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Disulfide-stabilized single-chain antibody-targeted superantigen: Construction of a prokaryotic expression system and its functional analysis 被引量:7
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作者 Jian-Li Wang Yu-Ling Zheng +3 位作者 RU Ma Bao-Li Wang Ai-Guang Guo Yong-Qiang Jiang 《World Journal of Gastroenterology》 SCIE CAS CSCD 2005年第31期4899-4903,共5页
AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial... AIM: To construct the expression vector of B3 (scdsFv)-SEA (D227A) and to identify its binding and cytotoxic ability to B3 antigen positive carcinoma cell lines.METHODS: This fusion protein was produced by a bacterial expression system in this study. It was expressed mainly in the inclusion body. The gene product was solubilized by guanidine hydrochloride, refolded by conventional dilution method, and purified using SP-sepharose cation chromatography.RESULTS: The expression vector B3 (scdsFv)-SEA-PETwas constructed, the expression product existed mainly in the inclusion body, the refolding product retained the binding ability of the single-chain antibody and had cytotoxic effect on HT-29 colon carcinoma cells. The stability assay showed that the resulting protein was stable at 37 ℃.CONCLUSION: This genetically engineered B3 (scdsFv)-SEA fusion protein has bifunction of tumor targeting and tumor cell killing and shows its promises as an effective reagent for tumor-targeted immunotherapy. 展开更多
关键词 B3 monoclonal antibody single-chain disulfide-stabilized Fv SUPERANTIGEN
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Screening and evaluation of human single-chain fragment variable antibody against hepatitis B virus surface antigen 被引量:8
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作者 Jian-Lin Zhang, Jian-Jin Guo, Zi-Yan Zhang, Yi-Xin Jing, Lin Zhang, Rui Guo, Ping Yan, Niu-Liang Cheng, Bo Niu and Jun Xie Department of Biochemistry and Molecular Biology, Shanxi Medical University ,Taiyuan 030001,China 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2006年第2期237-241,共5页
BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody... BACKGROUND: Phage display technology has become a vital tool in studies aimed at identifying molecules binding to a specific target. It enables the rapid generation and selection of high affinity, fully human antibody product candidates to essentially any disease target appropriate for antibody therapy. In this study, we prepared the recombinant single-chain fragment variable ( ScFv) antibody to hepatitis B virus surface antigen (HBsAg) by the phage display technology for obtaining a virus-targeting mediator. METHODS: mRNA was isolated from B-lymphocytes from a healthy volunteer and converted into cDNA. The fragment variables of heavy and light chain were amplified separately and assembled into ScFv DNA with a specially constructed DNA linker by polymerase chain reaction. The ScFv DNA was ligated into the phagmid vector pCANT-AB5E and the ligated sample was transformed into competent E. coli TG1. The transformed cells were infected with M13K07 helper phage to form a human recombinant