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

【摘要】：第一部分大鼠ATF3基因原核表達(dá)質(zhì)粒的構(gòu)建及其在大腸桿菌中的表達(dá)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(zhǎng)度片段的3個(gè)重組原核表達(dá)質(zhì)粒,并在大腸桿菌中表達(dá)。方法:PCR擴(kuò)增出大鼠ATF3基因及其截短片段,克隆至原核表達(dá)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(cè)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(dá)。通過(guò)SDS-PAGE,Western blot進(jìn)行分析和鑒定,并且經(jīng)過(guò)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(cè)定證實(shí)重組質(zhì)粒構(gòu)建正確。經(jīng)過(guò)IPTG誘導(dǎo)以后,三個(gè)重組質(zhì)粒在SDS-PAGE均未見(jiàn)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(dá)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(zhǎng)度片段的3個(gè)重組原核表達(dá)質(zhì)粒,其中pGEX-4T-1/ATF3能夠在宿主菌BL21中正確表達(dá)。 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(duì)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(lán)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(xì)胞SP2/0和小鼠脾細(xì)胞融合,ELISA法篩選出陽(yáng)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(xì)胞,快速定性試紙法對(duì)抗體的類型進(jìn)行鑒定,并且通過(guò)Western blot鑒定此單克隆抗體的特異性。結(jié)果:經(jīng)過(guò)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�？焖俣ㄐ栽嚰埛ㄨb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(shí)別細(xì)胞內(nèi)以及宿主菌BL21表達(dá)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(jìn)一步研究ATF3的生物學(xué)功能提供了有用的實(shí)驗(yàn)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(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前3條

1 顧關(guān)云;蔣昱;;植物抗腫瘤藥物研究概況Ⅷ[J];現(xiàn)代藥物與臨床;2009年03期

2 喬春霞;沈倍奮;;重組多克隆抗體——一類新的治療制劑[J];中國(guó)免疫學(xué)雜志;2009年01期

3 李洪福;李永輝;王勇;魏娜;譚銀豐;張俊清;;高良姜化學(xué)成分及藥理活性的研究[J];中國(guó)實(shí)驗(yàn)方劑學(xué)雜志;2014年07期

相關(guān)會(huì)議論文 前1條

1 李洪福;李永輝;王勇;魏娜;譚銀豐;張俊清;;高良姜化學(xué)成分及藥理活性研究進(jìn)展[A];第六屆海南省科技論壇“醫(yī)藥科技創(chuàng)新與藥品質(zhì)量安全”專題論壇、海南省藥學(xué)會(huì)2012年學(xué)術(shù)年會(huì)論文集[C];2012年

相關(guān)博士學(xué)位論文 前4條

1 錢衛(wèi)珠;新型抗IgE人源化單克隆抗體的結(jié)構(gòu)與功能研究[D];第二軍醫(yī)大學(xué);2011年

2 呂俊;LINGO-1多克隆抗體的制備及其被動(dòng)免疫治療大鼠急性脊髓損傷的實(shí)驗(yàn)研究[D];南方醫(yī)科大學(xué);2010年

3 王海燕;9-甲氧基喜樹堿（MCPT）抗腫瘤作用及其機(jī)制研究[D];華中科技大學(xué);2013年

4 張俊清;姜科藥用植物益智Alpinia oxyphylla資源化學(xué)研究[D];南京中醫(yī)藥大學(xué);2013年

相關(guān)碩士學(xué)位論文 前1條

1 郭寶鳳;25-乙�；�-7，，8二去氫—升麻醇-3-木糖苷對(duì)HepG2的細(xì)胞毒性及其作用機(jī)制初探[D];北京中醫(yī)藥大學(xué);2011年

本文編號(hào)：1970744