CN1145641A - 通过随机片段化和重装配进行的dna诱变 - Google Patents
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Abstract
描述了随机片段化后重装配DNA的方法,及其在体外、或体内用于核酸序列突变的应用。具体地讲,描述了生产编码突变蛋白质的核酸片段或多聚核苷酸的方法。本发明也涉及突变、杂合和选择的循环的方法,该方法使蛋白质在体内或体外发生定向分子进化。
Description
发明背景
发明领域
本发明涉及生产多核苷酸的方法,所说多核苷酸提供所需的表型和/或编码一种具有可选择的有利的预定特性的蛋白质。一方面,该方法可用于生产和选择编码突变蛋白质的核酸片段。
相关领域的描述
一种生物大分子例如蛋白质,DNA等的活性序列的复杂性已被称为其信息内容(“ IC”;5-9)。一种蛋白质的信息内容定义为活性蛋白对氨基酸的抗性,可以从用于描述一个具有相同功能的相关序列族所需要的不可变氨基酸(bits)的基本数目计数出(9,10)。对随机诱变敏感的蛋白质具有高信息内容。在1974年,制定该定义时,蛋白质的多样性仅表现为分类上的多样性。
分子生物学的发展例如分子文库已使我们可以鉴别更大量的可变碱基,甚至可以从随机文库中选择功能序列。大多数残基可以是变化的,但是通常不都是在同时发生变化,取决于上下文中的补体变化。所以一个含100个氨基酸的蛋白质不仅可能含有2000种不同的突变,而且可能有20100种的突变组合。
信息密度是单位序列长度上的信息含量。酶的活性位点倾向有高的信息密度。相反,酶的可变的连接点有低的信息密度(8)。
在模式文库中目前广泛应用的制造变异蛋白质的方法是易于产生错误的聚合酶链反应(11,12,19)和盒基因诱变(8,20,21,22,40,41,42),其中用合成的诱变寡核苷酸替代有待最佳化的特定区域。在两种情况下,在原始序列中一些位点周围产生了‘突变云’(4)。
易于产生错误的PCR使用了低精确性的多聚化条件以在一个长序列内导入低水平的随机点突变。利用易于产生错误的PCR对未知序列的片段的混合物进行诱变。但是,计算机模拟显示单独进行点诱变经常由于太慢而不能发生为连续序列演化所需要的模块变化。公开的易于产生错误的PCR方案不允许大于0.5-1.0kb的DNA片段进行增殖,从而限制了其在实际中的应用。此外易于产生错误的重复的循环导致中性突变积聚,这种积聚例如产生一个免疫源蛋白质。
在寡核苷酸定位诱变中,用合成的诱变寡核苷酸取代一个短序列。这种方法不会产生远缘突变的组合,因此不是组合的。相对于巨大的序列长度而言有限的文库大小是指使蛋白质最佳化不可避免地要进行许多个周期的选择。用合成的寡核苷酸进行诱变需要在每次选择周期之后对单个克隆进行测序,随后划分到族,任意地选择单一的族,将它减少到共有序列基元,重新合成该序列基元并重新插入到单个基因中,随后进行另一次选择。该方法构成了统计学上的峡口,该方法劳动强度大并且对于许多个周期的诱变是不实用的。
因此易于产生错误的PCR和寡核苷酸定位诱变适用于对序列进行精细调整的单个周期,当用于多个周期时很快受限制。
利用易于产生错误的PCR对未知序列的片段的混合物进行诱变(11,12)。但是公开的易于产生错误的PCR方案(11,12)遭受了聚合酶的低加工性能。因此,该方案不能导致一个平均大小的基因产生随机诱变。这种无能限制了易于产生错误的PCR的实用性。
易于产生错误的PCR另一个严重的限制是具有序列信息内容的反向突变的生长速率。对于一定的信息内容,文库大小,和诱变速率,反向突变到正向突变的平衡从统计学上阻碍了对进一步改善的选择性(统计学限度)。
最后易于产生错误的重复的循环导致中性突变积聚,这种积聚产生例如免疫原性但是没有结合亲和力。
因此易于产生错误的PCR由于太慢而不能发生为连续序列演化所需要的模块变化(1,2)。
在盒诱变中,通常用(部分)随机化的序列替代单链模板的序列模块。因此从统计学上说可以获得的最大信息内容受到随机序列数(即文库大小)的限制。该方法构成了统计学上的峡口,消除了通常不是最好的其它序列族,但是它具有更长周期的潜力。
此外,用合成的寡核苷酸进行诱变需要在每次选择周期之后对单个克隆进行测序(20),因此,该方法劳动强度大并且对于许多个周期的诱变是不实用的。
易于产生错误的聚合酶链反应和盒基因诱变最适合于并且被广泛地用于对相对低信息内容区域进行精细加工。一种明显例外的情况是利用易于产生错误的PCR进行许多个周期的扩增和选择可以从随机文库中选择一种RNA连接酶核糖酶(13)。
用于设计重组线性生物学序列例如蛋白质,RNA和DNA的手段并不象已经研制的手段那样有力量已逐渐显而易见。基于在越来越大的文库内研究越来越多的序列而寻找越来越好的突变体,以及增加诱变扩增和选择的循环数是必要的。但是正如上文中讨论的,当用于重复循环时广泛使用的现存的诱变方法有明显的限制。
通过天然的选择和有性的再生产大多数生物体发生演变。有性再生产可确保所选定的个体的后代的基因进行混合和组合。在有丝分裂期间,来自亲代的同源染色体沿其长度方向以互相并列和交叉部分的方式排列,因此交换了遗传材料。DNA进行这样的交换或混合使生物体可以快速地演化(1,2)。在有性重组中,由于在同源环境中所插入的序列具有实用性,因而一旦插入到新的序列中该插入的序列可能仍具有基本信息内容。
Marton等人(27)描述了在体外利用PCR监测具有直接重复序列的质粒中发生的重组。Marton等人公开了由于DNA的断裂或缺口而在PCR期间出现重组。这样产生重组分子。Meyerhans等人(23)也公开了在体外PCR期间DNA重组的出现。
术语应用分子演变(“AME”)是指为特定的有用目的应用演变的设计的规则系统。而对于多核苷酸(3,11-14),肽和蛋白质(噬菌体(15-17),lacI(18)和多核糖体已经报道了许多不同的AME的模式文库,在这些模式文库中已经使用随机交叉重组随意地制备重组文库。