phage antibody library. The volume and recombinant rate of the library were evaluated by bacterial colony count and restriction analysis. After two rounds of panning with HBsAg. the phage clones displaying ScFv of the antibody were selected by enzyme-linked immunosorbant assay ( ELISA) from the enriched phage clones. The antigen binding affinity of the positive clone was detected by competition ELISA. HB2151 E. coli was transfected with the positive phage clone demonstrated by competition ELISA for production of a soluble form of the anti-HBsAg ScFv. ELISA assay was used to detect the antigen binding affinity of the soluble anti-HBsAg ScFv. Finally, the relative molecular mass of soluble anti-HBsAg ScFv was measured by SDS-PAGE. RESULTS: The variable heavy ( VH ) and variable light (VL) and ScFv DNAs were about 340bp, 320bp and 750bp, respectively. The volume of the library was up to 2 × 106 and 8 of 10 random clones were recombinants. Two phage clones could strongly compete with the original HBsAb for binding to HBsAg. Within 2 strong positive phage clones, the soluble anti-HBsAg ScFv from one clone was found to have the binding activity with HBsAg. SDS-PAGE showed that the relative molecular weight of soluble anti-HBsAg ScFv was 32 kDa. CONCLUSION: The anti-HBsAg ScFv successfully produced by phage antibody technology may be useful for broadening the scope of application of the antibody. 展开更多
关键词 phage display technology phage antibody library hepatitis B virus surface antigen single-chain fragment variable
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Bioinformatics-led design of single-chain antibody molecules targeting DNA sequences for retinoblastoma 被引量:1
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作者 Guo-Gang Shang, Jun Yun 《International Journal of Ophthalmology(English edition)》 SCIE CAS 2011年第1期8-13,共6页
The development of single-chain Fv antibody (scFv) by recombinant gene expression is an important milestone for cancer therapy. Single-chain antibodies are reconstructed for cancer-targeted therapy to provide good pen... The development of single-chain Fv antibody (scFv) by recombinant gene expression is an important milestone for cancer therapy. Single-chain antibodies are reconstructed for cancer-targeted therapy to provide good penetration into tumor tissue and to improve their pharmacokinetics in vivo, offering a clinically valuable application. The relationship needs to be analyzed that there may be some variations between the structure and function of the fusion proteins, and the relationship between the structure and function of protein molecules was obtained through analyzing relevant literature at home and abroad as well as modeling analysis. Through our analysis of the interaction region between antibody and antigen, and of the binding sites for molecular conformation, it is clear that existing antibodies need to be modified at the DNA sequence level, enhancing the biological activity of the antibodies. Based on the view that bio-molecular computer models are closely integrated with biological experiments, a bio-molecular structure-activity relationship