利用噬菌體展示法篩選出對srr-1 蛋白具高度特異性結合能力的單鏈變異片段抗體

江復賢(FU-SIAN JIANG)

指導教授 ： 楊沂淵

共同指導教授 ： 呂思潔

臺北醫學大學/醫學院/醫學科學研究所/碩士(2010年)

https://doi.org/10.6831/TMU.2010.00085

摘要

B型鏈球菌是一種常見的致病菌，常引發新生兒腦膜炎，造成新生兒的死亡，B型鏈球菌可以穿過血腦屏障造成腦膜炎，而B型鏈球菌的srr-1蛋白在此機制扮演一個重要的角色，因此針對srr-1發展一個快速的檢驗方法是必需的。
在本實驗我們針對B型鏈球菌的膜蛋白srr-1利用基因重組的方法表現不同長度的片段。將蛋白大量的表現之後再純化，其中核苷酸序列961-1929(srr-1 N2N3)的片段是其中表現量最多的片段，利用高純度的N2N3蛋白打入萊亨雞的皮下，使其產生免疫反應產生抗srr-1的IgY，從蛋黃的部份將抗srr-1 IgY純化出來，收集4 次免疫反應的IgY，利用西方墨點法及ELISA測定IgY的效價。
將免疫雞隻的脾臟取出，抽取mRNA建立抗體重鏈及輕鏈的基因庫，將建立的基因庫送入噬菌體內，在噬菌體表面抗體蛋白經過4次panning篩選及富化對srr-1蛋白具高度特異性的scFv。從第四次panning所長的菌落中隨機挑選15個菌落以電泳分析phagemid DNA，從中挑選有接入scFv基因的phagemid送入TOP 10 F表現scFv抗體片段並以其分子量及anti-chicken light chain 抗體來作確認，利用ELISA和西方墨點法分析scFv抗體片段對N2N3蛋白的特異性結合能力。在之後的實驗這些重組srr-1蛋白、多株抗體IgY和單株抗體scFv會被測試抑制B型鏈球菌感染或生長的效果，以這些結果為基礎將能夠幫助我們發展快速的檢驗試劑或治療被B型鏈球菌感染的新生兒。

關鍵字

B型鏈球菌 ； 噬菌體展示法 ； 單鏈變異片段抗體

並列摘要

Group B Streptococcus (GBS) is the leading cause of bacterial meningitis in newborn infants. As GBS is able to invade, survive and cross the blood-brain barrier (BBB), srr-1 (serine-rich repeat-1) was identified as one of surface-expressed virulence factors that contribute to BBB penetration and the pathogenesis of meningitis. An effective method for rapid diagnosis for GBS should be developed. In this study, we cloned and expressed various lengths of srr-1 gene as his-fused proteins. However, only a fragment spanning nucleotides 961 to 1929 (srr-1 N2N3) could be easily expressed to a quantitative amount for subsequent experiment. After purified to a high degree of homogeneity, the purified protein was used to immunize chickens. A strong and specific humoral antibody response against srr-1 N2N3 was seen after 4 repeated challenges as characterized by Western blot and ELISA analysis. Total mRNAs was extracted from immunized chicken’s spleen to establish phage antibody libraries containing immunoglobulin heavy chain and light chain genes. The antibody libraries were then used to screen single chain variable fragment (scFv) anti-srr-1 N2N3 antibodies. In the fourth panning cycle, 3 of randomly selected 15 clones contain the scFv gene insert. We transformed the phagemid DNA into the TOP 10 F E. coli cells for scFv antibody expression. The identity of these recombinant scFv antibodies were confirmed by its molecular weight and anti-chicken light antibodies. Their binding specificity to srr-1N2N3 protein was analyzed by ELISA and Western blot methods. In the subsequent experiments, the recombinant srr-1 N2N3 protein, these polyclonal IgY or monoclonal scFv antibodies will be examined for their inhibitory effect on bacterial invasion and proliferation. Taken together, all the results based on the present study will help us to develop the early diagnostic or therapeutic agent for GBS infection in newborns.

並列關鍵字

Group B Streptococcus ； phage display ； scFv

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