【作者】 李婧；

【导师】 叶邦策；

【作者基本信息】 华东理工大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 许多细菌都能够合成并释放一种或多种信号分子,随着菌体密度的增加,信号分子的浓度也随之增加。当信号分子的浓度积累到一定阀值的时候,就会启动菌体中相关基因的表达,调控细菌的某些生物行为。在大部分革兰氏阴性致病菌中,信号分子的积累可以启动下游致病因子的表达。从苏云金芽孢杆菌Bt.4Q7中克隆得到的aiiA基因编码的AiiA蛋白可以降解群体感应信号分子,从而使得致病菌的致病性降低或者消失。AiiA蛋白的活性越高,对致病菌致病性的抑制作用也就越明显。本文通过PCR扩增,得到了aiiA4Q7、aiiA8010、aiiAWB4、aiiAWB12、aiiAT-HW3、aiiA6A3共6个aiiA同源基因；运用DNA shuffling分子进化技术,通过一步DNaseⅠ(Rnase Free)随机酶切、三轮无引物PCR、一轮有引物PCR过程,成功构建了aiiA基因突变库；以紫色色杆菌突变株CV026为报告菌,从中筛选得到了S29、S47、S21、S4、S36、S52、S7、S14、S63共9株具有较高AiiA蛋白酶活的突变株,并进一步得到了进化后的aiiAS29、aiiAS47、aiiAS21、aiiAS4、aiiAS36、aiiAS52、aiiAS7、aiiAS14、aiiAS63基因；aiiAS29和aiiAS36基因的测序结果完全相同,其余aiiA基因序列各不相同。制备了AiiAT-HW3、AiiA4Q7、AiiA8010、AiiAWB4/WB12、AiiAS29/S36、AiiAS47/S52、AiiAS21、AiiAS4、AiiAS7、AiiAS63共10个AiiA蛋白样品,通过测定各AiiA蛋白样品之间的相对酶活,得到了具有高酶活性的AiiA蛋白即AiiAs29／s36,其进化后的酶活性比原始AiiA4Q7提高了2.2倍,成功实现了对群体感应信号分子降解酶AiiA的分子进化。更多还原

【Abstract】 aiiA gene cloned from Bt.4Q7 (B. thuringiensis 4Q7) encodes an enzyme which inactivating QS (quorum sensing) signal molecules AHLs (N-acylhomoserine lactones) involved in regulation of divers biological functions especially the expression of pathogenic gene in the Gram negative bacteria. Phenotypic characterization could be abolished or reduced further more when the AHLs inactivated by AiiA with higher enzyme activity which was desired in our molecule evolution. Library of aiiA gene was constructed by DNA shuffling including steps DNase I digestion, three round PCR without primers and one round PCR with primers. Reporter strain CV026(Chromobacterium violaceum 026) was used for the high throughput screening. Nine mutant strains with high enzyme activity were abtained and the evolved aiiA genes were cloned and sequenced. The evolution of AiiA quorum sensing inhabitor was achieved as the activity of AiiAS29/S36 was increased to 2.2 times as the original AiiA4Q7 by the relatively enzyme activity assay of AiiA samples.更多还原