本文選題：Needle　切入點：ScFv　出處：《福建農(nóng)林大學》2012年碩士論文　論文類型：學位論文

【摘要】：副溶血弧菌（Vibrio parahaemolyticus）是一種嗜鹽的革蘭氏陰性致病菌，廣泛存在于海底沉積物、近海岸水體和海產(chǎn)品中，不僅會引發(fā)魚、蝦、貝類等海產(chǎn)品敗血癥，還會導致人食物中毒及腹瀉、惡心、嘔吐等典型胃腸炎疾病。目前對于副溶血弧菌的致病機理尚未研究清楚，缺乏有效的預(yù)防與治療手段。三型分泌系統(tǒng)（T3SS）是已知副溶血弧菌致病的主要因子之一，而T3SS毒力因子的運輸管道Needle在其致病過程中起著決定性作用。本研究基于DNA重組技術(shù)成功構(gòu)建抗Needle噬菌體抗體文庫，利用噬菌體展示技術(shù)成功淘選到抗Needle的特異性單鏈抗體，并以共表達技術(shù)純化獲得此抗體；為預(yù)防和治療由副溶血弧菌T3SS引起的海產(chǎn)品敗血癥提供了重要材料，為進一步探索副溶血弧菌的致病機制奠定了基礎(chǔ)。 通過分子克隆技術(shù)，將編碼Needle單聚體的vp1694基因成功構(gòu)建到pGEX-6p-1及pET-32a(+)載體上，，獲得融合表達的GST-vp1694、TRX-vp1694蛋白，經(jīng)純化測定濃度分別為0.237mg/mL、0.942mg/mL，利用GST-vp1694作為人工抗原免疫小鼠，TRX-vp1694作為檢測抗原檢測小鼠免疫的血清效價。 經(jīng)三次皮下免疫后，ELISA檢測小鼠血清效價達到16000，處死小鼠后提取脾臟總RNA，經(jīng)RT-PCR獲得cDNA第一鏈，以此鏈為模板，通過PCR獲得抗體重鏈可變區(qū)VH基因和輕鏈可變區(qū)基因VL，以(Gly4Ser)3作為Linker，通過交錯延伸PCR成功將VH、Linker和VL組裝成完整的scFv基因。再將scFv基因克隆到噬菌體載體pCANTAB-5E上，構(gòu)建了庫容量約為1.4×107cfu/mL的噬菌體抗體文庫。 通過噬菌體展示技術(shù)，經(jīng)過六輪的富集淘選，從噬菌體抗體庫中淘選到兩株驗證無誤的單鏈抗體菌株，分別將其命名為F-A7，F(xiàn)-E9，經(jīng)測序scFv片段長為729bp，含有VH-Linker-VL序列結(jié)構(gòu)，確為鼠源單鏈抗體，ELISA檢測發(fā)現(xiàn)anti-vp1694-scFv與TDH、TLH、VopQ、HrpA抗原結(jié)合不顯著，與vp1694抗原結(jié)合顯著，顯著性分析P0.01，具有較高的抗體特異性。 擴增噬菌體載體中的scFv基因，構(gòu)建到含Skp細菌伴侶蛋白的共表達載體pACYC-Duet-1-Skp中，獲得細胞內(nèi)可溶性表達的單鏈抗體，經(jīng)Ni2+-NTA親和純化，BCA測定抗體濃度為0.102mg/mL。利用純化的scFv進行親和力測定，Kaff≈1.07×108。此外，利用IMGT/V-QUEST數(shù)據(jù)庫在線分析軟件對anti-vp1694-scFv進行序列比對、CDR分區(qū)、同源性等生物信息學分析，發(fā)現(xiàn)anti-vp1694-scFv的VH鏈歸屬于鼠源胚系基因IGHV5、IGHJ2和IGHD2，VL歸屬于鼠源胚系基因IGKV4和IGKJ2。通過IMGT/Collier-de-Perles分析軟件對anti-vp1694-scFv的氨基酸結(jié)構(gòu)進行成功模擬，將為抗體的進一步改造提供參考。
[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.
【學位授予單位】：福建農(nóng)林大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R378

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