从理论上说,在一个100个氨基酸的蛋白质中存在2000个不同的单突变。一个含100个氨基酸的蛋白质可能有20100种的突变组合,该数目太大以致于不能用常规方法彻底地开发。研制一种使所有的这些可能的突变组合产生和对此筛选的系统是有利的。
Winter和其合作者(43,44)已经利用一个体外位点特异性重组系统将用于在噬菌体系统中表达的轻链抗体基因与重链抗体基因进行组合。但是,它们的系统依赖于重组的特定位点并且因而受到限制。Hayashi等人(48)报道了通过重叠延伸和PCR而在单链抗体中(scFv)发生的抗体CDR区域的同步诱变。
Caren等人(45)描述了利用体内随机重组而产生大批量的多位点突变的方法。但是这些方法需要两个不同的质粒文库发生重组,每个文库具有不同的选择性标记。因此这种方法仅限制在相当于所存在的选择性标记数目的有限数的重组,并且这些方法还伴随着使连接到选定的序列上的标记基因数目呈线性增加。
Calogero等人(46)和Galizzi等人(47)报道了在位于质粒上的两个同源但被截短的昆虫毒素基因进行体内重组可产生杂合基因。Radman等人(49)报道了在错配修复酶缺陷的缩主细胞中实质上错配的DNA序列进行体内重组,导致形成杂合分子。
研制一种生产突变蛋白的方法是有利的,利用该方法可以研制易于研究的突变核酸序列的大文库。本文描述的发明涉及点诱变的重复循环的使用,核酸混合以及对高度复杂线性序列例如通过随机重组的蛋白质的体外特定的分子演化的选择。
因此,研制一种用于生产突变DNA,RNA和蛋白质的大量文库以及为所需目的选择特定的突变体的方法是有利的。本文描述的发明涉及诱变的重复循环的使用,体内重组以及对高度复杂线性序列例如通过随机重组的蛋白质,DNA,RNA的体外特定的分子演化的选择。
根据下面的描述和参考附图,本发明的进一步的优点是显而易见的。
发明概述
本发明涉及用于制备选定的多核苷酸序列或选定的多核苷酸序列群体的方法,通常所说的序列是被扩增的和/或克隆的多核苷酸形式,从而选定的多核苷酸序列具有用于选择所需要的表型特性(例如,编码多肽,促进被连接的多核苷酸的转录,结合蛋白质等等)。鉴别具有所需结构或功能特性例如结合到预定的生物大分子(例如受体)上的多肽的方法涉及从多肽的大文库中筛选单个的文库成员,该成员具有该多肽的氨基酸所授予的所需的结构和功能特性。
本发明提供了用于生产适合于亲和作用筛选或表型筛选的目的多肽或目的抗体的文库的方法。该方法包括(1)获得第一批大量的选定的文库成员,含有目的多肽或目的抗体和编码所说目的多肽或目的抗体的相关多核苷酸,以及获得所说相关多核苷酸或其复制本,其中所说的相关多核苷酸含有实际上相同序列的一个区域,在所说多核苷酸或复制本中非强制性地引入突变,和(2)在适合于PCR扩增的条件下通常随机地将所说相关多核苷酸或复制本合并和分裂以形成其片段,进行PCR扩增和非强制性地诱变,从而使所说片段同源重组以形成一个重组多核苷酸杂合池(shuffling pool),从而在第一批大量选定的文库成员中不存在所说杂合池的重组多核苷酸的基本片段(例如,大于10%),所说杂合池组成了适合于亲和作用筛选的目的多肽或目的抗体的文库。非强制性地,该方法包括从杂合池中筛选文库成员的附加步骤,以便鉴别具有与预定的大分子,例如一种蛋白质受体,肽,寡糖,病毒颗粒,或其它预定的化合物或结构相结合或相互作用(例如催化抗体)的能力的单个杂合文库成员。目的多肽,抗体,模拟肽抗体,和从这样的文库中鉴别出的可变区序列可用于治疗,诊断,研究,和有关目的(例如催化剂,用于提高水溶液渗透性的溶质等等),和/或可用于一个或多个混合和/或亲和选择的其它的循环。对该方法进行修改,以便使选择步骤除具有与预定的分子亲和结合外还具有表型特性(例如催化活性,稳定性,抗氧化性,抗药性,或授予宿主细胞的可检测表型)。
在一个实施方案中,通过体外PCR将第一批大量选定的文库成员分裂成片段并且同源重组。
在一个实施方案中,体外将第一批大量选定的文库成员分裂成片段,将得到的片段转移到宿主细胞或生物体并且同源重组,体内形成杂合的文库成员。
在一个实施方案中,不将第一批大量选定的文库成员分裂成片段,但是将它克隆在一个脂质体的可复制的载体中或在该载体中扩增为直系重复片段,其中每个重复片段含有一个远缘品种的选定的文库成员序列,将所说载体转移到一个细胞中并且通过内-载体重组方法进行同源重组以便体内形成杂合的文库成员。
在一个实施方案中,提供了体外和体内杂合的组合方法以加强组合多样性。
本发明提供了用于生产适于亲和作用筛选的目的抗体文库的方法。该方法包括(1)获得第一批大量的选定的文库成员,含有目的抗体和编码所说和目的抗体的相关多核苷酸,以及获得所说相关多核苷酸或其复制本,其中所说的相关多核苷酸含有实际上相同的可变区域骨架序列的一个区域,和(2)在适合于PCR扩增的条件下通常随机地将所说相关多核苷酸或复制本合并和分裂以形成其片段,进行PCR扩增和非强制性地诱变,从而使所说片段同源重组以形成一个包含新的CDR组合的重组多核苷酸杂合池,从而在第一批大量选定的文库成员中不存在含有CDR组合的所说杂合池的重组多核苷酸的基本片段(例如,大于10%),所说杂合池组成了适合于亲和作用筛选的含有CDR预突变的目的抗体的文库。非强制性地,将杂合池用于亲和筛选以选出与预定的表位抗原决定基(抗原)结合的杂合文库成员以及从而选出了大量的选定的杂合文库成员。非强制性地,将选定的杂合文库成员进行杂合并且重复地筛选1-1000个循环或者按照需要直到获得具有所需结合亲和力的文库成员。
