CN116240221B - 噬菌体辅助自环化环状rna进化体系 - Google Patents
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Abstract
本发明公开了一种辅助自环化环状RNA进化噬菌体的自环化gIII基因,其特征在于其序列如SEQ ID No.6所示。还公开了辅助自环化环状RNA进化噬菌体及噬菌体辅助自环化环状RNA进化体系。本发明的噬菌体辅助自环化RNA的进化体系,诱变质粒MP6转化到宿主菌株中,使用M13噬菌体侵染宿主菌株,诱变质粒不断突变噬菌体基因组。高活性的自环化序列变体通过RNA环化使得pIII蛋白的N端与C端重组成完整的pIII蛋白;而低活性的自环化序列变体不能进行RNA环化,无法表达出有活性的pIII蛋白。新一代噬菌体基因组中携带高活性的自环化序列变体,进而进行下一步的侵染和突变进化,从而获得高活性自环化RNA。
Description
技术领域
本发明涉及一种噬菌体辅助自环化环状RNA进化体系,属于定向进化技术领域。
背景技术
环状RNA是一个共价闭合环状的RNA分子,具有稳定性好、翻译效率高和免疫原性低等特点,成为基因表达、核酸药物和疫苗的重要载体工具。体外RNA环化主要依赖于I型内含子的自剪切或者真核生物细胞内的自剪接;体外RNA环化依赖于T4 RNA连接酶或者I型内含子的自剪切。因此,I型内含子的自剪切在RNA环化上具有重要的作用。但是,现有的I型内含子的环化效率并不高,只有1-10%左右,极大地限制了环状RNA的大规模应用。
定向进化是近些年来兴起的生物技术,利用实验室规模的进化系统,可以在数月的时间内获得自然界中需要数十亿年的进化结果,是一项使用的蛋白或RNA改造技术。正因如此,定向进化技术获得了2018年的诺贝尔化学奖。噬菌体辅助菌株进化技术,又称为PACE技术(phage assisted continuous evolution),是哈佛大学David R.Liu于2011年开发的一种基于噬菌体的酶进化技术。这种技术的核心是将酶的活性与噬菌体的侵染能力进行偶联,即酶的活性越强,噬菌体的效价越高。再利用随机诱变的方法在进化过程不断的产生酶突变体,酶的基因整合到了噬菌体的基因组中,从而实现基因组上携带越高活性酶基因能够包装出更高活性噬菌体的目的。最终利用此方法筛选出高活性的酶变体。PACE具有操作简单、成本低廉和进化速度快等优点,因此广泛应用在各种酶进化的领域。但目前没有将PACE用于自环化RNA进化的报道。
发明内容
本发明的目的是提供一种噬菌体辅助自环化环状RNA进化体系,通过噬菌体定向进化的方式,获得高活性自环化RNA。
本发明采用的技术方案为:
一种辅助自环化环状RNA进化噬菌体的自环化gIII基因,其特征在于其序列如SEQIDNo.6所示。
本发明还公开了一种辅助自环化环状RNA进化噬菌体,其特征在于:所述噬菌体的pIII蛋白被拆分成C端和N端,所述pIII蛋白C端编码基因与pIII蛋白N端编码基因之间通过RBS序列连接,所述pIII蛋白的编码基因两端分别连有自环化序列。
优选的,所述pIII蛋白编码基因为自环化序列1-pIII蛋白C端编码基因-PBS-pIII蛋白N端编码基因-自环化序列2,其中自环化序列1如SEQ ID No.4所示,自环化序列2如SEQIDNo.5所示。
优选的,所述噬菌体中的gIII基因为SEQ ID No.6所示的序列。
优选的,所述噬菌体的基因组序列如SEQ ID No.7所示。
本发明还公开了一种噬菌体辅助自环化环状RNA进化体系,其特征在于包括:
一诱变质粒;
一上述的噬菌体;
一宿主菌。
优选的,所述诱变质粒为MP6质粒。
优选的,所述宿主菌含有F质粒,优选为大肠杆菌。
本发明的噬菌体辅助自环化RNA的进化体系,包含一个宿主菌株、一个基因组中自环化序列的M13噬菌体、一个诱变质粒。诱变质粒MP6转化到宿主菌株中,使用M13噬菌体侵染宿主菌株,诱变质粒不断突变噬菌体基因组。高活性的自环化序列变体通过RNA环化使得pIII蛋白的N端与C端重组成完整的pIII蛋白;而低活性的自环化序列变体不能进行RNA环化,无法表达出有活性的pIII蛋白。新一代噬菌体基因组中携带高活性的自环化序列变体,进而进行下一步的侵染和突变进化。利用这个方法,进化池中的噬菌体逐渐被基因组中含高活性自环化序列变体的子代噬菌体所替代,分离获得子代噬菌体即可获得高活性自环化RNA。
附图说明
图1完整pIII表达与自环化RNA序列活性偶联机制图。
图2进化过程中噬菌体效价变化。
图3环化效率检测示意图。
图4进化前后环化效率比较。
