本文選題：B淋巴細胞刺激因子 + 白喉類毒素　； 參考：《濟南大學》2011年碩士論文

【摘要】：免疫毒素(Immunotoxins,IT),又被稱之為生物導彈,其具有專門選擇性的破壞某一特異性標記細胞功能的一類融合蛋白,它是通過通過化學交聯(lián)的方式將具有高度特異性的單克隆抗體與具有強大殺傷作用的毒素分子構建而成。免疫毒素利用腫瘤細胞上導向分子的受體與細胞結(jié)合,進入細胞后由毒素發(fā)揮蛋白質(zhì)合成抑制作用,最終能夠?qū)е掳屑毎乃劳?其具備了特異性的識別的功能和毒素殺傷的功能效應,對傳統(tǒng)的治療腫瘤化療、放療的缺陷進行了彌補。 B淋巴細胞刺激因子(B lymphocyte stimulating factor,BAFF)屬腫瘤壞死因子家族新成員,因其在B細胞發(fā)育及自身免疫病中起重要作用而倍受關注。BAFF通過與其受體結(jié)合廣泛參與T、B淋巴細胞增殖和功能調(diào)節(jié),發(fā)揮其生物學效應。BAFF的缺乏可導致免疫功能低下,過表達則與多種自身免疫性疾病的發(fā)生和發(fā)展密切相關。 目的 為探討人sBAFF-DT388在B細胞惡性腫瘤及自身免疫性疾病中的治療作用,我們進行了人sBAFF-DT388融合蛋白表達載體的構建、重組蛋白的分離純化及其生物學活性的初步研究。 方法 1.根據(jù)優(yōu)化合成的hsBAFF-DT388基因序列設計引物,經(jīng)PCR擴增將目的基因片段插入到pMD19-T克隆載體,菌落PCR、限制性內(nèi)切酶酶切及DNA測序鑒定陽性克隆。 2.重組克隆載體經(jīng)NdeⅠ、XhoⅠ雙酶切后,瓊脂糖凝膠電泳分離目的基因,將其插入表達載體pColdⅡ相應酶切位點,轉(zhuǎn)化BL21感受態(tài)菌株,菌落PCR鑒定重組表達載體。 3.挑取單克隆菌落擴增培養(yǎng)至對數(shù)生長期,經(jīng)IPTG誘導后,SDS-PAGE分析和Western blot鑒定重組蛋白。 4.Ni2+-NTA層析柱純化重組蛋白,經(jīng)透析后,SDS-PAGE檢測重組蛋白。 5.利用PE熒光素標記重組蛋白,檢測其與受體結(jié)合能力。 6.細胞毒性實驗檢測重組蛋白對BAFF-R(+)B細胞株Hmy2.CIR細胞的殺傷作用。采用SPSS16.0統(tǒng)計軟件進行統(tǒng)計學分析。 結(jié)果 1.成功構建了人sBAFF-DT388重組質(zhì)粒pMD-hsBAFF-DT388,測序結(jié)果顯示克隆目的基因序列與優(yōu)化后的人sBAFF-DT388基因序列完全一致。 2.陽性重組表達載體轉(zhuǎn)化BL21感受態(tài)菌株后經(jīng)IPTG誘導,行SDS-PAGE分析結(jié)果顯示誘導后的陽性重組菌株在58.4KD處出現(xiàn)明顯蛋白條帶,符合目的蛋白大小,圖像掃描分析顯示獲得的目的蛋白約占菌體總蛋白的40%。Western blot結(jié)果顯示重組蛋白能與抗BAFF多克隆抗體和抗His-Tag多克隆抗體均能發(fā)生特異反應,表明獲得的重組蛋白為特異性hsBAFF-DT388融合蛋白。 3.重組蛋白經(jīng)Ni2+-NTA層析柱純化,經(jīng)凝膠圖像掃描分析,hsBAFF-DT388重組蛋白純度達90%以上。 4.熒光素標記重組蛋白后在倒置熒光顯微鏡下可觀察到BAFF-R(+)Hmy2.CIR細胞有較強紅色熒光出現(xiàn),而陰性對照組BAFF-R(-)U937細胞則未見熒光,表明重組蛋白與BAFF-R(+)Hmy2.CIR細胞表面受體具有特異性結(jié)合能力。 5.細胞毒實驗檢測重組蛋白對BAFF-R(+)B細胞株Hmy2.CIR細胞的殺傷作用,結(jié)果顯示重組蛋白對其具有較強的抑制效應,且抑制效應具有劑量依賴關系,即隨著重組蛋白濃度的增加抑制效應越強(P0.05)。而不同濃度的重組蛋白對陰性對照組BAFF-R(-) U937細胞抑制效應無統(tǒng)計學差異(P0.05)。 結(jié)論 利用PCR技術成功克隆了人sBAFF-DT388目的基因,序列分析顯示與優(yōu)化后的基因序列一完全致;重組技術構建pcoldⅡ-hsBAFF-DT388的原核表達載體,轉(zhuǎn)化BL21感受態(tài)菌株后獲得穩(wěn)定表達的工程菌株;初步探索優(yōu)化了重組pcoldⅡ-hsBAFF-DT388的表達條件、包涵體提取步驟及蛋白初步純化工藝;獲得了具有靶向B細胞活性的重組蛋白,為其在B細胞惡性腫瘤及自身免疫性疾病治療中的應用研究奠定了基礎。
[Abstract]:Immunotoxins (IT), also known as a biological missile, has a specific selective fusion protein that destroys the function of a specific marker cell. It is constructed by chemically crosslinking a highly specific monoclonal antibody and a toxin with strong killing effect. The binding of the receptor to the cell on the tumor cells and the cells of the cells, and after the entry of the cell to the cell, can play the inhibitory effect of the protein synthesis, and eventually lead to the death of the target cells. It has the specific recognition function and the function effect of the toxin killing, and makes up for the defects of the traditional therapy for the treatment of tumor and the radiotherapy.
B lymphocyte stimulating factor (B lymphocyte stimulating factor, BAFF) is a new member of the tumor necrosis factor family. Because of its important role in the development of B cell and autoimmune disease, it is concerned that.BAFF is widely involved in T, B lymphocyte proliferation and function regulation by combining with its receptor, and the lack of biological effect.BAFF can lead to the deficiency of its biological effect. Low immune function and over expression are closely related to the occurrence and development of various autoimmune diseases.
objective
In order to investigate the role of human sBAFF-DT388 in the treatment of B cell malignant tumors and autoimmune diseases, we have carried out the construction of human sBAFF-DT388 fusion protein expression vector, the separation and purification of recombinant protein and the preliminary study of its biological activity.
Method
1. the primers were designed based on the optimized hsBAFF-DT388 gene sequence, and the target gene fragment was inserted into the pMD19-T cloning vector by PCR amplification. The colony PCR, restriction endonuclease digestion and DNA sequencing were used to identify the positive clones.