model can be established in terms of molecular conformation, physical and chemical properties and the biological activity of single-chain antibodies. Two enlightenments are obtained from our analysis. On one hand, the structure-activity relationship is clear for new immune molecules at the gene expression level. On the other hand, a single-chain antibody molecule can be designed and optimized for the cancer-oriented treatment. In this article, we provided the theoretical and experimental basis for the development of single-chain antibodies appropriate for retinoblastoma therapy. 展开更多
关键词 single-chain antibody BIOINFORMATICS RETINOBLASTOMA structure-activity relationship
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Expression of secreted human single-chain fragment variable antibody against human amyloid beta peptide in Pichia pastoris
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作者 Jiong Cai Fang Li Shizhen Wang 《Neural Regeneration Research》 SCIE CAS CSCD 2008年第8期910-913,共4页
BACKGROUND:Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE: Based on successful screening of a... BACKGROUND:Studies have shown that monoclonal or polyclonal antibody injections of amyloid β peptide are effective in removing amyloid β peptide overload in the brain. OBJECTIVE: Based on successful screening of a human single-chain fragment variable antibody specific to amyloidβpeptide, this paper aimed to express recombinant human single-chain variable antibody against amyloid β peptide. DESIGN, TIME AND SETTING: A single sample experiment was performed at the Department of Nuclear Medicine, Peking Union Medical College Hospital, Chinese Academy of Medical Sciences and Peking Union Hospital (Beijing, China) from January to July 2006. MATERIALS: Human single-chain fragment variable antibody gene against amyloid β peptide was screened from a human phage-display antibody library. METHODS: Human single-chain fragment variable antibody gene was mutated to eliminate a BamHI restriction site and cloned into a T easy plasmid for pT-scFvAβ construction, which was identified by PCR amplification and endonuclease digestion. Plasmid pT-scFvAβ was cut by EcoRI and NotI endonucleases, and the antibody gene was cloned into pPIC9K plasmid to construct pPIC9K-scFvAβ expression vector, which was confirmed by gene sequencing. Linearized pPIC9K-scFvAβ was used to transform a Pichia pastoris GS115 cell line, and the recombinant was induced by 0.5% methanol to express human single-chain fragment variable antibody specific to amyloid β peptide. MAIN OUTCOME MEASURES: Protein electrophoresis was used to identify PCR products, gene sequencing was used to verify the pPIC9K-scFvA sequence, and SDS-PAGE was used to detect recombinant expression of human single-chain fragment variable antibody specific to amyloid β peptide in Pichia pastoris. RESULTS: Gene sequencing confirmed pPIC9K-scFvAβ orientation. Recombinants were obtained by linearized pPIC9K-scFvAβ transformation. After induction with 0.5% methanol, the recombinant yeast cells secreted proteins of 33-ku size. CONCLUSION: The expression vector pPIC9K-scFvAβ was successfully constructed. Human single-chain fragment variable antibody specific to amyloid β peptide was recombinantly expressed in Pichia pastoris. 展开更多