因此,本发明的一个方面提供了一种方法,包括将一个或多个突变导入到模板双链多核苷酸中,其中通过将一个或多个单链或双链寡核苷酸加到得到的双链片段群体上,已经将模板双链多核苷酸裂解为所需大小的随机片段,其中所说的寡核苷酸含有一个等同于模板多核苷酸的区域和异源于模板多核苷酸的区域;使得到的双链随机片段和寡核苷酸的混合物变性为单链片段;将得到的单链片段群体与一种聚合酶一起保温,保温条件是使所说的单链片段退火在相当于该单链片段的区域上并且形成诱变的双链多核苷酸;并且按照需要重复上述步骤。
另一方面,本发明涉及生产具有生物学活性的重组蛋白质的方法,包括通过在使所说模板多核苷酸裂解为具有所需大小的随机双链片段的条件下处理含有编码野生型蛋白质的双链模板多核苷酸的样本;将一个或多个单链或双链寡核苷酸加到得到的随机片段群体上,其中所说的寡核苷酸含有一个等同于模板多核苷酸的区域和异源于模板多核苷酸的区域;使得到的双链随机片段和寡核苷酸的混合物变性为单链片段;将得到的单链片段群体与一种聚合酶一起保温,保温条件是使所说的单链片段退火在等同区域上并且形成诱变的双链多核苷酸;按照需要重复上述步骤,以及然后从诱变的双链多核苷酸表达该重组蛋白质。
本发明的第三个方面涉及获得嵌合多核苷酸的方法,包括在使所说模板多核苷酸裂解为具有所需大小的随机双链片段的条件下处理含有不同双链模板多核苷酸的样本,其中所说的不同模板多核苷酸含有一个等同区域和异源区域;使得到的含有被处理样本的双链随机片段变性为单链片段;将得到的单链片段与一种聚合酶一起保温,保温条件是使所说的单链片段退火在等同区域上并且形成一种嵌合的双链多核苷酸序列,该序列含有模板多核苷酸序列;并且按照需要重复上述步骤。
本发明的第四个方面涉及复制模板多核苷酸的方法,包括通过体外将单链模板多核苷酸与由模板多核苷酸的裂解和变性产生的随机单链小片段结合,以及在核酸多聚酶存在下保温所说的核酸片段混合物,保温条件是形成双链模板多核苷酸群体。
本发明还提供了将体外和/或体内多核苷酸杂合方法用于使编码多肽的多核苷酸和/或含有转录调节序列的多核苷酸进行杂合。
本发明还提供了将多核苷酸杂合方法用于使病毒基因(例如衣壳蛋白,针尖糖蛋白,聚合酶,蛋白酶等等)或病毒基因组(例如副粘病毒科,正粘病毒科,疱疹病毒,反录病毒,呼肠病毒,鼻病毒等等)群体进行杂合。在一个实施方案中,本发明提供了使编码整个或部分免疫病毒蛋白的序列进行杂合以产生新组合的表位决定基以及由重组产生的新的表位决定基的方法;这种杂合的病毒蛋白可以含有表位决定基或表位决定基的组合,它们可能在病毒演变(例如流感病毒菌株的重组)的天然环境中产生。
本发明还提供了适于使多核苷酸序列杂合的方法,用于产生基因治疗载体以及复制缺陷型基因治疗构件体,例如可用于人的基因治疗,包括但不限于基于DNA的免疫接种的免疫接种载体,以及抗瘤形成的基因治疗和其它基因治疗方法。
附图简述
附图1图解比较由易于产生错误的PCR进行的诱变杂合;(a)起始文库;(b)在亲和选择的第一个周期中选定序列的集合体;(d)选定序列的体外重组(‘杂合’);(f)在杂合之后亲和选择的第二个周期中选定序列的集合体;(c)易于产生错误的PCR;(e)在易于产生错误的PCR之后亲和选择的第二个周期中选定序列的集合体。
附图2描述了1.0kb LacZα基因片段与10-50bp随机片段的重组。(a)具有LacZα基因的DNA片段经PCR扩增后凝胶电泳的照片。(b)在用DNA酶I(DNAseI)消化之后的DNA片段凝胶电泳下照片。(c)从被消化的LacZα基因DNA片段纯化的10-50bpDNA片段的凝胶电泳的照片;(d)在DNA重组进行指定数目的周期之后10-50bpDNA片段的凝胶电泳的照片;(e)在用引物进行PCR扩增之后重组混合物凝胶电泳的照片。
附图3图解描述了LacZα基因终止密码子突变体和其DNA序列。加方框的区域是异源区,作为标记。该终止密码子定位于较小的框中或者底下划线。“+”表示野生型基因,“-”表示该基因的突变区。
附图4图解描述一个合成的寡核苷酸导入或插入的LacZα基因的重组过程中。
附图6图解显示用scFv的抗兔IgG抗体(A10B)的抗体CDR杂合模型系统。
附图7描述在scFv的抗兔IgG抗体(A10B)的杂合DNA中观察到的CDRs的特定组合出现的频率。
附图8描述了在DNA杂合和每个选择周期之后scFv的抗兔IgG抗体的改善的亲和力。
附图9图解描绘了pBR322-Sfi-BL-LA-Sfi和通过直系重复区进行的体内质粒内重组,以及由重新构成一个功能性β-内酰胺酶质粒内重组产生氨苄青霉素抗性菌落的速率。
附图10图解描绘了pBR322-Sfi-2Bla-Sfi和通过直系重复区进行的体内质粒内重组,以及由重新构成一个功能性β-内酰胺酶质粒内重组产生氨苄青霉素抗性菌落的速率。
附图11描述了用于检测将多核苷酸片段导入到产生重组蛋白的细胞之后多个周期同源重组的效率的方法。
附图12图解描绘了通过基因盒在下列位点杂合而产生的载体文库:启动子,前导肽,终止子,可选择的抗药性基因,和复制原点。在每个位点的多条平行线表示对于那个盒具有盒多样性。
附图13图解显示在各个位点适合于通过杂合方法构建原核载体文库的盒的一些例子。
优选实施例的描述
本发明涉及用于在随机片段化之后核酸分子发生重组的方法以及将该方法用于DNA序列的诱变。还描述了用于生产编码具有增强的生物活性的突变蛋白的核酸片段的方法。尤其是,本发明还涉及重复循环的诱变,核酸杂合和可以制备具有增强的生物活性的突变蛋白的选择的方法。
本发明涉及产生DNA,RNA或蛋白质突变体的极大文库的方法。该方法在产生可选出所需核酸片段的有关DNA片段方面有特别的优点。特别是本发明还涉及重复循环的诱变,同源重组和可以制备具有增强的生物活性的突变蛋白的选择的方法。
但是在详细讨论本发明之前,首先定义下列术语。
定义
如本文所用,下列术语具有下列含义:
术语“DNA重组”用于指两个相同序列之间出现重组。
