抗速灭威单链抗体基因在巴斯德毕赤酵母中的表达及性质研究

选用巴斯德毕赤酵母（Pichia pastoris）系统表达特异性抗速灭威单链抗体（scFv）基因,以为速灭威特异性抗体的大量制备奠定基础。设计引物扩增阳性克隆scFv基因,亚克隆至表达载体pPICZαC,获得重组酵母表达质粒pPICZαC-scFv,线性化pPICZαC-scFv并高效电转P.pastoris（X-33）,对转化子进行抗性梯度筛选得到一株高效表达的X-33-Pp-SMW-12-6菌株。对获得的菌株先后进行表达条件的优化、优化条件下的诱导表达及单链抗体性质研究。结果表明：P.pastoris-scFv的质量接近其亲本E.coli-scFv的质量,X-33-Pp-SMW12-6在优化条件下的表达产量达28mg/L,比未优化前的产量提高了约8mg/L,经纯化后抗体纯度可达85%以上。因此,利用P.pastoris表达系统制备抗速灭威单链抗体比细菌表达系统更有效、更经济。     

抗黄曲霉毒素单链抗体基因的克隆与表达

为降低黄曲霉毒素大量样品的制备成本,在实验室已有的抗黄曲霉毒素单克隆抗体8F6的基础上,成功克隆得到了该单克隆抗体的重链（VH）和轻链可变区（VL）基因片段。通过重叠延伸PCR的方法将轻、重链可变区基因连接,并引入连接肽(Linker) 编码序列,构建VH-Linker-VL结构的单链抗体(ScFv)基因, 并将该基因克隆到噬菌体表达载体pCANTAB 5E 上,使单链抗体以噬菌体展示形式在大肠杆菌TG1 中表达。间接竞争ELISA方法检测到该ScFv对黄曲霉毒素B1的抑制率（IC50）值为0.57ng/mL,表明该单链抗体与亲本鼠单抗有相同的抗原结合特异性,且具有很高灵敏度。     

天然鼠源噬菌体单链抗体库的构建

噬菌体展示技术是一项利用丝状噬菌体体外表达外源基因的新技术。它模拟动物体内抗体的形成过程，将抗体片段呈现在噬菌体表面，建成噬菌体抗体库，用与亲和层析相类似的原理，从抗体库中筛选出特异性噬菌体抗体，为制备人和动物用抗体提供了极为有效的手段。单链抗体是利用基因工程技术，由一个DNA片段(Linker)将抗体重链、     

病毒性出血性败血症病毒单链抗体的原核表达及其功能鉴定

为制备病毒性出血性败血症病毒(VHSV)单链抗体(single chain variable fragment antibody,ScFv)并鉴定其生物学功能,本研究提取抗VHSV单抗1G5的杂交瘤细胞株总RNA并反转录获得cDNA模板,通过PCR扩增VHSV抗体的轻链可变区(VL)和重链可变区(VH)编码序列,将其拼接成单链抗体Sc Fv基因后插入载体pET28a中,构建原核表达重组质粒并在大肠杆菌中诱导表达。结果表明,单链抗体主要以可溶性形式表达,分子量约28 ku,能特异性识别VHSV病毒的G蛋白并对VHSV病毒具有体外中和活性,其对VHSV病毒的G蛋白亲和力(KD)达到1.4×10~(–8) M。单链抗体ScFv的制备为进一步研究VHSV的治疗性抗体、快速诊断试剂奠定了基础。     

Preparation of Monoclonal Antibody Against PthA-NLS and Cloning of the Relative ScFv Gene

The present study aimed at the preparation of monoclonal antibody against the recombinant PthA-NLS and the isolation of the relative ScFv （single chain variable fragment） genes, providing the possibility to better understand the pathogenesis mechanism via PthA, and developing proper construct for future experimentation to obtain citrus plants resistant to canker disease by transformation and plant antibody techniques. The recombinant polypeptide PthA-NLS was injected into Balb/c mice to produce monoclonal antibody. Total RNA was isolated from the hybridoma cell line 3D10H2 which secreted anti- PthA-NLS McAb, and the variable region genes were amplified with specific primers by RT-PCR and SOE-PCR （splicing by overlap extension）, and then the ScFv gene was isolated. The recombinant ScFv gene was cloned into pGEM-T and pET32a（＋） vector. The later plasmid was transferred into E. coli BL21 （DE3） and the expression of the recombinant protein was induced. Three cell lines producing monoclonal antibody against PthA-NLS were acquired and named 1C8H1, 2D12B6, and 3D8A10. The recombinant ScFv gene of about 750 bp was constructed. The sequencing results showed that the ScFv gene consists of a 360 bp heavy chain, a 342 bp light chain, and a 45 bp linker region. The recombinant fusion ScFv protein was expressed by IPTG induction, and a 44.5 kDa of recombinant fusion protein was obtained. In conclusion, we obtained three cell lines stably producing monoclonal antibody specifically bound to PthA-NLS, and the relative ScFv gene was constructed and successfully expressed in E. coli. These results may play an important role in further understanding the pathogenesis mechanism and in the development of possible citrus resistant to canker disease by genetic transformation and plant antibiobody.     