关键词 Alzheimer's disease β amyloid peptide single-chain fragment variable antibody
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Observation on the Efficiency of the Mongolian Gerbil Kidney Tissue Culture Inactivated Bivalent Vaccine for Hemorrhagic Fever with Renal Syndrome
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作者 董关木 朱智勇 +5 位作者 安祺 朱凤才 刘文雪 孔艳 杨立宏 俞永新 《Journal of Microbiology and Immunology》 2004年第2期115-119,共5页
The Z 10 and Z 37 strains of hemorrhagic fever with renal syndrome (HFRS) virus and the Mongolian gerbil ( Merions unguiculatus ) kidney cells were used to prepare the inactivated bivalent vaccine. A phase Ⅱ clinical... The Z 10 and Z 37 strains of hemorrhagic fever with renal syndrome (HFRS) virus and the Mongolian gerbil ( Merions unguiculatus ) kidney cells were used to prepare the inactivated bivalent vaccine. A phase Ⅱ clinical trial use of this vaccine was made in 750 Chinese volunteers. The results showed that the side reaction rate was 2.5% and the sero-conversion rate of neutralizing antibodies against Hantaan and Seoul viruses in the inoculated volunteers were 87.6% and 96.3% respectively. 展开更多
关键词 bivalent HFRS vaccine Clinical reaction IFA antibody Neutralization antibody
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Generation of high affinity human single-hain antibody against PreSl of hepatitis B virus from immune phage-display antibody library 被引量:5
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作者 Zhi-Chao Zhang, Xue-Jun Hu and Qing Yang Dalian, China State Key Laboratory of Fine Chemicals, Dalian Uni- versity of Technology, Dalian 116012, China 《Hepatobiliary & Pancreatic Diseases International》 SCIE CAS 2004年第1期77-81,共5页
BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filame... BACKGROUND: A single-chain antibody ( ScFv) phage display library was created by cloning antigen-binding re- gions of VH (variable domain) and VL gene repertoires as fusion proteins with a minor coat protein of filamentous phage, from which high affinity completely humanized ScFv against PreS1 of hepatitis B virus could be screened and characterized. METHODS: A combinatorial library of phage-display hu- man ScFv genes, which were derived from peripheral blood lymphocytes immunized by peptide PreS1 in vitro, was constructed. The library contained 7 × 108 clones. RESULTS: After 3 rounds panning, a high affinity (K = 10-7-10-8 mol/L) ScFv specific to PreS1 was obtained. Sequence analysis showed that the VH belonged to the VH4 family and Vλ to Vλ4. CONCLUSIONS: The described ScFv may provide a more satisfactory therapy. This application further illustrates that the method of in vitro antigen stimulation is expeditious for the source of human immune antibody library. 展开更多
关键词 hepatitis B virus PRES1 single-chain antibody immune antibody library panning
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Anti-amyloid beta single-chain Fv ameliorates behavioral impairment in Alzheimer's disease mice via adeno-associated virus delivery 被引量:1
