| 重组人颗粒溶素的纯化及其单克隆抗体的制备 |
| Alternative Title | The purification of recombinated granulysin and the preparation of its monoclonal antibody
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| 李新立 |
| Thesis Advisor | 居军
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| 2010-05-13
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| Degree Grantor | 兰州大学
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| Place of Conferral | 兰州
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| Degree Name | 硕士
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| Keyword | 颗粒溶素
纯化
单克隆抗体
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| Abstract | 目的：纯化原核表达的重组人颗粒溶素（GNLY），并制备其单克隆抗体（mAb），为GNLY
相关疾病的诊断提供一种新指标。
方法：Ni离子亲和层析法纯化重组人GNLY，用其免疫BALB/c小鼠，采用常规杂交瘤
技术制备相应mAb。间接ELISA法测杂交瘤细胞培养上清和腹水mAb的效价，ELISA
相加试验测定抗原表位，硫氰酸盐洗脱法测定相对亲和力指数，并行Ig亚类、特异性
及杂交瘤细胞分泌mAb的稳定性检测。
结果：纯化的重组GNLY融合蛋白，其纯度和含量分别为95%和0.8g/L。共筛选出能稳定分泌抗人GNLY mAb的杂交瘤细胞4株，分别命名为5E5、5G7、6C8和9C6。4株杂交瘤细胞培养上清及腹水中mAb的效价分别为1:100～1:3200和（0.2～8）×10-4。5E5杂交瘤细胞株分泌mAb的Ig亚类为IgM，其余3株mAb为IgG1。4株杂交瘤细胞体外连续培养2个月及液氮冻存6个月后复苏，分泌mAb的水平仍能达到之前的水平。4株mAb的相对亲和力指数为1.5～3.0mol/L；5G7、6C8和9C6 3株杂交瘤细胞株分泌mAb的相加指数（AI）<50%，而5E5分泌mAb与上述3株细胞分泌mAb的AI >50%。4株纯化后的mAb与GNLY抗原有较高的吸光度值，而与其他包被的抗原不反应。
结论：利用Ni离子亲和层析法获得可溶性重组GNLY；通过杂交瘤技术，获得4株能分泌特异性强、效价高的GNLY mAb杂交瘤细胞株。 |
| Other Abstract | Objective:To purify recombinated GNLY which was expressed by prokaryotic expression system,and to prepare the mAb against it.The mAb against GNLY will provide a new marker for GNLY related disease diagnosis.
Methods:The purification of recombinant human GNLY by Ni-ion affinity chromatography.Employing the recombinated GNLY fusion protein immuned BALB/c mice,using conventional hybridization technique to prepare its corresponding mAb.After purification of mAb,identified the mAb and hybridoma cells.Indirect ELISA to measure the titer of hybridoma supernatants and ascites mAb, ELISA determination of epitope,determination of relative affinity index by thiocyanate elution.And detected Ig sub-class,specificity and stability of hybridomas secreting mAb.Results:Purified soluble recombinant fusion protein GNLY was obtained by Ni-ion affinity chromatography,its purity and content were 95% and 0.8g/L, respectively.Four hybridoma cell lines can stable secret mAb aganist GNLY,named as 5E5, 5G7, 6C8 and 9C6.Their neutralizing titers were among 1:100～1:3200 and (0.1～8) ×10-4 in supernatant and ascites.The Ig sub-class of mAb secreted by 5E5 hybridoma cell was IgM, mAbs secreted by the other three hybridoma cells were all IgG1.Four mAbs continuous culture two months later in vitro and liquid nitrogen frozen six months recovery,the level of secretion of mAb able to reach the level before.Four mAb relative affinity index for the 1.5～3.0mol/L; 5G7,6C8 and 9C6 three mAbs strains secreting mAb of the additive index (AI) <50%,while the 5E5 mAb secreting cells with the three mAb of the additive index (AI)> 50%.Indirect ELISA analysis of the specificity of mAbs,four purified mAbs had higher aborbance values with GNLY,while did not respond with the people Gzms-A, Gzms-B,IFN-γ and TNF-α.
Conclusion:The soluble recombinant GNLY was purified by Ni-ion affinity chromatography. Using conventional hybridization technique,obtained four hybridoma cell lines which can stable secrete specific,high titer of mAb against GNLY. |
| URL | 查看原文
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| Language | 中文
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| Document Type | 学位论文
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| Identifier | https://ir.lzu.edu.cn/handle/262010/222176
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| Collection | 学院待认领
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| Affiliation | 临床医学院
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Recommended Citation
GB/T 7714 |
李新立. 重组人颗粒溶素的纯化及其单克隆抗体的制备[D]. 兰州. 兰州大学,2010.
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