本文關(guān)鍵詞：廣州管圓線蟲半乳糖凝集素-1基因的克隆、表達(dá)和凝集反應(yīng)　出處：《中國寄生蟲學(xué)與寄生蟲病雜志》2017年01期 　論文類型：期刊論文

  更多相關(guān)文章： 廣州管圓線蟲 半乳糖凝集素- 原核表達(dá) 凝集反應(yīng)

【摘要】：目的克隆、表達(dá)廣州管圓線蟲(Angiostrongylus cantonensis)半乳糖凝集素-1(galectin-1)基因并分析其凝集反應(yīng)。方法用Swiss Model分析galectin-1的三維結(jié)構(gòu)。提取廣州管圓線蟲雌性成蟲總RNA,根據(jù)galectin-1基因編碼序列(Gen Bank登錄號為JN133961.1)設(shè)計(jì)引物,RT-PCR擴(kuò)增目的基因,亞克隆至p ColdⅢ質(zhì)粒,轉(zhuǎn)化大腸埃希菌(Escherichia coli)BL21,取重組質(zhì)粒進(jìn)行雙酶切、PCR鑒定和測序。陽性質(zhì)粒經(jīng)0.1 mmol/L異丙基-β-D-硫代半乳糖苷(IPTG)誘導(dǎo)表達(dá),鎳柱親和層析法純化galectin-1重組表達(dá)蛋白后,十二烷基硫酸鈉-聚丙烯酰胺凝膠電泳(SDS-PAGE)分析重組蛋白表達(dá)情況。以廣州管圓線蟲全蟲免疫的小鼠血清為一抗,蛋白質(zhì)印跡(Western blotting)驗(yàn)證目的蛋白。將重組蛋白10倍稀釋為5.55×10~(-1)~5.55×10~(-5)ng/μl,顯微鏡觀察其與新鮮的ICR小鼠血液中紅細(xì)胞的凝集反應(yīng)。結(jié)果 Swiss Model分析結(jié)果顯示,galectin-1以非二聚體形式發(fā)揮作用。galectin-1基因RT-PCR擴(kuò)增產(chǎn)物約為850 bp,與預(yù)期結(jié)果一致。經(jīng)雙酶切、PCR和測序結(jié)果顯示,重組質(zhì)粒p ColdⅢ-galectin-1構(gòu)建成功。SDS-PAGE結(jié)果顯示,經(jīng)IPTG誘導(dǎo)獲得相對分子質(zhì)量約36 000的可溶性重組融合蛋白。Western blotting分析結(jié)果顯示,galectin-1蛋白能被全蟲免疫的小鼠血清識別。凝集反應(yīng)結(jié)果顯示,galectin-1蛋白與小鼠紅細(xì)胞發(fā)生明顯凝集反應(yīng)的最低濃度為5.55×10~(-4)ng/μl。結(jié)論克隆的galectin-1基因能在p ColdⅢ原核表達(dá)系統(tǒng)中呈可溶性表達(dá),并與小鼠紅細(xì)胞發(fā)生凝集反應(yīng)。
[Abstract]:Objective cloning. Expression of galactose agglutinin-1 galectin-1 (Angiostrongylus cantonensis). Methods the three-dimensional structure of galectin-1 was analyzed by Swiss Model. The total RNA of female adult of Angiostrongylus cantonensis was extracted. Primers were designed to amplify the target gene by RT-PCR according to the galectin-1 gene encoding sequence Gen Bank accession number is JN133961.1). The recombinant plasmid was subcloned into p Cold 鈪，

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