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蓝白斑筛选,一种很炫的东西

分子克隆实验是分子生物学中常见的一个实验。感兴趣的基因插入到表达载体中,而后转化进入宿主内,但是并非所有质粒都包含目的基因,怎样筛查出包含有目的基因的菌落呢,这样的菌落也就是我们所说的阳性克隆。早期的科学家发展了蓝白斑筛选这种狂拽炫酷的东西,只需通过菌落的颜色,我们能够轻松找出阳性克隆。今天,小编就为大家简单介绍下隐含在这种酷炫技术背后的秘密。

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蓝白斑筛选的原理

首先我们需要知道是什么让菌落显现出蓝白两色的。其实呢,白色吧,就是菌落本身的颜色,汗。是真的,看一下平板就知道了,菌落就是白色的。那蓝色呢?蓝色确实是比较神奇的。蓝色其实是β-半乳糖苷酶(β-galactosidase enzyme)分解X-gal产生的产物的颜色。X-gal?什么鬼。它的全名是5-溴-4-氯-3-吲哚-β-D-半乳糖苷(不要试图记住全名了,为了记住它,小编已经阵亡)。产物又是啥,产物是一种叫做5-溴-4-氯靛兰的蓝色东东。

大肠杆菌本身是能够表达β-半乳糖苷酶的(乳酸存在的前提下),所以正常的大肠杆菌无法进行蓝白斑筛选,有乳酸存在的条件下,所有菌落都会分解X-gal产生蓝色。能够用来进行蓝白斑筛选的菌株,我们管之叫作为β-半乳糖苷酶缺陷型菌株。缺陷就意味这不正常。也就是说能够进行蓝白斑筛选的菌株内的β-半乳糖苷酶的表达是不正常的(小编你好啰嗦)。β-半乳糖苷酶可以拆分成两个部分,N端和C端。β-半乳糖苷酶缺陷型菌株的基因组中含有表达β-半乳糖苷酶C端的基因,而N端(一个146个氨基酸的短肽,即α肽链)的基因被安放到了表达的载体中。N端基因经过改造,中间插入多克隆位点。这段经过改造的N端基因被称为lacZ基因(见图1)。

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图1 适用于蓝白斑筛选表达载体

LacZ基因是神一般的存在。神在何处,听小编慢慢道来。缺陷株基因组无法单独编码有活性的β-半乳糖苷酶,但当菌体中含有带lacZ的质粒后,质粒lacZ基因编码的α肽链(酶的N端)和菌株基因组表达的β-半乳糖苷酶的C端互补,具有与完整β-半乳糖苷酶相同的作用,具有分解X-gal生成蓝色物质的能力。这种现象也叫α-互补。操作中,添加IPTG(异丙基硫代-β-D-半乳糖苷)以激活lacZ中的β-半乳糖苷酶的启动子,在含有X-gal的固体平板培养基中菌落就会呈现蓝色。但是当MCS位点上插入DNA片段时,lacZ基因就失活了,它突变了,表达出来的东西连它的兄弟,β-半乳糖苷酶C端都不认识,没法完成α互补,β-半乳糖苷酶失活,无法分解X-gal,因此菌落就是白色的。好神奇啊?那么有看官要问了,β-半乳糖苷酶缺陷型菌株都有哪些?与之配套的表达质粒又有哪些呢?小编在这里简单列举几个常用的:β-半乳糖苷酶缺陷型菌株有DH5alpha、TOP10等;适用于蓝白斑筛选的载体有Puc系列(Puc18 ,Puc19)、M13mp系列等。

蓝白斑筛选的不足

经过上面啰哩叭嗦的解释,大家应该明白蓝白斑筛选的原理了。但是“再炫的技术,也会有自己的软肋”,这条至理名言同样适用于蓝白斑筛选 (这是哪里的至理名言,小编你瞎编的吧!? 额,是的)。蓝白斑筛选的不足之处在于,如果在表达载体内插入的基因太短同时没有破坏掉lacZ的阅读框,表达的α肽链可能还是有些活性的,菌落仍然会显蓝色。这就产生了传说中的假阴性。

最后解释下开放阅读框(open reading frame):开放阅读框是基因序列的一部分,包含一段可以编码蛋白的碱基序列。由于拥有特殊的起始密码子和直到可以从该段碱基序列产生合适大小蛋白才出现的终止密码子,该段碱基序列编码一个蛋白。(这句话完全来在百度百科,如有雷同,那也是来自百度百科)

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作者: 于浩然

本科及硕士毕业于天津大学,博士毕业于伦敦大学学院。现就职于浙江大学化学工程与生物工程学院,PI,博导。研究方向为蛋白质工程、生物催化剂、合成生物学等。



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