相反,术语“DNA杂合”用于指基本上同源但不相同的序列之间的重组,在一些实施方案中,DNA杂合是指非同源重组出现的交叉,例如借助于cre/lox和/或flp/frt系统等等。
术语“扩增”是指增加一个核酸片段的拷贝数。
术语“相同”或“等同”是指两个核酸序列具有相同序列或一个互补序列。因此“等同区域”是指一个核酸片段或多核苷酸的区域相同或互补于另一个多核苷酸或核酸片段。
术语“相应于”是指一个多核苷酸序列与整个或部分参照多核苷酸序列同源(即相同,演化上不严格相关),或者是指一个多肽序列等同于参照多肽序列。与之相区别,本文中使用的术语“互补于”是指该互补序列与整个或部分参照多核苷酸序列同源。为了描述,核苷酸序列“TATAC”对应于参照序列“TATAC”并且互补于参照序列“GTATA”。
下列术语用于描述两个或多个多核苷酸之间的序列关系:“参照序列”,“比较窗口”,“序列等同性”,“序列等同百分数”和“基本等同”。“参照序列”是指用作为序列比较的基础的一个选定的序列;参照序列可以是较大序列一个子集,例如在序列表给出的全长cDNA或基因序列的片段,例如图1或图2(b)的多核苷酸序列,或者可以包括完整的cDNA或基因序列。通常,参照序列是长度为至少20个核苷酸,通常长度为至少25个核苷酸,以及经常长度为至少50个核苷酸。由于两个多核苷酸可以各(1)含有两个多核苷酸之间相类似的一个序列(即,整个多核苷酸序列的一部分),(2)进一步可含有两个多核苷酸之间差异较大的一个序列,通常通过借助于“比较窗口”对两个多核苷酸序列进行比较以鉴别和比较定位区序列相似性通常可完成对两个(或多个)多核苷酸序列的比较。
本文中使用的“比较窗口”是指至少20个连接位点片段,其中多核苷酸序列相当于至少20个连接核苷酸的参照序列以及其中将两个序列最佳排列之后与参照序列(不含有叠加或缺失)相比比较窗口中多核苷酸序列的一部分可以含有20%或者更少的叠加或者缺失(即缺口)。采用Smith and Waterman(1981)Adv.App1.Math.2:482的位点同源规则,采用Needleman and Wunsch(1970)J.Mol.Biol.48:443的同源排列规则,根据Pearson and Lipman(1988)Proc.Natl.Acad.Sci.(U.S.A.)85:2444对相似方法的研究,这些规则的计算机应用(GAP,BESTFIT,FASTA,and TFASTA in the Wisconsin Genetic Software Package Release 7.0,GeneticsComputer Group,575 Science Dr.,Madison,WI),或者通过检查可以完成对比较窗口进行排列而使序列排列最佳,并且选出有各种方法产生的最佳排列(即产生比较窗口的最高同源百分数)。
术语“序列等同性”是指两个多核苷酸序列的比较窗口是相同的(即,以一个核苷酸接一个核苷酸为基础)。术语“序列等同百分数”是通过下列方法计数的:将两个最佳排列的序列的比较窗口进行比较,测定两个序列上出现等同核酸碱基(例如,A,T,C,G,U,或I)的位置的数目以得出错配位置的数目,将错配位置的数目除于比较窗口中总位置数目(即窗口大小),以及将得到的结果乘于100以产生序列等同百分数。本文中使用的术语“基本等同”是指多核苷酸序列的特性,其中该多核苷酸含有一个序列,在至少20个核苷酸位置,最常见的是至少25-50个核苷酸的比较窗口内,该序列与参照序列至少有80%的序列等同性,优选地至少85%等同以及通常为90-95%序列等同,最通常为99%序列等同,其中通过将参照序列与多核苷酸序列进行比较而计算出序列等同百分数,所说多核苷酸可以含有参照序列比较窗口内总共20%或更少序列的缺失或叠加。
保守的氨基酸组成是指可内部转化的具有相似侧链的残基。例如,具有脂族侧链的一组氨基酸是甘氨酸,丙氨酸,缬氨酸,亮氨酸,和异亮氨酸;具有脂族-羟基侧链的一组氨基酸是丝氨酸,和苏氨酸;具有含酰胺侧链的一组氨基酸是天冬氨酸和谷氨酰胺;具有芳香族侧链的一组氨基酸是苯丙氨酸,酪氨酸和色氨酸;具有碱性侧链的一组氨基酸是赖氨酸,精氨酸和组氨酸;具有含硫侧链第一组氨基酸是半胱氨酸和甲硫氨酸。优选的保守氨基酸组成基团是:缬氨酸-亮氨酸-异亮氨酸,苯丙氨酸-酪氨酸,赖氨酸-精氨酸,丙氨酸-缬氨酸,和天冬氨酸-谷氨酰胺。
术语“同源”是指一个单链核酸序列与一个互补单链核酸序列杂交。杂交程度取决于多个因素,包括序列之间等同数目和杂交条件例如温度和下文中讨论的盐浓度。优选地等同区域是大于约5bp,更优选地等同区域是大于约10bp。
术语“异源”是指一个单链核酸序列不能与另一个单链核酸序列后其互补酸序列杂交。因此,异源区域是指具有不能与另一个核酸或多核苷酸杂交的序列的区域的核酸片段或多核苷酸。例如这样的区域是突变区。
本文中使用的术语“关连”是指品种之间演化和功能上相关的一个基因序列。例如但不限于,在人基因组中,人CD4基因与鼠CD4基因是一个关连基因,因为这两个基因的序列和结构显示它们是高度同源的并且两个基因编码一个蛋白质,该蛋白质通过MHCII型限定的抗原识别而在作为标记的T细胞活化中起作用。
术语“野生型”是指不含有任何突变的核酸片段。一种“野生型”蛋白质是指在天然存在的活性水平上时该蛋白质是有活性的并且含有天然存在的氨基酸序列。
术语“相关的多核苷酸”是指该多核苷酸区域是相同的并且该多核苷酸区域是异源的。
术语“嵌合多核苷酸”是指该多核苷酸含有野生型区域以及被突变的区域。也是指该多核苷酸含有来自一个多核苷酸的野生型区域以及来自另一个相关多核苷酸的野生型区域。
术语“切割”是指用酶消化一个多核苷酸或裂解该多核苷酸。
本文中使用的术语“群体”是指成分例如多核苷酸,核酸片段或蛋白质的集合。一种“混合的群体”是指属于相同核酸或蛋白质族(即相关)但是其序列不同(即不相同)因此其活性不相同的成分的集合。