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作者 Jiong Cai Yanwei Zhong +1 位作者 Fang Li Shizhen Wang 《Neural Regeneration Research》 SCIE CAS CSCD 2011年第2期96-100,共5页
Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide (Aβ, anti-Aβ single-chain Fv, scFv) via adeno-associated virus (AAV) inhibits amyloid deposition in transgenic mice. However, the... Intracranial delivery of human Fc-deleted antibody specific to amyloid-β peptide (Aβ, anti-Aβ single-chain Fv, scFv) via adeno-associated virus (AAV) inhibits amyloid deposition in transgenic mice. However, the effects of AAV-mediated Fc-deleted antibody on animal behavior remain unclear. In this study, the anti-Aβ scFv antibody gone, isolated from phage display, was fused to the 5' end of the scFv antibody gone for antibody secretion by 2 rounds of polymerase chain reaction amplification. The fused antibody cDNA was cloned into a pSNAV2 plasmid under the control of the cytomegalovirus promoter. The sequence verified expression vector pSNAV2/scFv was transferred to BHK-21 ceils, and stable transfected BHK-21/scFv cells were established by G418 selection and infected with the recombinant herpes simplex virus rHSV/repcap for AAV production. Recombinant AAV was injected into the left quadriceps femoris of PDAPP transgenic mice. After 3 months, Morris water-maze results confirmed significantly improved cognitive function in a mouse model of Alzheimer's disease. Key Words: Alzheimer's disease; adeno-associated virus; amyloid-β peptide; single-chain antibody; neurodegenerative diseases; neural regeneration 展开更多
关键词 Alzheimer's disease adeno-associated virus amyloid-13 peptide single-chain antibody neurodegenerative diseases neural regeneration
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Fusion protein of single-chain variable domain fragments for treatment of myasthenia gravis
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作者 Fangfang Li Fanping Meng +4 位作者 Quanxin Jin Changyuan Sun Yingxin Li Honghua Li Songzhu Jin 《Neural Regeneration Research》 SCIE CAS CSCD 2014年第8期851-856,共6页
Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pa... Single-chain variable domain fragment (scFv) 637 is an antigen-specific scFv of myasthenia gravis. In this study, scFv and human serum albumin genes were conjugated and the fusion pro-tein was expressed in Pichia pastoris. The afifnity of scFv-human serum albumin fusion protein to bind to acetylcholine receptor at the neuromuscular junction of human intercostal muscles was detected by immunolfuorescence staining. The ability of the fusion protein to block myas-thenia gravis patient sera binding to acetylcholine receptors and its stability in healthy serum were measured by competitive ELISA. The results showed that the inhibition rate was 2.0-77.4%, and the stability of fusion protein in static healthy sera was about 3 days. This approach suggests the scFv-human serum albumin is a potential candidate for speciifc immunosuppressive therapy of myasthenia gravis. 展开更多
