小鼠B淋巴细胞活化刺激因子可溶性片段的酵母表达、纯化及生物学活性的初步鉴定

发布时间：2018-01-11 14:27

本文关键词：小鼠B淋巴细胞活化刺激因子可溶性片段的酵母表达、纯化及生物学活性的初步鉴定　出处：《暨南大学》2013年硕士论文　论文类型：学位论文

【摘要】：目的克隆小鼠B淋巴细胞活化刺激因子（B cell activating factor belonging tothe TNF family，BAFF）可溶性片段，表达并纯化出有生物学活性的小鼠可溶性（mouse soluble）msBAFF蛋白，为探讨小鼠BAFF可溶性蛋白作为B细胞杂交瘤生长促进剂和佐剂的潜在用途提供原料支持。方法分离BALB/c小鼠外周血单核细胞，提取总RNA；反转录后，以cDNA产物为模板扩增目的基因，将目的基因构建到克隆载体pMD18-T中，经转化DH5α感受态细胞后挑取阳性细菌抽提质粒进行酶切和测序鉴定。将测序鉴定为正确的质粒进行双酶切，回收目的片段，克隆至pPICZαA表达载体并转化DH5α感受态细胞，挑取阳性转化子提取质粒并酶切和测序鉴定；将鉴定正确的质粒导入巴斯德毕赤酵母GS115，使用Zeocin加压筛选高抗性的菌株；筛选到的阳性酵母工程菌进行甲醇诱导表达，表达产物以SDS-PAGE鉴定。依次使用硫酸铵沉淀及Q Sepharose XL阴离子交换柱纯化目的蛋白msBAFF。将纯化后的msBAFF与anti-IgM共刺激B淋巴细胞，使用CCK8试剂盒检测B淋巴细胞的增殖情况。将msBAFF与抗原共同免疫小鼠，检测小鼠免疫后的血清抗体效价，分析msBAFF对小鼠免疫后抗体产生的影响。结果琼脂糖凝胶电泳和基因测序结果显示PCR所得基因序列与预期相符，可知已克隆得到小鼠BAFF基因可溶性片段。酶切和测序鉴定结果显示目的DNA已正确插入到克隆和表达载体，SDS-PAGE结果显示酵母表达的目的蛋白分子量约20kDa，与预期相符，表明小鼠BAFF蛋白可溶性片段获得表达；阴离子纯化柱洗脱产物SDS-PAGE检测结果显示，0.3M NaCl可较好的将目的蛋白msBAFF从柱上洗脱下来。体外B细胞刺激实验中，CCK8法结果显示msBAFF与anti-IgM共刺激组的OD值明显高于对照组，表明该msBAFF蛋白具有促B细胞增殖的生物学活性。msBAFF与抗原共免疫小鼠一次、两次和三次后，ELISA检测其血清抗体效价，结果显示血清效价随着免疫次数的增加而增加，但是较高浓度的msBAFF对小鼠抗体产生具有抑制作用。结论本实验克隆得到了小鼠BAFF可溶性片段基因，经酵母表达并纯化后得到了较纯的小鼠BAFF可溶性多肽片段。体外B细胞刺激实验中，该蛋白可显著促进B细胞的增殖，说明该小鼠BAFF可溶性片段具有良好的促B细胞增殖活性。初步体内试验表明，较高浓度的msBAFF对小鼠抗体产生有抑制作用，其具体机理有待探讨。
[Abstract]:Objective to clone mouse B lymphocyte activation factor (B cell activating factor stimulated belonging tothe TNF family, BAFF) soluble fragment, expression and purification of mouse soluble bioactive msBAFF protein (mouse soluble), to investigate the soluble BAFF protein in mice as B cell hybridoma growth promoter and potential use of adjuvant and providing material support.
Methods blood mononuclear cells isolated from BALB/c mice peripheral, extraction of total RNA; after reverse transcription, cDNA products as template. The target gene to construct the gene cloned into vector pMD18-T and transformed to competent cells after DH5 alpha positive bacterial plasmids were identified by restriction enzyme digestion and sequencing. The sequencing is correct plasmids were digested, the target fragment was reclaimed and cloned into pPICZ expression vector and transformed into DH5 alpha A alpha competent cells, then the positive transformant Plasmid Extraction and enzyme digestion and sequencing; the identification of the correct plasmid into Pasteur Pichia pastoris GS115, using the Zeocin pressure screening high resistant strains; methanol induced expression of yeast the engineering bacteria screened and identified by SDS-PAGE. The expressed product by using ammonium sulfate precipitation and Q Sepharose XL anion exchange column to purify the protein msBAFF. msBAFF and anti-IgM will be purified The B lymphocytes were co stimulated. The proliferation of B lymphocytes was detected by CCK8 kit. MsBAFF and antigen were co immunized to mice, and the serum titer of mice after immunization was detected, and the effect of msBAFF on the antibody production after immunization in mice was analyzed.
The results of agarose gel electrophoresis and gene sequencing showed the PCR gene sequence consistent with expectations, we have cloned the mouse BAFF gene fragment. The soluble enzyme digestion and sequencing results showed that DNA has been correctly inserted into cloning and expression vector of target protein SDS-PAGE showed that yeast expression amount of about 20kDa, consistent with expectations that mouse BAFF protein expression and purification of soluble fragments obtained; anion column elution products SDS-PAGE assay showed that 0.3M NaCl could be the target protein msBAFF eluted from the column. B cells stimulated in vitro experiments, CCK8 results showed that the OD value of msBAFF and anti-IgM co stimulation group was significantly higher than the control group, indicating that the msBAFF protein possesses biological the activity of.MsBAFF antigen and B cell proliferative co immunized mice once, two times and three times, ELISA detected the serum antibody titer, the results show The serum titer increased with the increase of the number of immunization, but the high concentration of msBAFF inhibited the antibody production in mice.
Conclusion the cloned mouse BAFF gene by yeast soluble fragment, obtained after expression and purification of mouse BAFF soluble peptide fragments. Compared with pure B cells in vitro experiment, the protein can significantly promote the proliferation of B cells, indicating that the mouse soluble BAFF fragment with B cell proliferative activity. Results in test of high concentration of msBAFF has inhibitory effect on mouse antibody production, to evaluate the specific mechanism.

【学位授予单位】：暨南大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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