术语“特定的核酸片段”是指具有一定的端点并且具有一定的核酸序列的核酸片段。两个核酸片段具有两个不同的特定的核酸片段,这两个核酸片段中一个核酸片段具有相同于第二个核酸片段的一部分但是末端不同的序列。
术语“突变”是指野生型核酸序列内发生变化或肽序列内发生变化。这样的突变可以是点突变例如碱基转换或者转变。突变可以是缺失,插入或者复制。
在本文中使用的肽标记中,按照标准用法和规律左手方向是氨基末端方向,右手方向是羧基末端的方向。类似地,除非有特定的说明,单链多核苷酸序列的左手末端是5′末端;双链多核苷酸序列所左手末端是5′末端。5′3′新生RNA转录物的叠加方向是指转录方向;位于具有类似于RNA的序列的DNA链上并且是5′至RNA转录物的5′末端的序列区域称为“上游区域”;位于具有类似于RNA的序列的DNA链上并且是3′至编码RNA转录物的3′末端的序列区域称为“下游区域”。
本文中使用的术语“天然存在”用作为指物体时是指该物体可以在自然界中发现指定事实。例如,存在于可从天然来源分离的生物体(包括病毒)中并且人类并不想在实验室中将它修饰的多肽或多核苷酸序列是天然存在的。通常,术语天然存在是指存在于非病原(未发病)个体中的物体,例如典型物种。
本文中使用的术语“试剂”是指化合物,化合物的混合物,一系列空间定位的化合物(例如,VLSIPS肽列阵,多核苷酸列阵,和/或小分子组合列阵),生物学大分子,噬菌体肽陈列文库,噬菌体抗体(例如,scFv)陈列文库,多核糖体肽陈列文库,或从生物材料例如细菌,植物,真菌或动物(特别是哺乳动物)细胞或者组织。通过参与下文中描述的筛选方法,评价这些试剂作为抗瘤形成,抗炎性或者apoptosis调节子的活性。通过参与下文中描述的筛选方法,评价这些试剂作为特定蛋白质相互作用抑制剂的活性(即,选择性地抑制两种预定多肽之间的相互作用但是基本上不干扰细胞存活性的一种试剂)。
如本文中使用的,“基本上纯化”是指一个物种以优势种存在(即以摩尔为基础,它在该组合物中比其它单个大分子品种的含量更丰富),优选地,一种基本纯化组分是指其中该物种的含量至少为存在的所有大分子的50%(以摩尔计)的组合物。通常,一种基本上纯化的组合物含有该组合物中所有大分子的80-90%以上。更优选地,将该物种纯化至基本上匀质(采用常规方法不能检测出该组合物中的污染物),其中该组合物基本上由单一大分子组成。溶剂,小分子(<500道尔顿),基本离子不认为是大分子。
如本文中使用的,术语“生理条件”是指温度,pH,离子强度,粘度,和类似的生化参数,这些参数与活生物体相对应,和/或通常存在于培养的酵母活细胞或哺乳动物细胞内的条件。例如,在常规实验室培养条件下生长的酵母细胞的胞内条件是生理条件。通常用于体外转录的合适的体外反应条件是生理条件。一般地说,体外生理条件包括50-200mMNaCl或KCl,pH 6.5-8.5,20-45℃和0.001-10mM二价阳离子(例如,Mg++,Ca++);优选地,约150mMNaCl或KCl,pH7.2-7.6,5mM二价离子,通常包括0.01-1.0%非特异蛋白(例如,BSA)。通常存在约0.001-2%的非离子型去污剂(吐温,NP-40,Triton X-100),典型的是0.05-0.2%(v/v)。由常规方法的实践者选定特定的含水条件。对于常规应用,可以应用下列缓冲含水条件:10-250mMNaCl,5-50mM Tris HCl,pH 5-8,非强制性地加入二价阳离子,和/或金属螯合剂和/或非离子型去污剂和/或膜组分和/或消沫剂和/或闪烁材料。
本文中特定的杂交定义为在第一多核苷酸和第二多核苷酸(例如,具有独特的但是与第一多核苷酸基本相同的序列的多核苷酸)之间形成杂合体,其中在严谨杂交条件下第一多核苷酸优选与第二多核苷酸杂交,其中基本上不相关的多核苷酸序列在混合物中不形成杂合体。
如本文中使用的,术语“单链抗体”是指含有以多肽键连接的VH区和VL区,通常通过间隔肽(例如,[Gly-Gly-Gly-Gly-Ser]x)连接并且在氨基末端和/或羧基末端含有其它的氨基酸序列的一个多肽。例如,单链抗体含有用于连接到编码多核苷酸上的一个限制片段。作为举例,scFv是一个单链抗体。单链抗体通常是由一个或多个多肽片段组成的蛋白质,所说多肽片段至少含基本由免疫球蛋白超家族的基因编码的(例如,参见The Immunoglobulin Gene Superfamily,A.F.Woilliams and A.N.Barclay,in Immunoglogulin Genes,T.Honjo,F.W.Alt,and T.H.Rabbitts,eds.,(1989)Academic Press:San Diego,CA,pp.361-387,引入本文作为参考),最常见是由啮齿动物,非人灵长类,鸟类,猪,牛,绵羊,山羊,或者人重链或者轻链基因序列编码的10个相邻氨基酸。通常一个单链抗体含有大部分的免疫球蛋白超家族产物以致于保留了与特定的靶分子,通常一种受体或者抗原(表位决定基)结合的特性。
如本文中使用的,术语“互补决定区”和“CDR”是指本领域内认可的术语如由Kabat和Chothia例举的CDR定义,通常也称为极度可变区或极度可变环(Chothia and Lesk(1987)J.Mol.Biol.196:901;Chothia et al.(1989)Nature 342:877;E.A.Kabat et al.,Sequence of Proteins of Immunological Interest(National Institutesof Health,Bethesda,MD)(1987);and Tramontano et al.(1990)J.Mol.Biol.215:175)。通常可变区域含有天然存在的免疫球蛋白(例如1-110位氨基酸)的约105-115氨基酸氨基末端,尽管更短或更长的可变区域也适合于形成单链抗体。