关键词 nerve regeneration myasthenia gravis acetylcholine receptor anti-acetylcholine re-ceptor antibody single-chain variable domain fragment human serum albumin fusion protein immunosuppressive therapy autoimmune disease NSFC grant neural regeneration
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猪瘟活疫苗-猪口蹄疫O型、A型二价灭活疫苗联合免疫效果的评价 被引量:3
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作者 陈昌海 蒋锁俊 +4 位作者 邱冬 王小新 徐小艳 开妍 刘云 《畜牧与兽医》 CAS 北大核心 2023年第3期84-87,共4页
为建立一种既有效又简便的猪瘟活疫苗(CSF)和猪口蹄疫O型、A型二价灭活疫苗(FMD O-A)联合免疫方法,达到“一针多防”的目的,通过动物免疫试验,评价了CSF和FMD O-A联合免疫的可行性。采用左、右各一针同步2点接种试验猪群(联合免疫),以2... 为建立一种既有效又简便的猪瘟活疫苗(CSF)和猪口蹄疫O型、A型二价灭活疫苗(FMD O-A)联合免疫方法,达到“一针多防”的目的,通过动物免疫试验,评价了CSF和FMD O-A联合免疫的可行性。采用左、右各一针同步2点接种试验猪群(联合免疫),以2种疫苗分别单独免疫的猪群为对照,在免疫前0 d、免疫后14、40、60和90 d采集血清检测2种疫苗对应的抗体。经统计学分析发现,2种疫苗的联合免疫不影响其抗体的产生,并且能够保持抗体在猪群个体间的稳定分布。 展开更多
关键词 猪瘟耐热保护剂活疫苗 猪口蹄疫O型、A型二价灭活疫苗 联合免疫 抗体
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五种市售鸡传染性鼻炎灭活疫苗免疫效力比较 被引量:5
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作者 孙惠玲 苗得园 +5 位作者 陈晓峰 张培君 龚玉梅 唐桂运 Anne-Lise Saint-Gerand 《中国预防兽医学报》 CAS CSCD 北大核心 2005年第6期544-548,共5页
本研究对国内市场上几个主要鸡传染性鼻炎灭活疫苗生产厂家生产的灭活疫苗免疫鸡只进行血清HI抗体水平测定和攻毒,比较不同公司所产疫苗效力的效力差异,并评估A、C型二价灭活疫苗两次免疫鸡群对国内B型分离株:DL-1株和最近从免疫失败鸡... 本研究对国内市场上几个主要鸡传染性鼻炎灭活疫苗生产厂家生产的灭活疫苗免疫鸡只进行血清HI抗体水平测定和攻毒,比较不同公司所产疫苗效力的效力差异,并评估A、C型二价灭活疫苗两次免疫鸡群对国内B型分离株:DL-1株和最近从免疫失败鸡场分离到的SD-1株的交叉保护作用,分析免疫失败的原因。结果表明:A、D、E公司的疫苗免疫两次后,A型HI抗体阳性率分别为92.5%、100%和95%,滴度分别为33.9、55.1和59.9,攻毒保护率都是100%。C型HI抗体阳性率分别为72.5%3、8.5%和77.5%,滴度分别为11.4、2.7和27,攻毒保护率分别为80%、70%和80%。而B和C公司的疫苗免疫两次后A型HI抗体阳性率分别为59.4%7、7.1%,抗体滴度分别为5.4和21.8,攻毒保护率分别为50%和66.7%;C型HI抗体阳性率分别为54.1%和51.4%,抗体滴度分别为6.1和6.8,攻毒保护率分别为38.3%和50%。在五个疫苗产品中,以A、D、E的保护效力较好,B、C产品效力较差。另外,A、C型二价灭活疫苗免疫后不能对B型菌的攻击提供保护,其A、C型HI抗体阳性率、抗体滴度与对B型菌攻毒保护率无相关性。 展开更多
关键词 副鸡嗜血杆菌 鸡传染性鼻炎灭活疫苗 HI抗体
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用双价肾综合征出血热灭活疫苗加强接种HNT型单价疫苗免疫者的效果观察 被引量:3
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作者 朱智勇 李岩金 +4 位作者 陆群英 唐汉英 翁景清 李敏红 姚苹苹 《中国公共卫生》 CAS CSCD 北大核心 1999年第7期612-613,共2页
对1988年经过HNT型疫苗初免3针,并于1989年加强1针的7名志愿者,于1997年加强1针双价疫苗后14天采血,测定对HNT型和SEO型HFRS病毒的中和抗体。结果经加强双价疫苗的7名志愿者血清中,均检出HNT型... 对1988年经过HNT型疫苗初免3针,并于1989年加强1针的7名志愿者,于1997年加强1针双价疫苗后14天采血,测定对HNT型和SEO型HFRS病毒的中和抗体。结果经加强双价疫苗的7名志愿者血清中,均检出HNT型和SEO型病毒的中和抗体,其中对HNT病毒的中和抗体较高,对SEO型病毒的中和抗体也能达到规定的要求,表明双价HFRS灭活疫苗可用于过去接种HNT型疫苗人群的加强。 展开更多
关键词 肾综合征出血热 灭活疫苗 疫苗 接种 效果
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不同表型双价痢疾菌苗滴鼻免疫小鼠后黏膜和系统记忆反应观测 被引量:3
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作者 石辛甫 邢丽 +1 位作者 彭虹 高杰英 《免疫学杂志》 CAS CSCD 北大核心 2002年第6期421-425,共5页
目的 观测 5株 2类不同表型的双价痢疾菌苗滴鼻免疫后所引起的黏膜和系统的记忆反应 ,为痢疾菌苗的构建提供理论依据。方法 以小鼠滴鼻免疫为模型 ,3次免疫 (间隔 2周 )后 ,2 0周再次免疫 ,分别于 3次免疫及 2 0周加强免疫后第 7天 ,... 目的 观测 5株 2类不同表型的双价痢疾菌苗滴鼻免疫后所引起的黏膜和系统的记忆反应 ,为痢疾菌苗的构建提供理论依据。方法 以小鼠滴鼻免疫为模型 ,3次免疫 (间隔 2周 )后 ,2 0周再次免疫 ,分别于 3次免疫及 2 0周加强免疫后第 7天 ,收取鼻咽、肺、小肠及阴道冲洗液和血清 ,应用ELISA方法检测福氏、宋内氏特异性抗体。结果  2类双价菌苗株滴鼻免疫 3次后 ,多个黏膜部位及血清均产生了抗原特异性sIgA、IgG ,较PBS对照组有明显升高 ;2 0周后再次加强免疫 ,仍可诱导黏膜及系统的免疫反应 ,但较 3次免疫后水平明显降低。结论  2株双价痢疾菌苗滴鼻免疫小鼠后可诱导多个黏膜及系统的特异性抗体产生 ,并可产生一定时间的记忆反应 。 展开更多
关键词 双价痢疾菌苗 特异性抗体 滴鼻免疫 记忆反应 小鼠
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抗人大肠癌双价单链抗体基因的构建及表达 被引量:8
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作者 严丹丹 杨福辉 方瑾 《世界华人消化杂志》 CAS 北大核心 2006年第24期2395-2400,共6页
