【摘要】：目的克隆、原核表達(dá)藍(lán)氏賈第鞭毛蟲(Giardia lamblia,賈第蟲)的胞外核酸酶編碼區(qū),并對(duì)其蛋白產(chǎn)物進(jìn)行活性鑒定。方法對(duì)賈第蟲胞外核酸酶(GeNuc)蛋白進(jìn)行生物信息學(xué)分析,根據(jù)分析結(jié)果以C2株賈第蟲基因組DNA為模板擴(kuò)增獲得GeNuc去信號(hào)肽段編碼區(qū)序列,雙酶切連入原核表達(dá)載體pET-28a(+),將酶切和測(cè)序驗(yàn)證正確的重組質(zhì)粒轉(zhuǎn)化E.coli Rosetta(DE3),經(jīng)IPTG誘導(dǎo)表達(dá)融合蛋白,SDS-PAGE及Western blot鑒定蛋白產(chǎn)物。Ni-NTA親和層析純化GeNuc蛋白,經(jīng)復(fù)性后驗(yàn)證其對(duì)質(zhì)粒DNA的水解能力。結(jié)果成功克隆了長(zhǎng)約800bp的GeNuc編碼區(qū)并構(gòu)建了原核表達(dá)載體pET-28a(+)-GeNuc,測(cè)序結(jié)果顯示C2株GeNuc序列與WB株相同;在大腸桿菌中誘導(dǎo)表達(dá)獲得了相對(duì)分子量約30.8kDa的融合蛋白;復(fù)性后的純化GeNuc蛋白具有降解雙鏈DNA的能力,但活性較商品化DNaseⅠ低。結(jié)論證明了GeNuc的存在,為GeNuc抗體的制備及賈第蟲致病機(jī)制的研究提供了實(shí)驗(yàn)材料。
[Abstract]:Objective to clone and express the extracellular nuclease coding region of Giardia lamblia (Giardia lamblia, in prokaryotic cells, and to identify the activity of its protein product. Methods the extracellular nuclease (GeNuc) protein of Giardia giardia was analyzed by bioinformatics analysis. According to the analysis results, the coding region of GeNuc designaling peptide was amplified by using genomic DNA of C2 strain Giardia as template. The recombinant plasmid was transformed into E.coli Rosetta (DE3) by restriction endonuclease digestion and sequencing, and the fusion protein was induced by IPTG. The protein products were identified by SDS-PAGE and Western blot. GeNuc protein was purified by Ni-NTA affinity chromatography and its hydrolysis ability to plasmid DNA was verified by renaturation. Results the GeNuc coding region of about 800bp was successfully cloned and the prokaryotic expression vector pET-28a ()-GeNuc, was constructed. The results showed that the GeNuc sequence of C2 strain was the same as that of WB strain. The fusion protein with relative molecular weight of about 30.8kDa was induced and expressed in E. coli, and the purified GeNuc protein after renaturation had the ability to degrade double-stranded DNA, but its activity was lower than that of commercial DNase I. Conclusion the existence of GeNuc is proved, which provides experimental materials for the preparation of GeNuc antibody and the study of pathogenic mechanism of Giardia.
【作者單位】： 華北理工大學(xué)附屬醫(yī)院檢驗(yàn)科;華北理工大學(xué)生命科學(xué)學(xué)院;
【基金】：國(guó)家自然科學(xué)基金(No.31471954) 河北省青年科學(xué)基金(No.C2012401039)聯(lián)合資助~~
【分類號(hào)】：R382.21

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 權(quán)彤彤;;膽汁內(nèi)查見藍(lán)氏賈第鞭毛蟲1例[J];西南國(guó)防醫(yī)藥;2009年06期

2 梁毅,曾美琴,吳曼秋;藍(lán)氏賈第鞭毛蟲膽道感染16例報(bào)告[J];新醫(yī)學(xué);1974年11期

3 王才中;;滾動(dòng)玻瓶培養(yǎng)藍(lán)氏賈第鞭毛蟲的評(píng)價(jià)[J];國(guó)外醫(yī)學(xué)(寄生蟲病分冊(cè));1984年01期

4 王又紅;藍(lán)氏賈第鞭毛蟲基因組序列研究[J];國(guó)外醫(yī)學(xué)(寄生蟲病分冊(cè));1999年03期

5 陳小寧,盧思奇,王f3星,王峰,李春榮,郝祥俊;藍(lán)氏賈第鞭毛蟲承德株分子生物學(xué)研究[J];承德醫(yī)學(xué)院學(xué)報(bào);2000年04期

6 盧思奇,李繼紅,王鳳云,張亞平,文建凡;藍(lán)氏賈第鞭毛蟲磷酸丙糖異構(gòu)酶基因種內(nèi)差異研究(英文)[J];Chinese Medical Journal;2002年05期

7 溫少芳;盧思奇;;藍(lán)氏賈第鞭毛蟲遺傳多樣性研究進(jìn)展[J];國(guó)際醫(yī)學(xué)寄生蟲病雜志;2006年01期

8 曹軍皓;陳真真;容東寧;李芳;;胸水檢出藍(lán)氏賈第鞭毛蟲一例[J];檢驗(yàn)醫(yī)學(xué);2006年05期

9 田喜鳳;衛(wèi)茹;楊志宏;盧思奇;;甲硝唑?qū)w外藍(lán)氏賈第鞭毛蟲形態(tài)結(jié)構(gòu)的損傷[J];中國(guó)寄生蟲學(xué)與寄生蟲病雜志;2006年05期

10 楊志宏;田喜鳳;衛(wèi)茹;;藍(lán)氏賈第鞭毛蟲的基因及基因型[J];熱帶病與寄生蟲學(xué);2007年01期

相關(guān)會(huì)議論文 前1條

1 王厚芳;柳杰;孫芾;;隱孢子蟲抗原和藍(lán)氏賈第鞭毛蟲抗原的快速檢測(cè)方法及臨床應(yīng)用[A];中華醫(yī)學(xué)會(huì)第八次全國(guó)檢驗(yàn)醫(yī)學(xué)學(xué)術(shù)會(huì)議暨中華醫(yī)學(xué)會(huì)檢驗(yàn)分會(huì)成立30周年慶典大會(huì)資料匯編[C];2009年

相關(guān)碩士學(xué)位論文 前1條

1 巨紅妹;藍(lán)氏賈第鞭毛蟲三種基因功能研究載體的構(gòu)建[D];大連大學(xué);2013年

，

本文編號(hào)：2473487