發(fā)布時間：2018-06-03 00:24

  本文選題：ATF3 + 融合蛋白��； 參考：《南京醫(yī)科大學》2009年碩士論文

【摘要】：第一部分大鼠ATF3基因原核表達質(zhì)粒的構(gòu)建及其在大腸桿菌中的表達與純化 目的:構(gòu)建包含大鼠激活轉(zhuǎn)錄因子3(activating transcription factor 3,ATF3)基因不同長度片段的3個重組原核表達質(zhì)粒,并在大腸桿菌中表達。方法:PCR擴增出大鼠ATF3基因及其截短片段,克隆至原核表達載體pGEX-4T-1,重組質(zhì)粒pGEX-4T-1/ATF3, pGEX-4T-1/ATF3(129-546bp)和pGEX-4T-1/ATF3 (237-546bp)經(jīng)酶切鑒定、核酸序列測定后轉(zhuǎn)化大腸桿菌(E.coli)BL21和rosetta,異丙基-β-D-硫代半乳糖苷(isopropylβ-D-thiogalactopyranoside,IPTG)誘導(dǎo)表達。通過SDS-PAGE,Western blot進行分析和鑒定,并且經(jīng)過谷胱苷肽瓊脂糖的層析柱層析純化。結(jié)果:經(jīng)酶切鑒定和核酸序列測定證實重組質(zhì)粒構(gòu)建正確。經(jīng)過IPTG誘導(dǎo)以后,三個重組質(zhì)粒在SDS-PAGE均未見到預(yù)期的目的條帶,但是Western blot結(jié)果表明,質(zhì)粒pGEX-4T-1/ATF3能在BL21中表達分子量為48KD的融合蛋白。融合蛋白用谷胱苷肽層析柱純化,SDS-PAGE顯示,純化蛋白純度較低。結(jié)論:成功構(gòu)建了包含ATF3不同長度片段的3個重組原核表達質(zhì)粒,其中pGEX-4T-1/ATF3能夠在宿主菌BL21中正確表達。 第二部分抗大鼠ATF3單克隆抗體的制備與鑒定 目的:制備針對大鼠激活轉(zhuǎn)錄因子3(activating transcription factor 3,ATF3)蛋白的單克隆抗體。方法:根據(jù)軟件合成偶聯(lián)有匙孔絨血藍蛋白(key hole limpet hemocyanin,KLH)的ATF3第168-181位氨基酸多肽,免疫Balb/c小鼠,體外將骨髓瘤細胞SP2/0和小鼠脾細胞融合,ELISA法篩選出陽性克隆,經(jīng)有限稀釋法以及三次亞克隆后篩選出能分泌抗ATF3蛋白第168-181位氨基酸多肽單克隆抗體的雜交瘤細胞,快速定性試紙法對抗體的類型進行鑒定,并且通過Western blot鑒定此單克隆抗體的特異性。結(jié)果:經(jīng)過三次亞克隆后得到一株能穩(wěn)定分泌抗大鼠ATF3蛋白的單克隆抗體。快速定性試紙法鑒定此單克隆抗體的亞型為IgG1,輕鏈為κ鏈。Western blot結(jié)果顯示這株單抗能夠特異性識別細胞內(nèi)以及宿主菌BL21表達的ATF3蛋白。結(jié)論:成功制備了一株抗大鼠ATF3蛋白的單克隆抗體,為進一步研究ATF3的生物學功能提供了有用的實驗工具。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2009
【分類號】：R392

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