et le sérogroupe: 93 % des souches appartenant aux groupes O1, O2, O4, O6, O7, O14, O15, O18, O22, O75 et O83 possèdent ces opérons, et seulement 37 % des souches appartenant à d'autres sérogroupes (P < 0,001) les possèdent. Ces résultats confirment l'utilité de notre typage MRHA pour l'identification (présomptive) des souches pathogènes de E. coli porteuses de divers facteurs de virulence. Ainsi, 85 % des souches possédant à la fois les opérons pap et sfa codant les adhésines sont du type III ou IVa (de MRHA) en relation avec la virulence des souches responsables d'infections urinaires et de bactériémies.     

Heterologous expression of full-length capsid protein of porcine circovirus 2 in Escherichia coli and its potential use for detection of antibodies

A capsid protein of porcine circovirus 2 (PCV 2) serves as a diagnostic antigen for the detection of PCV 2-associated disease known as a postweaning multisystemic wasting syndrome (PMWS). In this report, a bacterial expression system was developed for the expression and purification of the full-length PCV 2 capsid (Cap) protein from a codon-optimized cap gene. Replacement of rare arginine codons located at the 5′ end of the cap reading frame with codons optimal for E. coli was found to overcome the poor expression of the viral protein in the prokaryotic system. The Cap protein was purified to greater than 95% homogeneity by using a single cation-exchange chromatography at a yield of 10 mg per litre of bacterial culture. Despite the failure of the E. coli-expressed Cap protein to self-assemble into virus-like particles (VLPs), the immunization of mice with recombinant Cap yielded antibodies with the same specificity as those raised against native PCV 2 virions. In addition, the antigenic properties of the purified Cap protein were employed in a subunit-based indirect ELISA to monitor the levels of PCV 2 specific antibodies in piglets originating from a herd which was experiencing PCV 2 infection. These results pave the way for a straightforward large-scale production of the recombinant PCV 2 capsid protein and its use as a diagnostic antigen or a PCV 2 subunit vaccine.