人sBAFF-DT388融合蛋白在大肠杆菌表达及其活性研究

发布时间：2018-04-21 17:48

本文选题：B淋巴细胞刺激因子 + 白喉类毒素　；参考：《济南大学》2011年硕士论文

【摘要】：免疫毒素(Immunotoxins,IT),又被称之为生物导弹,其具有专门选择性的破坏某一特异性标记细胞功能的一类融合蛋白,它是通过通过化学交联的方式将具有高度特异性的单克隆抗体与具有强大杀伤作用的毒素分子构建而成。免疫毒素利用肿瘤细胞上导向分子的受体与细胞结合,进入细胞后由毒素发挥蛋白质合成抑制作用,最终能够导致靶细胞的死亡,其具备了特异性的识别的功能和毒素杀伤的功能效应,对传统的治疗肿瘤化疗、放疗的缺陷进行了弥补。 B淋巴细胞刺激因子(B lymphocyte stimulating factor,BAFF)属肿瘤坏死因子家族新成员,因其在B细胞发育及自身免疫病中起重要作用而倍受关注。BAFF通过与其受体结合广泛参与T、B淋巴细胞增殖和功能调节,发挥其生物学效应。BAFF的缺乏可导致免疫功能低下,过表达则与多种自身免疫性疾病的发生和发展密切相关。目的为探讨人sBAFF-DT388在B细胞恶性肿瘤及自身免疫性疾病中的治疗作用,我们进行了人sBAFF-DT388融合蛋白表达载体的构建、重组蛋白的分离纯化及其生物学活性的初步研究。方法 1.根据优化合成的hsBAFF-DT388基因序列设计引物,经PCR扩增将目的基因片段插入到pMD19-T克隆载体,菌落PCR、限制性内切酶酶切及DNA测序鉴定阳性克隆。 2.重组克隆载体经NdeⅠ、XhoⅠ双酶切后,琼脂糖凝胶电泳分离目的基因,将其插入表达载体pColdⅡ相应酶切位点,转化BL21感受态菌株,菌落PCR鉴定重组表达载体。 3.挑取单克隆菌落扩增培养至对数生长期,经IPTG诱导后,SDS-PAGE分析和Western blot鉴定重组蛋白。 4.Ni2+-NTA层析柱纯化重组蛋白,经透析后,SDS-PAGE检测重组蛋白。 5.利用PE荧光素标记重组蛋白,检测其与受体结合能力。 6.细胞毒性实验检测重组蛋白对BAFF-R(+)B细胞株Hmy2.CIR细胞的杀伤作用。采用SPSS16.0统计软件进行统计学分析。结果 1.成功构建了人sBAFF-DT388重组质粒pMD-hsBAFF-DT388,测序结果显示克隆目的基因序列与优化后的人sBAFF-DT388基因序列完全一致。 2.阳性重组表达载体转化BL21感受态菌株后经IPTG诱导,行SDS-PAGE分析结果显示诱导后的阳性重组菌株在58.4KD处出现明显蛋白条带,符合目的蛋白大小,图像扫描分析显示获得的目的蛋白约占菌体总蛋白的40%。Western blot结果显示重组蛋白能与抗BAFF多克隆抗体和抗His-Tag多克隆抗体均能发生特异反应,表明获得的重组蛋白为特异性hsBAFF-DT388融合蛋白。 3.重组蛋白经Ni2+-NTA层析柱纯化,经凝胶图像扫描分析,hsBAFF-DT388重组蛋白纯度达90%以上。 4.荧光素标记重组蛋白后在倒置荧光显微镜下可观察到BAFF-R(+)Hmy2.CIR细胞有较强红色荧光出现,而阴性对照组BAFF-R(-)U937细胞则未见荧光,表明重组蛋白与BAFF-R(+)Hmy2.CIR细胞表面受体具有特异性结合能力。 5.细胞毒实验检测重组蛋白对BAFF-R(+)B细胞株Hmy2.CIR细胞的杀伤作用,结果显示重组蛋白对其具有较强的抑制效应,且抑制效应具有剂量依赖关系,即随着重组蛋白浓度的增加抑制效应越强(P0.05)。而不同浓度的重组蛋白对阴性对照组BAFF-R(-) U937细胞抑制效应无统计学差异(P0.05)。结论利用PCR技术成功克隆了人sBAFF-DT388目的基因,序列分析显示与优化后的基因序列一完全致;重组技术构建pcoldⅡ-hsBAFF-DT388的原核表达载体,转化BL21感受态菌株后获得稳定表达的工程菌株;初步探索优化了重组pcoldⅡ-hsBAFF-DT388的表达条件、包涵体提取步骤及蛋白初步纯化工艺;获得了具有靶向B细胞活性的重组蛋白,为其在B细胞恶性肿瘤及自身免疫性疾病治疗中的应用研究奠定了基础。
[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.

【学位授予单位】：济南大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392

【参考文献】