本文關(guān)鍵詞：金黃色葡萄球菌β-溶血素的表達、純化及中和性抗體的制備　出處：《軍事醫(yī)學》2016年02期 　論文類型：期刊論文

  更多相關(guān)文章： 葡萄球菌 金黃色 β-溶血素 溶血活性 抗體制備

【摘要】：目的利用大腸桿菌重組表達金黃色葡萄球菌外分泌毒素β-溶血素(β-hemolysin,Hlb)及其突變體HlbH-149-N,檢測其生物學活性,并制備其功能性抗體,探討Hlb蛋白在金葡菌感染中的生物學意義。方法以金葡菌NCTC-8325基因組為模板,利用PCR技術(shù)擴增目的基因hlb,進一步構(gòu)建重組表達載體p ET28a-hlb,并轉(zhuǎn)至大腸桿菌BL21(DE3),利用點突變技術(shù)構(gòu)建重組表達載體p ET28a-hlbH-149-N,經(jīng)IPTG誘導表達目的蛋白,通過Ni2+柱親和純化獲得Hlb蛋白及突變體HlbH-149-N蛋白;通過溶血實驗檢測Hlb及突變體HlbH-149-N蛋白的生物學活性,并制備Hlb特異性抗體,檢測抗體的中和活性。結(jié)果獲得了純度較高的重組Hlb蛋白和突變體HlbH-149-N蛋白,溶血活性實驗結(jié)果表明,重組Hlb蛋白能夠裂解綿羊紅細胞,而突變體HlbH-149-N蛋白不能裂解綿羊紅細胞;不同物種紅細胞對Hlb敏感性不同;制備了抗Hlb蛋白的多克隆抗體(anti-Hlb),ELISA和Western印跡實驗結(jié)果表明,anti-Hlb能特異結(jié)合Hlb蛋白和突變體HlbH-149-N蛋白,同時發(fā)現(xiàn),anti-Hlb可有效抑制Hlb對綿羊紅細胞的裂解作用。結(jié)論獲得了具有良好溶血活性的重組Hlb蛋白以及無溶血活性的突變體HlbH-149-N蛋白,并制備了具有中和活性的特異性抗體,為深入研究Hlb在金葡菌致病過程中的作用奠定了基礎(chǔ)。
[Abstract]:Objective to detect the biological activity of Staphylococcus aureus exotoxin -hemolysin (Hlb) and its mutant HlbH-149-N by recombinant E.coli, and to prepare its functional antibody, and to explore the biological significance of Hlb protein in Staphylococcus aureus infection. Methods Staphylococcus aureus genomic NCTC-8325 as template, HLB amplification of the target gene by PCR technology to construct the recombinant expression vector p ET28a-hlb, and transferred to E.coli BL21 (DE3), using ET28a-hlbH-149-N technology to construct the recombinant expression vector p mutation induced by IPTG protein expression, Hlb protein and mutant HlbH-149-N protein purified by Ni2+ affinity; biological activity by hemolysis test to detect the expression of Hlb and mutant HlbH-149-N protein, and preparation of Hlb antibody, neutralizing antibody. Results the recombinant Hlb protein and mutant HlbH-149-N protein with high purity, hemolytic activity test results showed that the recombinant Hlb protein could lyse sheep red blood cells, while HlbH-149-N mutant protein can cleavage of sheep red blood cells; erythrocytes of different species in different sensitivity to Hlb; polyclonal antibody against Hlb protein was prepared (anti-Hlb), and ELISA Western blot results showed that anti-Hlb can bind Hlb protein and mutant HlbH-149-N protein, also found that anti-Hlb can effectively inhibit the lysis of sheep erythrocytes Hlb. Conclusion a recombinant Hlb protein with good hemolytic activity and a HlbH-149-N protein without hemolytic activity were obtained, and a specific antibody with neutralization activity was prepared, which laid a foundation for further research on the role of Hlb in the pathogenesis of Staphylococcus aureus.
【作者單位】： 軍事醫(yī)學科學院基礎(chǔ)醫(yī)學研究所;
【基金】：國家自然科學基金面上資助項目(31370170,31170074)
【分類號】：R392
【正文快照】： 金黃色葡萄球菌(Staphylococcus aureus,金葡菌)是一種自然界中廣泛存在的革蘭陽性菌,可引起人和動物的局部化膿性感染、肺炎、偽膜性腸炎、心包炎以及敗血癥、膿毒敗血癥等,同時也是造成人類食物中毒的常見致病菌之一[1]。金葡菌主要通過產(chǎn)生多種致病因子而致病,這些致病因子

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