【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)果成功克隆了長約800bp的GeNuc編碼區(qū)并構(gòu)建了原核表達(dá)載體pET-28a(+)-GeNuc,測序結(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

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