具体实施方式
下面结合实施例对本发明作进一步的说明,但实施例的描述不对本发明的保护范围产生任何限制。
除非另有定义,本文所使用的所有的技术术语和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同,本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
下列实施例中所用的物质或仪器,如果未进行特殊说明的话,均可以从常规的商用渠道获取。
实施例1噬菌体辅助自环化环状RNA进化方法的设计。
在本实施例中,我们设计了噬菌体辅助内含肽进化方法。为了将自环化序列的活性与噬菌体的增殖能力偶联,我们将噬菌体的pIII蛋白拆分成pIII-N(氨基酸序列见SEQID NO:1)和pIII-C(氨基酸序列见SEQ ID NO:2),将pIII-C置于pIII-N的前面,两者之间用RBS(DNA序列见SEQ ID NO:3)相连。再将自环化序列插入到重组pIII基因的上游和下游(DNA序列见SEQ ID NO:4、SEQ ID NO:5和SEQ ID NO:6)。示意图见图1,当突变后的自环化序列活性低时,无法生成环状pIII RNA,因此没有pIII蛋白的表达,无法包装出有侵染活性的子代噬菌体,在进化过程中处于劣势。但突变后的自环化序列活性高时,高效生成环状pIII RNA,pIII蛋白的表达水平升高,包装出有侵染活性的子代噬菌体,在进化过程中处于优势(见示意图2)。诱变质粒我们使用了MP6质粒(addgene#69669)。
实施例2噬菌体辅助自环化RNA自动进化流程
在本实施例中,我们公布了噬菌体辅助内含肽进化流程:
(1)进化菌株的制备。S1030大肠杆菌菌株中转入和诱变质粒MP6,含2g/L葡萄糖的50mg/L链霉素和氯霉素双抗性LB固体培养基中进行过夜培养,挑取单克隆到含2g/L葡萄糖的50mg/L链霉素和氯霉素双抗性LB液体培养基中培养至OD值在0.6左右。
(2)初代噬菌体的制备。构建好的M13基因组质粒(DNA序列见SEQ ID NO:7)转入到含pJC175e(Addgene#79219)的S1030菌株中,使用双层琼脂糖平板法分离M13噬菌体噬菌斑,挑取单个噬菌体到进化菌株中,培养后进行测序验证。
(3)低活性噬菌体进化。将获得的噬菌体按照1:1000侵染进化菌株,当自环化序列变体活性较低时,生成的pIII蛋白少,组装出来的有侵染活性的M13噬菌体少。通过加入50mM L-阿拉伯糖诱导诱变质粒中诱变基因的表达,产生自环化序列变体。培养6h后,取上清加入等体积的含pJC175e的S1030菌株中,继续培养6h。离心取上清,作为下一代噬菌体。
(4)高活性噬菌体进化。将获得的噬菌体按照1:1000侵染宿主菌,当自环化序列变体活性较高时,生成的pIII蛋白多,组装出来的有侵染活性的M13噬菌体多。通过加入50mML-阿拉伯糖诱导诱变质粒中诱变基因的表达,产生自环化序列突变体。突变后高活性自环化序列的噬菌体基因组侵染宿主菌后会组装更多的噬菌体,在进化池中占据比例优势。突变后低活性自环化序列的噬菌体基因组侵染宿主菌后会组装更少的噬菌体,在进化池中占据比例劣势。从而实现高活性内含肽的定向进化。
(5)进化平衡。当内含肽活性达到一定阈值,与完整pIII的表达解偶联。即自环化序列的活性变化无法大幅度影响pIII的表达,进化池中的噬菌体效价在短时间内即可达到效价阈值时,进化进入平衡阶段,进化结束。
(6)环化效率检测。环化效率检测的示意图如图3所示。提取进化后的宿主菌株RNA,利用RT-qPCR检测gIII基因的环化效率,结果如图4所示。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (6)
1.一种辅助自环化环状RNA进化噬菌体的自环化gIII基因,其特征在于其序列如SEQID No.6所示。
2.一种辅助自环化环状RNA进化噬菌体,其特征在于:所述噬菌体的基因组序列如SEQID No.7所示。
3.一种噬菌体辅助自环化环状RNA进化体系,其特征在于包括:
一诱变质粒;
一权利要求2所述的噬菌体;
一宿主菌。
4.根据权利要求3所述的噬菌体辅助自环化环状RNA进化体系,其特征在于:所述诱变质粒为MP6质粒。
5.根据权利要求3或4所述的噬菌体辅助自环化环状RNA进化体系,其特征在于:所述宿主菌含有F质粒。
6.根据权利要求3或4所述的噬菌体辅助自环化环状RNA进化体系,其特征在于:所述宿主菌为大肠杆菌。
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