目的:构建抗人大肠癌重组双价单链抗体ND- 1sc(Fv)2的融合基因,并在大肠杆菌中表达.方法:采用基因重组技术借助GGGGS Linker序列,将2个相同的ND-1scFv基因共价连接,构建pET-28a(+)ND-1sc(Fv)2表达载体,并在大肠杆菌中表达双价单链抗体... 目的:构建抗人大肠癌重组双价单链抗体ND- 1sc(Fv)2的融合基因,并在大肠杆菌中表达.方法:采用基因重组技术借助GGGGS Linker序列,将2个相同的ND-1scFv基因共价连接,构建pET-28a(+)ND-1sc(Fv)2表达载体,并在大肠杆菌中表达双价单链抗体的融合蛋白.表达产物用Ni-NTA resin亲和层析方法纯化,SDS- PAGE和Western blotting检测表达的目的蛋白.间接免疫荧光和ELISA检测抗体的免疫活性.结果:序列测定表明:ND-1sc(Fv)2基因全长1473 bp,包含了2个729 bp的scFv序列和15 bp的GGGGS序列.SDS-PAGE和Western blotting检测显示,融合蛋白的Mr55 000,与预期值一致.ND-1sc(Fv)2表达产物以不溶性包涵体形式存在,经亲和层析纯化后蛋白纯度达90%,间接免疫荧光及ELISA检测表明,ND-1sc(Fv)2保留了亲本抗体的免疫活性,对表达有相应抗原的靶细胞具有特异结合活性,其免疫活性明显高于ND-1scFv.结论:成功地构建了抗人大肠癌双价单链抗体ND-1sc(Fv)2,并在大肠杆菌中高效表达.融合蛋白具有良好的免疫活性. 展开更多
关键词 双价单链抗体 ND-1 大肠癌 免疫活性
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鳗弧菌O1/O2二价灭活疫苗免疫大菱鲆的抗体持续期和免疫保护期 被引量:3
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作者 李杰 李淑芳 +5 位作者 丁山 唐磊 李贵阳 莫照兰 李杰 陈娟 《中国水产科学》 CAS CSCD 北大核心 2019年第2期397-403,共7页
本研究分析了鳗弧菌(Vibrio anguillarum)O1/O2血清型二价灭活疫苗免疫大菱鲆后的抗体持续期和免疫保护期。以鳗弧菌O1血清型VAM003株和O2血清型VAM007株为抗原制备了福尔马林灭活二价疫苗,将疫苗按照三种剂量(10~7 cells/尾、10~8 cel... 本研究分析了鳗弧菌(Vibrio anguillarum)O1/O2血清型二价灭活疫苗免疫大菱鲆后的抗体持续期和免疫保护期。以鳗弧菌O1血清型VAM003株和O2血清型VAM007株为抗原制备了福尔马林灭活二价疫苗,将疫苗按照三种剂量(10~7 cells/尾、10~8 cells/尾、10~9 cells/尾)以腹腔注射途径免疫大菱鲆,在免疫后3 d、7 d、14 d、30 d、60 d、90 d、120 d、150 d,用血清凝集实验检测了免疫鱼血清的VAM003和VAM007抗体效价,用攻毒实验检测了疫苗的免疫保护率(RPS)。结果显示,在免疫后7 d三个剂量组的大菱鲆均产生了特异抗体,并获得27%~60%的RPS。三个剂量组大菱鲆的O1血清型抗体持续期分别>90 d (10~7 cells/尾组)、>150 d (10~8 cells/尾组)、>150 d (10~9cells/尾组),而三个剂量组大菱鲆的O2血清型抗体持续期均>150 d。三个剂量组的大菱鲆获得的免疫保护持续期均>150 d;以RPS>75%为有效免疫保护,各剂量组大菱鲆抵抗O1血清型病原感染的有效免疫保护期为:14~120d(10~7 cells组)、14~120 d (10~8 cells/尾)、14~150 d (10~9 cells/尾),抵抗O2血清型病原感染的有效免疫保护期为:14~60 d (10~7 cells组)、14~120 d (10~8 cells/尾)、14~120 d (10~9 cells/尾)。研究结果表明鳗弧菌二价灭活疫苗可为大菱鲆提供有效而稳定的免疫保护,获得的抗体持续期和免疫保护期为该疫苗的临床中试研究提供了基础。 展开更多
关键词 鳗弧菌O1/O2血清型二价灭活疫苗 大菱鲆 抗体持续期 免疫保护期
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双价痢疾菌苗免疫小鼠后抗体记忆反应的观测 被引量:1
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作者 石辛甫 徐辉 +3 位作者 彭虹 陈志华 邢丽 高杰英 《中国免疫学杂志》 CAS CSCD 北大核心 2001年第3期130-131,134,共3页
目的 :FSM 2 117和FS 5 416是我室构建的具有不同生物表型的福氏、宋内氏双价痢疾菌苗株 ,前者不表达侵袭蛋白 ,无侵袭力 ,而后者表达侵袭蛋白 (Ipa) ,具有侵袭力。拟观测两株菌苗FSM 2 117(Ipa- )、FS 5 416 (Ipa+ )免疫后所引起的肠... 目的 :FSM 2 117和FS 5 416是我室构建的具有不同生物表型的福氏、宋内氏双价痢疾菌苗株 ,前者不表达侵袭蛋白 ,无侵袭力 ,而后者表达侵袭蛋白 (Ipa) ,具有侵袭力。拟观测两株菌苗FSM 2 117(Ipa- )、FS 5 416 (Ipa+ )免疫后所引起的肠粘膜局部和系统中的免疫记忆反应 ,为研制痢疾菌苗提供理论依据。方法 :以小鼠灌胃免疫为模型 ,3次免疫后 ,间隔 3个月再次免疫 ,第 7天取血清和小肠冲洗夜 ,应用ELISA方法检测福氏、宋内氏特异性抗体。结果 :两菌苗株免疫组 ,血清中及局部抗原特异性sIgA、IgG抗体明显升高。两株菌苗与对照组相比 ,都具有显著差异。结论 :两株双价痢疾菌苗免疫小鼠后 。 展开更多
关键词 双价痢疾菌苗 特异性抗体 记忆反应 小鼠 细菌性痢疾
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地鼠肾原代细胞双价肾综合征出血热疫苗免疫效果的研究 被引量:2
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作者 安祺 韩亮 +3 位作者 董关木 刘文雪 孔艳 杨立宏 《微生物学免疫学进展》 2002年第4期14-17,共4页
对新近研制成功的地鼠肾原代细胞肾综合征出血热双价疫苗 (汉滩型 +汉城型 )进行了Ⅱ期临床扩大观察 ,考核其对人体的安全性和中和抗体反应。将观察人群分别选择在中国南方和北方两个点 ,每个点接种 5 89人和6 0 0人 ,观察其接种对象的... 对新近研制成功的地鼠肾原代细胞肾综合征出血热双价疫苗 (汉滩型 +汉城型 )进行了Ⅱ期临床扩大观察 ,考核其对人体的安全性和中和抗体反应。将观察人群分别选择在中国南方和北方两个点 ,每个点接种 5 89人和6 0 0人 ,观察其接种对象的副反应程度和采集血清样品测定其荧光抗体并以蚀斑减少中和法测定中和抗体 ,考核疫苗效果。观察结果显示 ,在观察的 2 5 3人中分别有 6人呈现轻度局部副反应和 1人 37.5℃以下的低热全身反应 ,总反应率分别为 2 .77% (7/ 2 5 3)。IFAT抗体阳转率对Ⅰ型病毒为 94.85 % (184/ 194) ,对Ⅱ型病毒为 89.6 9% (174/194) ,ELISA抗体阳转率为 99.44 % (179/ 180 ) ,GMT132 5。PRNT抗体阳转率Ⅰ型为 92 .5 5 % (87/ 94) ,GMT10 .17,Ⅱ型为 93.6 2 % (88/ 94) ,GMT11.0 8。经过Ⅱ期临床人体观察结果显示 ,该双价疫苗仅有轻度局部反应和良好的中和抗体应答。证明该疫苗对人体安全 。 展开更多