免疫球蛋白轻链或重链可变区由“主链”区组成,该主链区被三个极度可变区,也称为CDR’s打断。该主链区和CDR’s的长度已被精确地定义(参见“Sequencesof Proteins of Immunological Interest,”E.Kabat et al.,4th Ed.,U.S.Department ofHealth and Human Services Bethesda,MD(1987))。在一个品种内不同轻链或重链的主链区序列是非常保守的。如在本文中使用的,“人骨架区”是一个主链区,它基本上等同(约85%或更多,通常90%-95%或更多)于天然存在的人免疫球蛋白的主链区。作为组成型轻链和重链的组合主链区的一种抗体的主链区可用作为定位和排列CDR’s。该CDR’s主要具有结合到抗原的表位决定基上的作用。
如本文中使用的,术语“可变片段”是指新生肽的一部分,该肽含有一个随机的,假随机的,或限定的核心序列。可变片段可以含有变异的和非变异的残基位置,可以限制一个变异残基位置上残基变异的程度;两种选择由实践者的意见来决定。通常,可变片段的长度是约5-20个氨基酸残基(例如8-10),但是可变片段也可以更长并且可含有抗体部分或受体蛋白,例如抗体片段,核酸结合蛋白,受体蛋白等等。
如本文中使用的,“随机肽序列”是指由两个或多个氨基酸单体组成并且由随机过程构建的氨基酸序列。一种随机肽可包括主链或支架基元,它可以含有非变异序列。
如本文中使用的,“随机肽文库”是指编码一组随机肽的一组多核苷酸序列,并且指由这些多核苷酸序列编码的该组随机肽,以及含有这些随机肽的融合蛋白。
如本文中使用的,术语“假随机”是指具有有限的可变性的一组序列,以至于例如一个位置上的残基变异程度不同于另一个位置上的残基变异程度,但是任何假随机位置都被允许有一定的残基变异程度,但变异程度是有限的。
如本文中使用的,术语“限定的序列骨架”是指以非随机方法,通常以试验数据和结构资料为基础选出的一组限定的序列;例如限定的序列骨架含有被预测为可形成β-片层结构的一组氨基酸序列或者含有一个亮氨酸拉链型七个一套的重复基元,一个锌--指状物区域,其它为变异区。“限定的序列核心”是变异性有限的一套序列。(1)一个20个常规氨基酸的序列的完全随机的十聚体可以是(20)10序列,(2)一个20个常规氨基酸的序列的假随机的十聚体可以是(20)10序列,但是在特定位置和/或整个序列显示偏向于特定残基,(3)限定的序列核心是一组序列,如果每个残基位置允许是所说20个常规氨基酸(和/或允许的非常规氨基/亚氨基酸)的任一种则该组序列数低于可能序列数的最大数目。通常限定的序列核心在选定单个文库成员序列的片段上或整个长度内含有变异和非变异残基位点和/或含有变异残基位点,这些位点可含有选自于限定的氨基酸残基亚组的一个残基等等。选定的序列核心可以指氨基酸序列或多核苷酸序列。为了说明而不是为了限制,对序列(NNK)10和(NNM)10选定了序列核心,其中N代表A,T,G,或C;K代表G或T,和M代表A或C。
如本文中使用的,“表位决定基”是指一个抗原或其它大分子上能够形成结合作用的组分,它与一种抗体的可变区结合位置发生相互作用。通常这样的结合作用显示为与CDR的一个或多个氨基酸残基发生分子内接触。
如本文中使用的,“受体”是指与一个给定的配体有亲和力的分子。受体可以是天然存在或合成的分子。可以使用非变化状态的受体或者与其它种的结合体形式的受体。可以直接或借助于一个特定的结合物质将受体共价或非共价吸附到结合成员上。受体的例子包括但不限于与特定的抗原决定基(例如病毒上的,细胞,或其它材料)发生反应的抗体,包括单克隆抗体和抗血清,细胞膜受体,碳水复合物和糖蛋白,酶和激素受体。
如本文中使用的,“配体”是指由特定的受体识别的一种分子,例如随机肽或变异片段序列。如本领域内任一熟练技术人员将认识到,一个分子(分子大分子复合物)即可以是受体又可以是配体。通常具有较小分子量的结合配对体被称为配体,具有较大分子量的结合配对体被称为受体。
如本文中使用的,“连接头”或“间隔区”是指分子或连接两个分子的分子团,例如DNA结合蛋白和一种随机肽,并且将两个分子以优选的构型放置,(例如使该随机肽结合到与DNA结合蛋白有基本空间障碍的一个受体上)的一个分子或一组分子。
如本文中使用的,术语“可操作连接”是指多核苷酸元素以一定的功能关系进行连接。当置于功能关系中时一种核酸与另一种核酸序列可操作连接。例如如果实现了该编码序列的转录则一种启动子或增强子可操作连接到一编码序列上。可操作连接是指通常被连接的DNA序列是相邻的,并且如果必须要连接二个蛋白质编码区,它们是相邻的并且在阅读框架内。
方法论
核酸杂合方法是用于核酸片段或多核苷酸集合体的体外或体内同源重组的方法。将相关核酸序列或多核苷酸序列的混合物随机地裂解成片段,并且重新装配以产生重组核酸分子或多核苷酸的文库或混合群体。
与盒诱变相反,单独的杂合和易于产生错误的PCR可以将序列集合体盲目地突变(除引物外没有序列信息)。
最好是用抗体遗传工程的实例来解释在重复选择时使用本发明的诱变杂合比仅用易于产生错误的PCR具有优点。在附图1中显示了下文中描述的DNA杂合方法。起始文库由各种来源的相关序列(即来自天然mRNA的抗体)组成或者由任何类型的单抗体基因诱变(包括杂合)产生。在第一轮亲和选择之后得到了选定的互补决定区的集合(“CDRs”)(图1)。在该图中浓的CDRs使抗体分子与抗体的亲和力增强。杂合方法使所有的CDR1s和所有的CDR2s和所有的CDR3s等等自由组合(图1)。
