抗弓形虫重组SAG1抗原单克隆抗体的制备与鉴定

目的制备抗弓形虫SAG1单克隆抗体(McAb)。方法将pET32(a)-trSAG1重组质粒转化EscherichiacoliBL21(DE3),用0.1mmol/LIPTG诱导表达,超声破菌后获得可溶性表达产物,通过Ni-NTA一步法纯化,以纯化的rSAG1为抗原,免疫BALB/c小鼠,采用杂交瘤技术制备McAb,用ELISA和有限稀释法筛选出分泌高滴度McAb杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,Western blot分析其特异性。结果筛选出能稳定分泌抗rSAG1单克隆抗体的5株杂交瘤细胞株,抗体重链均为IgG1,轻链均为κ链;Western blot分析显示,5株单抗均能与弓形虫速殖子超声抗原中的天然SAG1发生特异性结合。结论制备的抗rSAG1杂交瘤细胞株能分泌识别SAG1的高特异性单抗,为进一步研究其在弓形虫病免疫诊断中的应用奠定了基础。     

抗弓形虫重组SAG1抗原单克隆抗体的制备与鉴定

目的制备抗弓形虫SAG1单克隆抗体（McAb）. 方法将pET32（a）-trSAG1重组质粒转化Escherichia coli BL21（DE3）,用0.1 mmol/L IPTG诱导表达,超声破菌后获得可溶性表达产物,通过Ni-NTA一步法纯化,以纯化的rSAG1为抗原,免疫BALB/c小鼠,采用杂交瘤技术制备McAb,用ELISA和有限稀释法筛选出分泌高滴度McAb杂交瘤细胞株,测定其免疫球蛋白亚类及其效价,Western blot分析其特异性. 结果筛选出能稳定分泌抗rSAG1单克隆抗体的5株杂交瘤细胞株,抗体重链均为IgG1, 轻链均为κ链;Western blot 分析显示,5株单抗均能与弓形虫速殖子超声抗原中的天然SAG1发生特异性结合. 结论制备的抗rSAG1杂交瘤细胞株能分泌识别SAG1的高特异性单抗,为进一步研究其在弓形虫病免疫诊断中的应用奠定了基础.     

弓形虫主要表面抗原1单链抗体与绿色荧光蛋白的融合表达及其生物活性初步观察

目的 构建、表达和纯化刚地弓形虫（Toxoplasma gondii）主要表面抗原1（SAG1）单链抗体S1与绿色荧光蛋白（GFP）的融合蛋白，体外观察其与弓形虫速殖子的特异性结合能力。 方法 分别设计引物，构建原核表达质粒pET-26b-GFP和pET-26b-GFPS1，测序验证后转化大肠埃希菌BL21（DE3），以异丙基-β-D-硫代半乳糖苷（IPTG)诱导表达，荧光显微镜观察融合基因中GFP的表达情况。用Ni²⁺螯合柱亲和纯化融合蛋白GFPS1，十二烷基磺酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）检测融合蛋白GFPS1。荧光显微镜观察融合蛋白GFPS1与弓形虫速殖子体外孵育后单链抗体S1对弓形虫速殖子的靶向作用。 结果 测序结果显示，弓形虫SAG1单链抗体S1与GFP的融合基因构建到原核表达载体pET-26b（+）中；IPTG诱导表达后，在荧光显微镜下可观察到E.coli BL21发出的特异性绿色荧光。SDS-PAGE检测到表达的融合蛋白GFPS1相对分子质量（Mr）约为53 000，多以包涵体形式存在。免疫荧光检测可见弓形虫速殖子表膜周围发出特异性的绿色荧光，表明纯化的GFPS1可特异性地结合到弓形虫速殖子表膜。 结论 弓形虫主要表面抗原1（SAG1）单链抗体S1与弓形虫速殖子表膜有较强的结合能力。     

