本文關(guān)鍵詞：單鏈抗體637與人血清白蛋白融合蛋白的真核表達(dá)及功能鑒定　出處：《延邊大學(xué)》2007年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 單鏈抗體 融合蛋白 表達(dá) 重癥肌無(wú)力

【摘要】： 目的：將已經(jīng)構(gòu)建的重癥肌無(wú)力抗乙酰膽堿受體主要免疫原區(qū)單鏈抗體637(ScFv)與人血清白蛋白(HSA)重組基因(ScFv-HSA)進(jìn)行誘導(dǎo)表達(dá)，確定融合蛋白的分子量，檢測(cè)融合蛋白與人肋間肌乙酰膽堿受體的親和性。方法：將攜帶有重組基因表達(dá)載體的畢赤酵母菌GS115菌株復(fù)蘇后，利用抗生素G-418篩選高拷貝重組子，用YPD培養(yǎng)基傳代培養(yǎng)，再經(jīng)BMGY、BMMY表達(dá)培養(yǎng)基培養(yǎng)3天，每24小時(shí)添加100％甲醇使甲醇終濃度為1％誘導(dǎo)表達(dá)。培養(yǎng)基上清液室溫經(jīng)14000g離心后，利用斑點(diǎn)雜交試驗(yàn)(dot blot)初步判定上清液中的融合蛋白；采用十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析融合蛋白的分子量；應(yīng)用間接免疫熒光技術(shù)確定融合蛋白與人肋間肌乙酰膽堿受體的親和性。結(jié)果：培養(yǎng)基上清液中有融合蛋白的表達(dá)，分子量約為97.4 KD，間接免疫熒光試驗(yàn)顯示在人肋間肌細(xì)胞間有熒光出現(xiàn)。結(jié)論：已經(jīng)從真核細(xì)胞表達(dá)載體成功表達(dá)出融合蛋白，并且此融合蛋白可以與人肋間肌乙酰膽堿受體結(jié)合。
[Abstract]:Objective: to construct the recombinant gene scFv-HSA-ScFv-HSAand the constructed single chain antibody (scFv) of the main immunogenetic region of myasthenia gravis against acetylcholine receptor (ACHR) and human serum albumin (HSA). The expression was induced. The molecular weight of the fusion protein was determined and the affinity of the fusion protein to the acetylcholine receptor of human intercostal muscle was determined. Methods: the recombinant gene expression vector of Pichia pastoris GS115 strain was resuscitated. High-copy recombinant clones were screened by antibiotic G-418 and cultured on YPD medium, and then cultured on BMGYYBMMY expression medium for 3 days. The final concentration of methanol was 1% when 100% methanol was added every 24 hours. The medium supernatant was centrifuged by 14000g at room temperature. The fusion protein in supernatant was preliminarily identified by dot blot assay. The molecular weight of the fusion protein was analyzed by SDS-PAGE with sodium 12 alkyl sulfate and polyacrylamide gel electrophoresis. Indirect immunofluorescence technique was used to determine the affinity of the fusion protein to the acetylcholine receptor in human intercostal muscle. Results: the fusion protein was expressed in the supernatant of culture medium with a molecular weight of 97.4 KD. Indirect immunofluorescence assay showed the presence of fluorescence in human intercostal muscle cells. Conclusion: fusion protein has been successfully expressed from eukaryotic expression vector. And the fusion protein can bind to acetylcholine receptor in human intercostal muscle.
【學(xué)位授予單位】：延邊大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2007
【分類號(hào)】：R392

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本文編號(hào)：1389227