特异性抗IBDV单链抗体基因的构建及序列分析

从抗 IBDV杂交瘤细胞系 SJS中分离总 RNA,经 RT-PCR体外扩增重、轻链可变区基因 ,通过一连接肽 (Giy4 Ser) 3基因拚接形成 Sc Fv基因 ,经 IBDV抗原亲和筛选出特异性阳性克隆进行核苷酸序列测定及氨基酸序列推导。所获得的特异性抗 IBDV单链抗体基因为 VH-Linker-VL 结构 ,其含有编码 (Giy4 Ser) 3的连接肽基因及维持抗体结构所必须的半胱氨酸残基 ,编码产物具有与亲本抗体相同的抗原结合活性和特异性 ,从而为研制具有应用潜力的高表达工程化抗体奠定了基础。     

抗牛传染性鼻气管炎病毒囊膜gD糖蛋白的单链抗体在昆虫细胞中表达及活性检测

为了获得具有生物学活性的牛传染性鼻气管炎病毒（infectious bovine rhinotracheitis virus,IBRV） gD蛋白特异性单链抗体,试验采用PCR方法扩增杂交瘤细胞的cDNA单链抗体重链可变区（VH）和轻链可变区（VL）基因,通过重叠延伸PCR及Linker序列获得完整的单链抗体基因,将单链抗体基因克隆至杆状病毒载体pFB-LIC-Bse中,构建重组转移载体pFB-VH-VL,将其转化至大肠杆菌DH10Bac感受态细胞中制备重组杆粒rBacmid-VH-VL,将重组杆粒rBacmid-VH-VL转染至Sf9细胞中获得携带单链抗体基因的重组杆状病毒。该重组杆状病毒在Sf9细胞中表达了分子质量约为30 ku的单链抗体蛋白。Western blotting结果显示,重组单链抗体蛋白能被His标签抗体特异性结合,也能特异性结合gD蛋白。间接免疫荧光试验结果显示,该单链抗体蛋白能够识别感染牛肾细胞MDBK中的IBRV。本研究在昆虫细胞中成功表达了具有识别IBRV的gD蛋白特异性单链抗体,为牛传染性鼻气管炎的诊断与治疗奠定了基础。     

单链抗体的研究进展

抗体在疾病的诊断、治疗和预防中发挥着重要的作用,其生产制备经历了抗血清多克隆抗体和单克隆抗体(Monoclonalantibodies,McAb)阶段,现已进入了基因工程抗体的时代。基因工程抗体主要包括单链抗体(ScFv)、Fab抗体、F(ab`)2抗体等小分子抗体、嵌合抗体和改形抗体。本文就单链抗体的研究进展作了综述。     

Expression and Biological Activity of Single Chain Antibody Against gD Glycoprotein of Infectious Bovine Rhinotracheitis Virus in Insect Cells

For preparation of bioactive single chain fragment variable (ScFv) which was targeted against envelope glycoprotein D (gD) of infectious bovine rhinotracheitis virus (IBRV),VH and VL genes were amplified from the cDNA of lymphocyte hybridoma, then VH and VL genes were integrated with a Linker by SOE-PCR,and the ScFv gene was cloned into the baculovirus vector pFB-LIC-Bse, which was transformed into Escherichia coli DH10Bac competent cells to construct a recombinant bacmid rBacmid-VH-VL. Finally,ScFv was expressed by transfected rBacmid-VH-VL into insect Sf9 cells, its molecular weight was about 30 ku. The result of Western blotting suggested that ScFv generated in Sf9 cells was specifically binds to His antibody,the protein also showed high affinity to gD of IBRV. The result of indirect immunofluorescence assay showed that ScFv could recognize IBRV which infected bovine kidney cells (MDBK). The study indicated that the recombinant ScFv was expressed in Sf9 cells, and could bind to gD of IBRV specifically. The result could provide the basis for the diagnosis and treatment of infection of IBRV.     

Selection of Beet Necrotic Yellow Vein Virus Specific Single-chain Fv Antibodies from a Semi-synthetic Combinatorial Antibody Library

Methods for the generation of monoclonal antibodies against plant viruses are limited because current hybridoma techniques do not allow efficient exploitation of the immune repertoire. Moreover, the immunization procedures often lead to a bias towards an immunodominant contaminant in the immunogen preparation and not to the plant virus itself. The selection of six different single-chain antibody variable fragments (scFv) against beet necrotic yellow vein virus from a semi-synthetic human combinatorial antibody library showed the feasibility of the phage display system. No bias towards minor contaminants in the purified virus preparation was observed in ELISA, as all the selected scFvs reacted only with beet necrotic yellow vein virus infected plant homogenates. In addition, two of the isolated beet necrotic yellow vein virus-specific scFvs could be produced in E. coli as a scFv fusion protein with alkaline phosphatase, and were applied in ELISA as specific ready to use antibody-enzyme conjugates. Because of their specificity, these antibodies have potential to be used as reagents in sensitive diagnostic assays for routine testing for beet necrotic yellow vein virus in sugar beets.