抗日本血吸虫噬菌体抗体库的筛选和阳性克隆的鉴定

目的　研制抗日本血吸虫循环抗原SjMAg的单链抗体。　方法　用日本血吸虫成虫代谢抗原SjMAg包板,对已构建的抗日本血吸虫噬菌体抗体库进行3轮富集,然后用ELISA法从富集的次级抗体库中筛选阳性克隆,最后借助ELISA、SDSPAGE和Western印迹等方法,根据阳性克隆表达产物与其它4种吸虫抗原反应的情况,进行特异性鉴定。　结果　从随机挑取的72个克隆中筛选到阳性克隆6个,经交叉筛选和鉴定最终获得特异性抗日本血吸虫阳性克隆2个。　结论　用固相富集和ELISA筛选法可以从抗体库中有效地筛选到抗日本血吸虫循环抗原的阳性克隆,获得的抗体克隆可用于抗日本血吸虫循环抗原单链抗体的制备。     

从人源大容量天然抗体库中筛选到抗禽流感病毒H5N1单链抗体

目的从人源大容量天然抗体库中筛选到抗禽流感病毒H5N1单链抗体。方法运用噬菌体表面呈现技术,用纯化的H5N1禽流感血凝素蛋白HA（A/Anhui/1/2005）对人源大容量天然抗体库进行富集筛选,对获得的阳性克隆进行序列分析,并将其克隆入携带人源Fc段的表达载体,实现scFv-Fc融合抗体的分泌型表达。用ELISA、IFA对所获人源单抗的功能特性进行鉴定。结果经过3轮富集筛选,共获得了82株特异性结合H5N1禽流感病毒HA的人源scFv单链抗体,分别属于5个不同的抗体序列,ELISA、IFA表明从抗体库筛选获得的5株人源单抗与禽流感病毒H5N1及其HA具有较好的特异性,而与甲1型和甲3型人流感病毒无交叉反应。结论从人源大容量天然抗体库中利用高通量的筛选策略在短时间内获得抗禽流感病毒H5N1人源单抗,对于禽流感的紧急预防和治疗具有重要意义。     

丙型肝炎病毒非结构蛋白NS5抗独特型人源单链可变区抗体的筛选与鉴定

目的：制备丙型肝炎病毒（HCV）非结构蛋白NSS（NS5）的抗独特型单链可变区抗体scFv(抗－Id scFv)，为研制HCV NS5的抗－Id scFv疫苗奠定基础。方法：采用噬菌体表面展示技术，将HCV NS5单克隆抗体固相包被于Nunc板，从噬菌体单链可变区抗体库中经过5轮“吸附－洗脱－扩增”筛选过程，随机挑选80个克隆，利用酶联免疫吸附试验（ELISA）、交叉反应和竞争抑制实验，对其进行免疫学检测，获得与HCV NS5单克隆抗体结合活性较强的抗－Id scFv阳性克隆，并对HCV NS5特异性抗－Id scFv的编码序列进行序列测定分析。结果：筛选得到的HCV NS5抗－Id scFv片段由786bp组成，具有结合HCV NS5单克隆抗体的生物学活性和特异性。结论：用噬菌体抗体库技术能够成功地获得HCV NS5的抗－Id scFv。本实验结果为开展用抗－Id scFv防治丙型肝炎的研究创造了条件。     

全人源肝癌噬菌体单链抗体基因的克隆及活性分析

从噬菌体单链抗体库中筛选克隆全人源肝癌抗体基因并进行活性鉴定.PCR鉴定阳性重组菌中人肝癌ScFv的插入率,以肝癌细胞SMMC-7721为抗原对所建抗体库进行4轮"吸附-洗脱-扩增"的亲和筛选.将筛选后的ScFv采用ELISA法鉴定其与人肝癌细胞的结合活性.ScFv基因插入率为70%.在亲和筛选过程中,肝癌噬菌体单链抗体得到富集,收获率逐轮提高,第4轮为第一轮的381倍.利用噬菌体抗体库技术筛选出了肝癌噬菌体单链抗体,且筛选后的抗体片段与人肝癌细胞有特异性的结合活性.     

