大鼠ATF3基因克隆与表达及单克隆抗体的制备

发布时间：2018-06-03 00:24

本文选题：ATF3 + 融合蛋白　；参考：《南京医科大学》2009年硕士论文

【摘要】：第一部分大鼠ATF3基因原核表达质粒的构建及其在大肠杆菌中的表达与纯化目的:构建包含大鼠激活转录因子3(activating transcription factor 3,ATF3)基因不同长度片段的3个重组原核表达质粒,并在大肠杆菌中表达。方法:PCR扩增出大鼠ATF3基因及其截短片段,克隆至原核表达载体pGEX-4T-1,重组质粒pGEX-4T-1/ATF3, pGEX-4T-1/ATF3(129-546bp)和pGEX-4T-1/ATF3 (237-546bp)经酶切鉴定、核酸序列测定后转化大肠杆菌(E.coli)BL21和rosetta,异丙基-β-D-硫代半乳糖苷(isopropylβ-D-thiogalactopyranoside,IPTG)诱导表达。通过SDS-PAGE,Western blot进行分析和鉴定,并且经过谷胱苷肽琼脂糖的层析柱层析纯化。结果:经酶切鉴定和核酸序列测定证实重组质粒构建正确。经过IPTG诱导以后,三个重组质粒在SDS-PAGE均未见到预期的目的条带,但是Western blot结果表明,质粒pGEX-4T-1/ATF3能在BL21中表达分子量为48KD的融合蛋白。融合蛋白用谷胱苷肽层析柱纯化,SDS-PAGE显示,纯化蛋白纯度较低。结论:成功构建了包含ATF3不同长度片段的3个重组原核表达质粒,其中pGEX-4T-1/ATF3能够在宿主菌BL21中正确表达。第二部分抗大鼠ATF3单克隆抗体的制备与鉴定目的:制备针对大鼠激活转录因子3(activating transcription factor 3,ATF3)蛋白的单克隆抗体。方法:根据软件合成偶联有匙孔绒血蓝蛋白(key hole limpet hemocyanin,KLH)的ATF3第168-181位氨基酸多肽,免疫Balb/c小鼠,体外将骨髓瘤细胞SP2/0和小鼠脾细胞融合,ELISA法筛选出阳性克隆,经有限稀释法以及三次亚克隆后筛选出能分泌抗ATF3蛋白第168-181位氨基酸多肽单克隆抗体的杂交瘤细胞,快速定性试纸法对抗体的类型进行鉴定,并且通过Western blot鉴定此单克隆抗体的特异性。结果:经过三次亚克隆后得到一株能稳定分泌抗大鼠ATF3蛋白的单克隆抗体。快速定性试纸法鉴定此单克隆抗体的亚型为IgG1,轻链为κ链。Western blot结果显示这株单抗能够特异性识别细胞内以及宿主菌BL21表达的ATF3蛋白。结论:成功制备了一株抗大鼠ATF3蛋白的单克隆抗体,为进一步研究ATF3的生物学功能提供了有用的实验工具。
[Abstract]:The construction of prokaryotic expression plasmid of rat ATF3 gene and its expression and purification in Escherichia coli Aim: to construct three recombinant prokaryotic expression plasmids containing different length fragments of rat activated transcription factor 3(activating transcription factor 3 (ATF3) gene and express them in Escherichia coli. Methods the rat ATF3 gene and its truncated fragment were amplified and cloned into prokaryotic expression vector pGEX-4T-1. The recombinant plasmids pGEX-4T-1 / ATF3, pGEX-4T-1 / ATF3129-546bpand pGEX-4T-1/ATF3 237-546bpwere digested. The nucleic acid sequence was sequenced and transformed into E. coli E. coli BL21 and Rossetta- 尾 -thiogaltopyranoside IPTG. the expression was induced by isopropyl 尾 -D-thiogaltopyranoside IPTG. It was analyzed and identified by SDS-PAGEG blot and purified by glutathione agarose chromatography. Results: the recombinant plasmid was constructed correctly by restriction enzyme digestion and nucleic acid sequencing. After induction by IPTG, none of the three recombinant plasmids had the desired target bands in SDS-PAGE, but the Western blot results showed that the fusion protein with molecular weight of 48KD could be expressed in BL21 by plasmid pGEX-4T-1/ATF3. SDS-PAGE showed that the purified protein was of low purity. Conclusion: three recombinant prokaryotic expression plasmids containing different length fragments of ATF3 were successfully constructed, in which pGEX-4T-1/ATF3 could be correctly expressed in host strain BL21. The second part: preparation and identification of monoclonal antibody against rat ATF3 Aim: to prepare monoclonal antibodies against rat activated transcription factor 3(activating transcription factor 3 (ATF 3) protein. Methods: the amino acid peptide of ATF3 at position 168-181 was synthesized by software, and the positive clones were screened by fusion Elisa of myeloma cells SP2/0 and mouse spleen cells in vitro. The hybridoma cells secreting monoclonal antibodies against amino acid peptides at position 168-181 of ATF3 protein were screened by limited dilution method and three subclones, and the types of antibodies were identified by rapid qualitative test paper method. The specificity of the monoclonal antibody was identified by Western blot. Results: after three subclones, a monoclonal antibody which could secrete rat ATF3 protein stably was obtained. The monoclonal antibody was identified as IgG1 by rapid qualitative assay. The light chain was 魏 chain. Western blot showed that the monoclonal antibody could specifically recognize the ATF3 protein expressed in the cell and BL21 of the host strain. Conclusion: a monoclonal antibody against rat ATF3 protein was successfully prepared, which provides a useful experimental tool for further study on the biological function of ATF3.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2009
【分类号】：R392

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