关键词 地鼠 肾原代细胞 肾综合征出血热 免疫效果 双价疫苗 临床观察 中和抗体
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不同连接肽的双价单链抗体基因的构建及表达 被引量:5
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作者 严丹丹 方瑾 宋今丹 《细胞生物学杂志》 CSCD 2007年第2期272-276,共5页
采用基因重组技术分别借助不同长度的连接肽[G4S和(G4S)3],将两个相同的抗人大肠癌单链抗体基因ND-1scFv共价连接,构建表达载体pET-28a(+)ND-1sc(Fv)2,并在大肠杆菌BL21中表达ND-1sc(Fv)2的融合蛋白。应用Ni2+亲和层析方法对表达产物进... 采用基因重组技术分别借助不同长度的连接肽[G4S和(G4S)3],将两个相同的抗人大肠癌单链抗体基因ND-1scFv共价连接,构建表达载体pET-28a(+)ND-1sc(Fv)2,并在大肠杆菌BL21中表达ND-1sc(Fv)2的融合蛋白。应用Ni2+亲和层析方法对表达产物进行纯化,SDS-PAGE、免疫荧光法(IFA)和ELISA对纯化后的蛋白质进行纯度和免疫活性分析。结果表明成功构建了表达载体pET-28a(+)ND-1sc(Fv)2,并在大肠杆菌中获得高效表达,其表达产物以不溶性包涵体形式存在。纯化后ND-1sc(Fv)2-5、ND-1sc(Fv)2-15的蛋白质纯度分别为90%和86%。IFA及ELISA结果表明,二者均保留了亲本抗体的免疫活性,对表达在人大肠癌细胞上的肿瘤相关抗原LEA具有特异结合活性,其免疫活性均明显高于ND-1scFv,其中ND-1sc(Fv)2-15的免疫活性更接近于亲本单抗ND-1,该抗体有望成为大肠癌临床导向诊断和治疗的理想载体。 展开更多
关键词 双价单链抗体 ND-1 连接肽 大肠癌 表达
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成年珍禽禽流感免疫抗体消长规律及免疫程序的研究 被引量:2
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作者 邓家波 杨晓梅 +7 位作者 王强 李兴玉 余建秋 廖党金 牛李丽 林毅 严慧娟 谢晶 《动物医学进展》 CSCD 北大核心 2012年第9期31-36,共6页
为了弄清动物园成年珍禽的禽流感免疫抗体消长规律,用禽流感(H5+H9)二价灭活疫苗免疫接种动物园的8种珍禽,分别在免疫接种前后不同时间无菌采血,检测血清中H5和H9亚型禽流感病毒血凝抑制(HI)抗体。结果表明,大部分珍禽首次免疫接种后都... 为了弄清动物园成年珍禽的禽流感免疫抗体消长规律,用禽流感(H5+H9)二价灭活疫苗免疫接种动物园的8种珍禽,分别在免疫接种前后不同时间无菌采血,检测血清中H5和H9亚型禽流感病毒血凝抑制(HI)抗体。结果表明,大部分珍禽首次免疫接种后都能产生H5和H9亚型禽流感病毒HI抗体,但不同种类免疫反应不同。孔雀等4种珍禽在首次免疫接种后400d,H5、H9亚型禽流感病毒的抗体仍能维持较高的水平;白冠长尾雉等4种珍禽首次免疫后,H5、H9亚型禽流感病毒抗体水平不高,且维持时间很短,在进行H5+H9二价灭活苗加强免疫后,能有效提高H5、H9亚型禽流感病毒免疫抗体,免疫抗体均能维持7个月。通过对免疫抗体的监测,对两种不同剂量的比较研究,并结合动物园珍禽的实际情况,本研究初步建立了成年珍禽的禽流感的免疫程序。 展开更多
关键词 珍禽 禽流感二价灭活苗 HI抗体 免疫抗体消长 免疫程序
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人源鼻咽癌双价抗独特型抗体疫苗的制备及活性鉴定 被引量:1
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作者 王甲甲 郭锋杰 +1 位作者 李跃辉 李官成 《细胞与分子免疫学杂志》 CAS CSCD 北大核心 2010年第7期667-670,共4页
目的:构建全人源鼻咽癌双价抗独特型抗体原核表达载体,并对其产物作初步鉴定。方法:利用分子克隆技术依次将G22,150插入到原核表达载体pET25b(+)中,经菌落PCR,双酶切验证并测序。阳性重组子转化入表达菌株E.coliBL21(DE3),... 目的:构建全人源鼻咽癌双价抗独特型抗体原核表达载体,并对其产物作初步鉴定。方法:利用分子克隆技术依次将G22,150插入到原核表达载体pET25b(+)中,经菌落PCR,双酶切验证并测序。阳性重组子转化入表达菌株E.coliBL21(DE3),经IPTG诱导表达后,表达的重组蛋白经Histag纯化试剂盒纯化后复性。用Western blot方法鉴定G22-150双价蛋白的表达,ELISA方法分析其蛋白活性,并进一步观察其对外周血单个核细胞的刺激增殖情况。结果:构建的原核表达载体pET25b—G22—150经测序验证,序列正确。双价蛋白G22—150以包涵体形式高效表达,经纯化后纯度达90%以上。Westernblot鉴定表达的蛋白相对分子质量(Ms)约42000,与预期相符。ELISA鉴定复性后的蛋白已恢复活性,并能在体外刺激外周血单个核细胞的增殖。结论:成功获得了有活性的双价抗独特型抗体,为临床对鼻咽癌进行主动免疫治疗提供了理论基础。 展开更多
关键词 鼻咽癌 抗独特型抗体疫苗 双价 原核表达
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基因工程双价抗体在肿瘤中的研究进展 被引量:4
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作者 王甲甲 李官成 《国际病理科学与临床杂志》 CAS 2008年第6期508-511,共4页
基因工程抗体技术的发展加速了单链抗体的应用,但其稳定性差,亲和力低,功能单一,体内清除过快等特点影响了它的广泛应用。双价抗体作为一种新型小分子抗体,具有双价的结合位点,能够使抗原分子上的两个表位交联或使两个分子连接,可以模... 基因工程抗体技术的发展加速了单链抗体的应用,但其稳定性差,亲和力低,功能单一,体内清除过快等特点影响了它的广泛应用。双价抗体作为一种新型小分子抗体,具有双价的结合位点,能够使抗原分子上的两个表位交联或使两个分子连接,可以模拟完整的单克隆抗体的抗原抗体反应,其构建方法有亮氨酸拉链法、利用部分抗体恒定区法、连接肽法、利用双聚化结构法、knobs into holes技术等,在乳腺癌、直肠癌、淋巴瘤等的诊治方面均有很好的应用价值。 展开更多
关键词 双价抗体 基因工程 肿瘤
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