丙型肝炎病毒核心蛋白人源单链可变区抗体的筛选与鉴定

目的 筛选、鉴定抗丙型肝炎病毒（HCV）核心蛋白的人源单链可变区抗体（ScFv)。方法 采用噬菌体表面展示技术，以重组的HCV核心蛋白为包被抗原，从噬菌体单链可变区抗体库中经过3轮“吸附－洗脱－扩增”筛选过程，获得抗原结合活性较强的HCV核心蛋白特异性人源单链可变区抗体片段阳性克隆，并对其进行免疫学及核苷酸序列测定。结果 筛选得到的ScFv片段具有抗HCV核心蛋白的特异性，基因序列分析结果表明符合人源单链可变区抗体基因序列的结构特征。结论 利用噬菌体抗体库技术，成功获得HCV核心蛋白的特异性人源单可变区抗体的编码基因。     

乙型肝炎病毒表面抗原人源单链可变区抗体基因的克隆与鉴定

目的 筛选,鉴定抗乙型肝炎病毒（ＨＢＶ）表面抗原（ＨＢｓＡｇ）蛋白的人源单链可变区抗体（ＳｃＦｖ）的编码基因,为细胞内表达小分子单链抗体的研究及抗ＨＢＶ的基因治疗研究奠定基础。方法 采用噬菌体表面展示技术,以氯化铯超速离心法纯化的ＨＢｓＡｇ蛋白为固相抗原,从噬菌体单链可变区抗体半合成库中经过５轮“吸附－洗脱－扩增”淘洗过程,获得抗原结合活性较强的ＨＢｓＡｇ人源单链可变区抗体阳性克隆,并对其进行免疫     

随机肽库筛选弓形虫特异性抗原表位诱导保护性免疫的研究

目的　从噬菌体随机肽库中筛选出模拟弓形虫特异性抗原表位的短肽分子 ,并探讨其对弓形虫的保护性效果。方法　以纯化的弓形虫免疫兔血清IgG为配基 ,亲和筛选法富集特异性噬菌体 ,随机挑取噬菌体克隆用夹心ELISA法和Dot-ELISA法检测其特异性 ,混合 4个阳性克隆免疫小鼠 ,1月后攻击感染 ,观察小鼠发病时间和死亡时间。结果　经 3轮筛选 ,特异性噬菌体得到了有效富集 ,第 3轮洗脱噬菌体的产量为第 1轮的 16 7倍 ,随机挑取 2 7个噬菌体经Dot -Bloting和夹心ELISA法检测 ,有 2 4个能与弓形虫免疫兔血清及单克隆抗体特异反应。用致死量弓形虫攻击感染经阳性克隆免疫的小鼠 ,存活时间明显长于对照组。结论　免疫筛选噬菌体随机多肽库可获得具有保护性的弓形虫抗原表位 ,为弓形虫疫苗研制提供了新途径。     

抗弓形虫靶向抗菌肽的构建及其效应的初步评价

目的构建弓形虫主要表面抗原1单链抗体S1与爪蟾抗菌肽（magainin）的融合基因,在大肠杆菌中诱导表达,观察纯化的靶向抗菌蛋白抗弓形虫感染的效果. 方法根据弓形虫主要表面抗原1单链抗体S1的编码序列设计引物从噬菌粒S1/pIT-2中扩增到S1基因;以其为模板,加入接头抗菌肽序列,采用重叠PCR法扩增得到S1基因与爪蟾抗菌肽的融合基因（S1M）,将其克隆到原核表达载体pET-32c中,构建成S1与爪蟾抗菌肽基因的重组表达质粒S1M/pET-32c;测序验证后转化E.coli BL21,以IPTG诱导表达,用Ni^2＋螯合柱亲和纯化融合蛋白S1M,SDS-PAGE检测融合基因的表达和纯化的融合蛋白S1M;分别采用体内和体外试验观察纯化的靶向抗菌蛋白抗弓形虫感染效果. 结果测序结果表明,成功地将弓形虫主要表面抗原1单链抗体S1与爪蟾抗菌肽的融合基因构建到原核表达载体pET-32c中;SDS-PAGE显示IPTG诱导表达的融合蛋白S1M大小约为43 kDa,与预期的大小相一致,以包涵体的形式存在;通过变性条件下Ni^2＋亲和柱纯化获得S1M重组融合蛋白;体内和体外试验证实,经靶向抗菌蛋白处理过的弓形虫对小鼠的致病能力下降,应用靶向抗菌蛋白的弓形虫感染小鼠的存活时间与对照组相比有明显的提高. 结论以抗弓形虫速殖子人源单链抗体作为靶向分子,以爪蟾抗菌肽作为效应分子,构建成功的靶向抗菌蛋白通过体内、外试验证实,具有一定的抗弓形虫感染的作用,虽然还不能完全杀灭弓形虫,但是在弓形虫生物治疗药物的研制方面进行了有益的探索,为弓形虫病的生物治疗提供了新的思路.     