该方法不同于PCR,不同之处在于它是一个逆转链反应。在PCR中,聚合酶起始位点的数目和分子数目呈指数增长。但是聚合酶起始位点的序列和该分子的序列基本保持相同。相反在随机片段的核酸重组或杂合方法中,起始位点的数目和随机片段的数目(但不是大小)同时降低。对于从整个质粒衍生的片段理论上的关键点是一种单链,大多联体分子。
由于在同源区域出现了交叉,因此首先在相同序列族成员之间出现重组。这阻碍了严重不相容的(例如抗相同抗原的不同表位决定基)的CDRs的组合。观察到在相同反应中多个序列族可以杂合。此外杂合保存了相对位置序列,以至于例如在CDR2位置上未发现CDR1。
稀有的杂合体含有大量的最好的(例如最高亲和力)CDRs并且基于其优异的亲和力可以选出这些稀少的杂合体(图1)。
以高达1006不同方法可以将来自100个不同的选定的抗体序列的集合体的CDRs排列。在DNA序列单个文库中不能体现这大量的排列。因此,根据序列长度和所需序列的多样性决定需要进行的DNA杂合和选择的多次循环。
相反易于产生错误的PCR在同样的相关序列内保留了所有选定的CDRs(图1),产生了更小的突变云。
在本发明方法中使用的模板多核苷酸可以是DNA或RNA。根据重组或重装配的基因或DNA片段的大小模板具有不同的长度。优选地模板多核苷酸大小从50bp--50kb。在本发明的方法中可以使用含有编码所需蛋白质的所需核酸的完整载体,事实上已经成功地使用。
通过利用PCR反应进行扩增(美国专利№4683201和4683195)或其它扩增或克隆方法可以获得模板多核苷酸。但是,在分裂成片段之前从PCR产物中除去游离引物可以获得更有效的结果。不能合适地除去引物可以导致产生低频率的交叉克隆。
通常模板多核苷酸应该是双链的。要求双链核酸分子可以确保得到的单链核酸片段的区域互相互补,由此可以杂交以形成双链分子。
在该步骤中将具有等同于模板多核苷酸的区域和异源于模板多核苷酸的区域的单链或双链核酸片段加到模板多核苷酸上。还涉及在该步骤中将两个不同但相关的多核苷酸模板混合。
将双链多核苷酸模板和任何增加的双链或单链片段随机地消化为约5bp--5kb或更大的片段。优选地随机片段的大小为约10bp--1000bp,更优选地随机片段的大小为约20bp--500bp。
另一种可选的方法,还涉及在本发明的方法中使用具有多个缺口的双链核酸。一个缺口是在双链核酸的一条链上有一断裂。所说缺口之间的距离优选的是5bp--5kp,更优选的是10bp--1000bp。
可用许多不同的方法消化核酸片段。可用核酸酶,例如DNA酶I或RNA酶消化核酸片段。采用超声处理方法或通过一个有小洞的管可以随机的剪切核酸。
还涉及用一种或多种限制性酶部分消化核酸,以至于从统计学上保留了交叉点。
任何一种特定核酸片段的浓度不会大于总核酸重量的百分之一,更优选的任何一种特定核酸片段的浓度不会大于总核酸重量的0.1%。
在该混合物中不同的特定核酸片段的数目至少为约100,优选的至少约500,更优选至少约1000。
在该步骤中将合成的或天然的单链或双链核酸片段加到随机双链片段上以便提高该核酸片段混合物的不均匀性。
在该步骤还涉及将随机断裂的双链核酸片段群体混合或组合。
如果需要在模板多核苷酸中插入突变,则以相当于总核酸重量20倍的过量加入具有等同于模板多核苷酸的区域和具有异源于模板多核苷酸的区域的单链或双链核酸片段,更优选地,加入相当于总核酸重量10倍的过量的单链核酸片段。
如果需要不同但相关的模板多核苷酸的混合物,以低于1∶100的比例,更优选地以低于1∶40的比例将来自每个模板的核酸片段群体组合。例如,按照需要将野生型多核苷酸与突变多核苷酸群体回交以去除中性突变(例如,产生所选定的表型特性发生非本质变化的突变)。在一个实施例中,加到随机消化的突变多核苷酸上的随机消化的野生型多核苷酸片段的比例是约1∶1-约100∶1,并且更优选的是从1∶1-40∶1。
将随机发生片段的混合群体变性以形成单链核酸片段并且随后重退火。只有具有与其它单链核酸片段同源的区域的那些单链核酸片段将会退火。
通过加热,随机核酸片段可以变性。本领域内技术人员可以确定使双链核酸完全变性所必须的条件。优选地温度是80℃-100℃,更优选地温度是90℃-96℃。可用于使核酸片段变性的其它方法包括压力(36)和pH。
通过冷却可将核酸片段重退火。优选地温度是20℃-75℃,更优选地温度是40℃-65℃。如果基于仅四个基本碱基同源性的平均值需要高频率的交叉,通过使用低退火温度可以加强重组,尽管该方法变为更复杂。出现变性的程度将依赖于单链核酸片段群体之间同源性的程度。
通过加入聚乙二醇(“PEG”)或盐可以加速变性恢复。优选的盐浓度是从0mM-200mM,更优选的盐浓度是从10mM-100mM。所说盐可以是KCl或NaCl。优选地PEG浓度是0%-20%,更优选地5%-10%。
接着在核酸聚合酶和dNTP′s(即dATP,dCTP,dGTP和dTTP)存在下保温退火的核酸片段。核酸聚合酶可以是Klenow片段,Taq聚合酶或本领域内已知的其它DNA聚合酶。
重组所使用的方法取决于应该产生回交的同源性的基本程度。如果等同区域较大,可将Taq聚合酶与45-65℃的退火温度一起使用。如果等同区域较小,可将Klenow聚合酶与20-30℃的退火温度一起使用。本领域技术人员可以改变退火的温度以增加回交的数目。
可以在退火之前,与退火同时或退火之后将聚合酶加到随机核酸片段上。
在聚合酶存在下变性,变性恢复和保温的循环是指核酸的杂合或重组。将该循环重复预定的时间。优选的将该循环重复2-50次,更优选的重复10-40次。
得到的核酸是约50bp--约100kb的较大的双链多核苷酸,优选的较大多核苷酸是500bp--50kb。
该较大的多核苷酸片段可以含有互相串联的多个拷贝的核酸片段,该核酸片段的大小相同于模板多核苷酸。然后将该多联体片段消化为多拷贝的模板多核苷酸。结果产生大小几乎相同于模板多核苷酸的核酸片段群体。该群体是一个混合群体,其中在杂合之前已经向模板多核苷酸加入了具有等同区和异源区的单链或双链核酸片段。