2. the recombinant cloning vector was cut through Nde I and Xho I, and the target gene was separated by agarose gel electrophoresis. It was inserted into the expression vector pCold II corresponding enzyme cutting site, transformed the BL21 receptive strain, and the colony PCR was used to identify the recombinant expression vector.
3. the colony was amplified and cultured to logarithmic growth phase. After induction by IPTG, the recombinant protein was identified by SDS-PAGE analysis and Western blot.
The recombinant protein was purified by 4.Ni2+-NTA chromatography column, and the recombinant protein was detected by SDS-PAGE.
5. the recombinant protein was identified by PE fluorescein and its binding ability with the receptor was detected.
6. cytotoxicity test was used to detect the killing effect of recombinant protein on Hmy2.CIR cells of BAFF-R (+) B cell line. Statistical analysis was performed by SPSS16.0 statistical software.
Result
1. the recombinant plasmid pMD-hsBAFF-DT388 of human sBAFF-DT388 was successfully constructed, and the sequencing results showed that the sequence of the target gene of the clone was identical with the optimized sequence of human sBAFF-DT388 gene.
2. positive recombinant expression vector transformed BL21 receptive strain and was induced by IPTG. The result of SDS-PAGE analysis showed that the positive recombinant strain appeared at 58.4KD, which conformed to the size of the target protein. The image scanning analysis showed that the obtained target protein accounted for the 40%.Western blot result of the total protein of the bacteria to show the recombinant protein. It can react with anti BAFF polyclonal antibody and anti His-Tag polyclonal antibody, indicating that the recombinant protein is a specific hsBAFF-DT388 fusion protein.
3. the recombinant protein was purified by Ni2+-NTA chromatography and the purity of recombinant hsBAFF-DT388 protein was over 90% after scanning and scanning.
After 4. fluorescein was labeled with recombinant protein, the strong red fluorescence of BAFF-R (+) Hmy2.CIR cells could be observed under the inverted fluorescence microscope, while the BAFF-R (-) U937 cells in the negative control group had no fluorescence, indicating that the recombinant protein had a specific binding ability to the BAFF-R (+) Hmy2.CIR cell surface receptor.
5. cytotoxicity test was used to detect the killing effect of recombinant protein on BAFF-R (+) B cell line Hmy2.CIR cells. The results showed that the recombinant protein had strong inhibitory effect on it, and the inhibitory effect was dose-dependent, that is, the inhibitory effect was stronger with the increase of the concentration of recombinant protein (P0.05). The recombinant protein of different concentrations was BAFF in the negative control group. The inhibitory effect of -R (-) U937 cells was not statistically different (P0.05).
conclusion
The target gene of human sBAFF-DT388 was cloned successfully by PCR technology. Sequence analysis showed that the gene sequence was completely induced by the optimized gene sequence. The recombinant technique was used to construct the prokaryotic expression vector of pCold II -hsBAFF-DT388, and the recombinant strain of BL21 receptor was transformed into a stable expression strain. The preliminary probe optimized the expression bar of the recombinant pCold II -hsBAFF-DT388. Parts, inclusion body extraction and protein preliminary purification process, recombinant protein with target B cell activity were obtained, which laid the foundation for its application in the treatment of B cell malignant tumor and autoimmune disease.

【學位授予單位】：濟南大學
【學位級別】：碩士
【學位授予年份】：2011
【分類號】：R392

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