抗副溶血弧菌Needle单链抗体的制备与鉴定

发布时间：2018-03-06 12:03

本文选题：Needle　切入点：ScFv　出处：《福建农林大学》2012年硕士论文　论文类型：学位论文

【摘要】：副溶血弧菌（Vibrio parahaemolyticus）是一种嗜盐的革兰氏阴性致病菌，广泛存在于海底沉积物、近海岸水体和海产品中，不仅会引发鱼、虾、贝类等海产品败血症，还会导致人食物中毒及腹泻、恶心、呕吐等典型胃肠炎疾病。目前对于副溶血弧菌的致病机理尚未研究清楚，缺乏有效的预防与治疗手段。三型分泌系统（T3SS）是已知副溶血弧菌致病的主要因子之一，而T3SS毒力因子的运输管道Needle在其致病过程中起着决定性作用。本研究基于DNA重组技术成功构建抗Needle噬菌体抗体文库，利用噬菌体展示技术成功淘选到抗Needle的特异性单链抗体，并以共表达技术纯化获得此抗体；为预防和治疗由副溶血弧菌T3SS引起的海产品败血症提供了重要材料，为进一步探索副溶血弧菌的致病机制奠定了基础。通过分子克隆技术，将编码Needle单聚体的vp1694基因成功构建到pGEX-6p-1及pET-32a(+)载体上，，获得融合表达的GST-vp1694、TRX-vp1694蛋白，经纯化测定浓度分别为0.237mg/mL、0.942mg/mL，利用GST-vp1694作为人工抗原免疫小鼠，TRX-vp1694作为检测抗原检测小鼠免疫的血清效价。经三次皮下免疫后，ELISA检测小鼠血清效价达到16000，处死小鼠后提取脾脏总RNA，经RT-PCR获得cDNA第一链，以此链为模板，通过PCR获得抗体重链可变区VH基因和轻链可变区基因VL，以(Gly4Ser)3作为Linker，通过交错延伸PCR成功将VH、Linker和VL组装成完整的scFv基因。再将scFv基因克隆到噬菌体载体pCANTAB-5E上，构建了库容量约为1.4×107cfu/mL的噬菌体抗体文库。通过噬菌体展示技术，经过六轮的富集淘选，从噬菌体抗体库中淘选到两株验证无误的单链抗体菌株，分别将其命名为F-A7，F-E9，经测序scFv片段长为729bp，含有VH-Linker-VL序列结构，确为鼠源单链抗体，ELISA检测发现anti-vp1694-scFv与TDH、TLH、VopQ、HrpA抗原结合不显著，与vp1694抗原结合显著，显著性分析P0.01，具有较高的抗体特异性。扩增噬菌体载体中的scFv基因，构建到含Skp细菌伴侣蛋白的共表达载体pACYC-Duet-1-Skp中，获得细胞内可溶性表达的单链抗体，经Ni2+-NTA亲和纯化，BCA测定抗体浓度为0.102mg/mL。利用纯化的scFv进行亲和力测定，Kaff≈1.07×108。此外，利用IMGT/V-QUEST数据库在线分析软件对anti-vp1694-scFv进行序列比对、CDR分区、同源性等生物信息学分析，发现anti-vp1694-scFv的VH链归属于鼠源胚系基因IGHV5、IGHJ2和IGHD2，VL归属于鼠源胚系基因IGKV4和IGKJ2。通过IMGT/Collier-de-Perles分析软件对anti-vp1694-scFv的氨基酸结构进行成功模拟，将为抗体的进一步改造提供参考。
[Abstract]:Vibrio parahaemolyticus (Vibrio parahaemolyticus) is a halophilic gram-negative pathogen widely found in seafloor sediments, coastal waters and seafood, causing not only sepsis of fish, shrimp and shellfish, but also food poisoning and diarrhea. Nausea, vomiting and other typical gastroenteritis diseases. At present, the pathogenetic mechanism of Vibrio parahaemolyticus is not clear, and there is no effective prevention and treatment. Type 3 secretion system (T3SS) is one of the main known pathogenic factors of Vibrio parahaemolyticus (Vibrio parahaemolyticus). The Needle of T3SS virulence factor transport pipeline plays a decisive role in the pathogenicity of T3SS. In this study, we successfully constructed an anti Needle phage antibody library based on DNA recombinant technology, and successfully panned the specific single chain antibody against Needle by phage display technique. The antibody was purified by co-expression, which provided important materials for the prevention and treatment of septicemia caused by Vibrio parahaemolyticus T3SS, and laid a foundation for further exploring the pathogenic mechanism of Vibrio parahaemolyticus. By molecular cloning, the vp1694 gene encoding Needle monomers was successfully constructed into pGEX-6p-1 and pET-32a () vector, and the fusion expressed GST-vp1694 TRX-vp1694 protein was obtained. The serum titers of mice immunized with GST-vp1694 as artificial antigen were determined to be 0.237 mg / mL and 0.942 mg / mL, respectively. The serum titers of mice immunized with TRX-vp1694 were detected by using TRX-vp1694 as antigen. After three times of subcutaneous immunization, Elisa was used to detect the titer of serum of mice up to 16000. The spleen total RNAs were extracted after the mice were killed. The first chain of cDNA was obtained by RT-PCR, and the chain was used as a template. The VH gene of heavy chain variable region and the variable region gene of light chain were obtained by PCR. Using Gly4Serf3 as Linker, the complete scFv gene was successfully assembled by interleaving PCR, and then the scFv gene was cloned into the phage vector pCANTAB-5E. A phage antibody library with a capacity of 1.4 脳 107 cfur / mL was constructed. By means of phage display technique, two scFv strains were obtained from phage antibody library after six rounds of enrichment and panning. They were named F-A7F- F-E9, and the scFv fragment was 729bp, which contained VH-Linker-VL sequence structure. It was found that the binding of anti-vp1694-scFv to vp1694 antigen was not significant, but to vp1694 antigen was significant (P 0.01), which showed that anti-vp1694-scFv had a high antibody specificity. ScFv gene was amplified from phage phage vector and constructed into co-expression vector pACYC-Duet-1-Skp containing Skp bacterial chaperone protein to obtain soluble single-chain antibody (scFv) expressed in cells. The antibody concentration determined by Ni2 NTA affinity purification was 0.102 mg / mL. The affinity of purified scFv was determined by using IMGT/V-QUEST database. In addition, the sequence alignment and homology of anti-vp1694-scFv were analyzed by IMGT/V-QUEST database on-line analysis software. It was found that the VH chain of anti-vp1694-scFv was attributed to the mouse embryogenic genes IGHV5GHJ2 and IGHD2VL to the murine embryogenic genes IGKV4 and IGK J22.The amino acid structure of anti-vp1694-scFv was successfully simulated by IMGT/Collier-de-Perles software, which would provide a reference for further modification of the antibody.
【学位授予单位】：福建农林大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R378

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