Human scFv antibody fragments specific for hepatocellular carcinoma selected from a phage display library

AIM: To identify the scFv antibody fragments specific for hepatocellular carcinoma by biopanning from a large human naive scFv phage display library. METHODS: A large human naive scFv phage library was used to search for the specific targets by biopanning with the hepatocellular carcinoma cell line HepG2 for the positive-selecting and the normal liver cell line L02 for the counter-selecting. After three rounds of biopanning, individual scFv phages binding selectively to HepG2 cells were picked out. PCR was carried out for identification of the clones containing scFv gene sequence. The specific scFv phages were selected by ELISA and flow cytometry. DMA sequences of positive clones were analyzed by using Applied Biosystem Automated DNA sequencers 3 730. The expression proteins of the specific scFv antibody fragments in F.coli HB2151 were purified by the affinity chromatography and detected by SDS-PAGE, Western blot and ELISA. The biological effect of the soluble antibody fragments on the HepG2 cells was investigated by observing the cell proliferation. RESULTS: Two different positive clones were obtained and the functional variable sequences were identified. Their DNA sequences of the scFv antibody fragments were submitted to GenBank (accession nos: AY686498 and AY686499). The soluble scFv antibody fragments were successfully expressed in E.coli HB2151. The relative molecular mass of the expression products was about 36 ku, according to its predicted M, value. The two soluble scFv antibody fragments also had specific binding activity and obvious growth inhibition properties to HepG2 cells. CONCLUSION: The phage library biopanning permits identification of specific antibody fragments for hepatocellular carcinoma and affords experiment evidence for its immunotherapy study.     

Preparation of human single chain Fv antibody against hepatitis C virus E2 protein and its identification in immunohistochemistry

AIM：To screen human single chain Fv antibody(scFv)against hepatitis C virus E2 antigen and identify its application in immunohistochemistry.METHODS:The phage antibody library was panned by HCVE2 antigen,Which was coated in microtiter plate.After five rounds of bipanning,56phage clones were identified specific toHCVE2antigen,The selected scFv clones were digested by SfiI/NotI and DNAwas sequenced.Then it was subcloned into the vector pCANTAB5Efor expression as E-tagged soluble scFv.The liver tissue sections from normal person and patients with choronic hepatitis B and chronic hepatitis C were immunostained with HCVE2 scFv antibody.RESULTS:The data of scFv-E2DNAdigestion and DNA sequencing showed that the scFv gene is composed of 750bp.ELISAand immunohistochemistry demonstrated that the human single chain Fv antibody against hepatitisCE2 antigen has a specific binding character with hepatitis virus E2antigen and paraffin-embedded tissue,but did not react with liver tissues from healthy persons or patients with chronic hepatitis B.CONCLUSION:We have successfully screened and identified HCVE2 scFv and the scFv could be used in the immunostaining of liver tissue sections from patients with chronic hepatitisC.     

Production of a human single-chain variable fragment antibody against esophageal carcinoma

AIM: To construct a phage display library of human singlechain variable fragment (scFv) antibodies associated with esophageal cancer and to preliminarily screen a scFvantibody against esophageal cancer.METHODS: Total RNA extracted from metastatic lymph nodes of esophageal cancer patients was used to construct a scFv gene library. Rescued by M13K07 helper phage, the scFv phage display library was constructed, esophageal cancer cell line Eca 109 and normal human esophageal epithelial cell line (NHEEC) were used for panning and subtractive panning of the scFv phage display library to obtain positive phage clones. Soluble scFv was expressed in E.coliHB2151 which was transfected with the positive phage clone, then purified by affinity chromatography.Relative molecular mass of soluble scFv was estimated by Western blotting, its bioactivity was detected by cell ELISA assay. Sequence of scFv was determined using the method of dideoxynucleotide sequencing.RESULTS: The size of scFv gene library was approximately 9×10^6 clones. After four rounds of panning with Eca109 and three rounds of subtractive panning with NHEEC cells,25 positive phage clones were obtained. Soluble scFv was found to have a molecular mass of 31 ku and was able to bind to Ecal09 cells, but not to HeLa and NHEEC cells.Variable heavy (VH) gene from one of the positive clones was shown to be derived from the γ chain subgroup IV of immunoglobulin, and variable light (VL) gene from the κ chain subgroup I of immunoglobulin.CONCLUSION: A human scFv phage display library can be constructed from the metastatic lymph nodes of esophageal cancer patients. A whole human scFv against esophageal cancer shows some bioactivity.     

