本文選題：短的上腭、肺及鼻咽上皮克隆1(SPLUNC1) + 定點(diǎn)突變��； 參考：《北京市結(jié)核病胸部腫瘤研究所》2010年碩士論文

【摘要】： 目的:真核表達(dá)SPLUNC1蛋白和制備鑒定SPLUNC1單克隆抗體內(nèi)容:1.SPLUNC1真核表達(dá)載體的構(gòu)建;2.m-SPLUNC1-Ig和h-SPLUNC1-Ig融合蛋白的表達(dá)及SPLUNC1蛋白的體外活性分析;3.SPLUNC1單克隆抗體的制備和鑒定分析。 方法:從肺腺癌組織中擴(kuò)增SPLUNC1基因,利用大引物法和巢式PCR實(shí)現(xiàn)基因定點(diǎn)突變,順利構(gòu)建PDH3-SPLUNC1的表達(dá)載體,同時(shí)構(gòu)建PDM3-SPLUNC1的表達(dá)載體。利用Lipofectamine2000轉(zhuǎn)染CHO/dhfr-細(xì)胞,利用二氫葉酸還原酶基因選擇系統(tǒng)加用透析血清及MTX進(jìn)行篩選分別獲得持續(xù)表達(dá)m-SPLUNC1-Ig和h-SPLUNC1-Ig融合蛋白的細(xì)胞株。采用ELISA和免疫印跡實(shí)驗(yàn)分析h-SPLUNC1-Ig融合蛋白與LPS結(jié)合,了解h-SPLUNC1-Ig體外的生物活性。采用常規(guī)的免疫方法免疫Balb/c小鼠,取效價(jià)最高的Balb/c小鼠行下一步融合實(shí)驗(yàn),利用有限稀釋法獲得穩(wěn)定分泌SPLUNC1抗體的雜交瘤,分析其親和力、亞類、表位以及與天然蛋白結(jié)合情況。 結(jié)果:順利完成了基因的定點(diǎn)突變,實(shí)現(xiàn)了PDH3-SPLUNC1和PDM3-SPLUNC1兩種表達(dá)載體的構(gòu)建,同時(shí)獲得了一種無需切膠純化的基因定點(diǎn)突變方法;獲得了分別表達(dá)m-SPLUNC1-Ig和h-SPLUNC1-Ig兩種融合蛋白的穩(wěn)定表達(dá)株,能夠持續(xù)穩(wěn)定地表達(dá)融合蛋白,體外活性分析發(fā)現(xiàn)h-SPLUNC1-Ig融合蛋白具有體外活性,能夠與革蘭氏陰性菌細(xì)胞壁的脂多糖成分結(jié)合;單抗制備過程中獲得了6株單克隆抗體,亞類均為IgG1,能夠與天然SPLUNC1蛋白結(jié)合。 結(jié)論:獲得了具有體外活性的SPLUNC1融合蛋白和SPLUNC1單克隆抗體,能夠與天然的SPLUNC1蛋白結(jié)合。
[Abstract]:Aim: to express SPLUNC1 protein and to prepare and identify SPLUNC1 monoclonal antibody. 1. Construction of Eukaryotic expression Vector of SPLUNC1. Expression of 2.m-SPLUNC1-Ig and h-SPLUNC1-Ig Fusion protein and activity Analysis of SPLUNC1 protein in Vitro. 3. Preparation and characterization of monoclonal antibody against SPLUNC1. Methods: SPL1 gene was amplified from lung adenocarcinoma tissue. The expression vector of PDH3-SPLUNC1 was successfully constructed by using large primer method and nested PCR to construct the expression vector of PDH3-SPL1, and the expression vector of PDM3-SPL1 was constructed at the same time. The cell lines expressing m-SPLUNC1-Ig and h-SPLUNC1-Ig fusion proteins were obtained by transfection of CHO / dhfr- cells with Lipofectamine2000, dihydrofolate reductase gene selection system and dialysis serum and MTX, respectively. The binding of h-SPL1-Ig fusion protein to LPS was analyzed by Elisa and Western blotting to study the bioactivity of h-SPL1-Ig in vitro. Balb / c mice were immunized with routine immune methods. Balb / c mice with the highest titer were selected for the next fusion experiment. Hybridomas secreting the antibody against SPLUNC1 were obtained by finite dilution method, and their affinity and subclasses were analyzed. Epitopes and their binding to natural proteins. Results: the site-directed mutation of the gene was successfully completed and the expression vectors PDH3-SPLUNC1 and PDM3-SPLUNC1 were constructed. At the same time, a method of site-directed mutation was obtained. The stable expression strains of m-SPLUNC1-Ig and h-SPLUNC1-Ig were obtained, which can express the fusion protein continuously and stably. The in vitro activity analysis of h-SPLUNC1-Ig fusion protein showed that h-SPLUNC1-Ig fusion protein had in vitro activity. It could bind to lipopolysaccharide from the cell wall of Gram-negative bacteria, and six monoclonal antibodies were obtained during the preparation of monoclonal antibodies, all of which were IgG1, which could bind to natural SPLUNC1 protein. Conclusion: the fusion protein of SPLUNC1 and the monoclonal antibody against SPLUNC1 have been obtained, which can bind to the natural SPLUNC1 protein.
【學(xué)位授予單位】：北京市結(jié)核病胸部腫瘤研究所
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2010
【分類號(hào)】：R392.1

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本文編號(hào)：2036405