Production and characterization of human monoclonal anti-idiotype antibodies to anti-dsDNA antibodies

Anti-dsDNA autoantibodies are the hallmark of systemic lupus erythematosus (SLE) and frequently correlate with disease activity. In this study we report the isolation and characterization of human anti-Id monoclonal antibody fragments as single-chain Fv fragments (scFv) against anti-dsDNA antibody. The anti-Id monoclonal antibodies, specific for anti-dsDNA antibodies, have been cloned from phage display antibody scFv libraries derived from a patient with SLE. The V gene repertoires were derived from the RNA obtained from the B cells of an SLE patient with anti-Ro/SSA and anti-La/SSB antibodies. Affinity-purified anti-dsDNA antibodies were used for selection of bacterial clones producing specific scFv antibody fragments against anti-dsDNA antibodies and little reactivity with normal IgG and other IgG antibodies by ELISA. The anti-Id antibody recognizes a public idiotope that is broadly cross-reactive with polyclonal and monoclonal anti-dsDNA antibodies. This binding was largely inhibited by dsDNA antigen. The anti-Id antibody inhibited anti-dsDNA binding to dsDNA antigen in immunoassays and in the Crithidia luciliae assay. The anti-Id scFv antibody fragments derived from human genes could modulate the pathogenicity of anti-dsDNA autoantibodies and may have therapeutic implications in SLE. They may also be used as probes in studies of the structure of the idiotype.     

Human antibodies with specificity for the C2 domain of factor VIII are derived from VH1 germline genes

A serious complication in hemophilia care is the development of factor VIII (FVIII) neutralizing antibodies (inhibitors). The authors used V gene phage display technology to define human anti-FVIII antibodies at the molecular level. The IgG4-specific, variable, heavy-chain gene repertoire of a patient with acquired hemophilia was combined with a nonimmune, variable, light-chain gene repertoire for display as single-chain variable domain antibody fragments (scFv) on filamentous phage. ScFv were selected by 4 rounds of panning on immobilized FVIII light chain. Sequence analysis revealed that isolated scFv were characterized by V(H) domains encoded by germline genes DP-10, DP-14, and DP-88, all belonging to the V(H)1 gene family. All clones displayed extensive hypermutation and were characterized by unusually long CDR3 sequences of 20 to 23 amino acids. Immunoprecipitation revealed that all scFv examined bound to the C2 domain of FVIII. Furthermore, isolated scFv competed with an inhibitory murine monoclonal antibody for binding to the C2 domain. Even though scFv bound FVIII with high affinity, they did not inhibit FVIII activity. Interestingly, the addition of scFv diminished the inhibitory potential of patient-derived antibodies with C2 domain specificity. These results suggest that the epitope of a significant portion of anti-C2 domain antibodies overlaps with that of the scFv isolated in this study. (Blood. 2000;95:558-563)     

从人源性噬菌体抗体库中筛选人抗HBsAg的Fab噬菌体抗体

目的从人源性噬菌体抗体库中筛选人抗HBsAgFab噬菌体抗体。方法用固相化HBsAg对所构建的噬菌体抗体库进行三轮淘筛,从中筛选人抗HBsAgFab噬菌体抗体。结果第三轮淘筛后洗脱下来的噬菌体,较第一轮增加80倍,含有抗体轻链基因和重链基因的克隆,也由淘筛前的17%增至100%,经ELISA证实,筛选到的抗HBsAg噬菌体抗体对HBsAg具有良好的结合活性,而与牛血清白蛋白和HAVAg等无关抗原均不反应。结论HBsAg对抗体库的淘筛,富集了表面呈现抗HBsAg的单克隆抗体的噬菌体。