然后将这些片段克隆到合适的载体中并将该连接混合物用于转化细菌。在克隆之前利用各种各样的方法包括PCR(美国专利№4683195和4683202)扩增单链合适片段而不是通过消化多联体可以从较大的多联体核酸片段获得单链核酸片段。
用于克隆的载体不是关键的,条件是它可以接受所需大小的DNA片段。如果需要该DNA片段进行表达,该克隆载体应该进一步含有与该DNA片段插入位点相连接的转录和转译信号以使该DNA片段在宿主细胞中表达。优选的载体包括pUC质粒系列和pBR质粒系列。
得到的细菌群体包括含有具有随机突变的许多重组DNA片段。对该混合群体进行检测以鉴别所需的重组核酸片段。选择方法取决于所需DNA片段。
例如如果需要为具有与配体的结合效力增强的蛋白质编码的DNA片段,采用本领域内已知的方法(即panning,亲和层析)检测由该群体或文库中每个DNA片段表达的蛋白质结合到配体上的能力。如果需要编码抗药性增加蛋白质的DNA片段,检测由该群体或文库中每个DNA片段表达的蛋白质授予宿主生物体抗药性的能力。本领域内技术人员借助于所需蛋白质的有关知识,很容易检测该群体以鉴别授予该蛋白质所需特性的DNA片段。
本发明涉及本领域内技术人员可以使用有关噬菌体展示系统,在该系统中在噬菌体的表面表达融合蛋白形式的蛋白质片段(Pharmacia,Milwaukee WI)。在导致融合蛋白转录的位点处将重组DNA分子克隆到噬菌体DNA中,该融合蛋白的一部分是由重组DNA分子编码的。含有重组核酸分子的噬菌体在细胞中进行复制或转录。该融合蛋白的前导序列指导该融合蛋白运输到噬菌体颗粒的顶端。因此由重组DNA分子部分编码的融合蛋白陈列于噬菌体颗粒上,采用上面描述的方法可以检测和选择。
本发明进一步涉及用来自第一群体的亚群体的核酸片段进行多个循环的核酸杂合,所说亚群体含有编码所需重组蛋白的DNA。按这种方式,可以获得有较高结合亲和力或酶促活性的蛋白质。
本发明还涉及用野生型核酸片段和来自第一或随后轮次的核酸杂交的核酸亚群体的混合物可以进行多个循环的核酸杂合,以便从亚群体中去除静止突变。
可以利用任何来源的纯化形式的核酸作为起始核酸。因此该方法可以利用DNA或RNA包括信使RNA,其中DNA或RNA可以是单链或双链。另外,可以利用含有各一条链的DNA-RNA杂合体。依据待诱变的核酸序列的大小,所说核酸序列可以是各种长度。优选的特定的核酸序列是50-50000bp。还涉及在本发明的方法中可使用含有编码所需蛋白质的核酸的完整载体。
可以从任何来源例如从质粒如pBR322,从克隆DNA或RNA或从来自细菌,酵母,病毒和高等生物如植物或动物的天然DNA或RNA获得核酸。可以从血液或组织材料中提取DNA或RNA。通过利用多核苷酸链反应(PCR)进行扩增可以获得模板多核苷酸(美国专利№4683202和4683195)。另一种方法,在存在于细胞中的载体中含有该核苷酸,并且通过培养该细胞和采用本领域内已知的方法从该细胞中提取核酸可以获得足够量的核酸。
采用本发明的方法可以利用任何特定的核酸序列生产突变群体。只需要在本发明方法之前存在或制造特定核酸序列的突变序列的小群体。
可采用许多不同的方法制备含有突变的特定核酸序列的起始小群体。采用易于产生错误的PCR可制备突变。易于产生错误的PCR利用低精确性的聚合条件以在整个序列长度内随机导入低水平的点突变。另一种方法,采用寡核苷酸定位诱变方法可将突变导入到模板多核苷酸中。在寡核苷酸定位诱变方法,利用限制性酶消化从该多核苷酸上去除短序列多核苷酸并且用一个合成多核苷酸取代,其中该合成的多核苷酸的各个碱基已经从原始序列发生变化。还可采用化学诱变方法改变该多核苷酸。化学诱变包括例如亚硫酸钠,亚硝酸羟胺,肼或甲酸。作为核酸前体类似物的其它试剂包括亚硝基胍,5-溴代尿嘧啶,2-氨基嘌呤或者吖啶。通常将这些试剂加到PCR反应中替代核酸前体从而使该序列突变。还可以使用插入试剂例如副核黄素,吖啶黄素,喹吖因等等。通过用X射线或紫外光照射也可以获得多核苷酸序列的随机诱变。通常,将如此诱变的质粒DNA或DNA片段导入到大肠杆菌中并且繁殖成突变质粒集合或文库。
另一种可用的方法找到了天然存在的特定核酸的混合小群体,它们由相同基因的不同等位基因或者不同相关种类的相同基因(即同族基因)组成。另一种可选择的方法,它们是一个种类存在的相关DNA序列,例如免疫球蛋白基因。
一旦产生了特定核酸分子的混合群体,可直接使用该多核苷酸或者利用本领域内熟知的技术将它插入到合适克隆载体中。
载体的选择依赖于多核苷酸序列的大小和本发明方法使用的宿主细胞。本发明的模板可以是质粒,噬菌体,粘粒,噬菌粒,病毒(例如反录病毒,副流感病毒,疱疹病毒,呼肠病毒,副粘病毒等等)或其选定的部分(例如包膜蛋白,针尖糖蛋白,衣壳蛋白)。例如,粘粒和噬菌粒是优选的,其中待诱变的特定核酸序列较大,因为这些载体能够稳定地繁殖大核酸片段。
如果将特定的核酸序列的混合群体克隆到一个载体中,通过将该各个载体插入到宿主细胞中并且使该宿主细胞扩增载体可以扩增该群体。这种方法称为克隆扩增,因为当核酸序列的绝对数增加时,突变数没有增加。
实用性
可以在未知序列的集合体中实施本发明的DNA杂合方法。通过在任何特定位点上将任何序列混合物加入到重组混合寡核苷酸(具有与被重组的序列同源的末端),而将该序列混合物掺入到另一序列混合物中,因此本发明还涉及将合成寡核苷酸,PCR片段或者整个基因的混合物在限定的位置上混合到另一序列中。一个序列(混合物)的插入不依赖于一个序列插入到该模板的另一部分中。因此重组程度所需同源性,和文库的多样性可独立地和同时随重组DNA的长度而变化。
将两种基因混合的方法可用于使来自鼠杂交瘤的抗体具有人的特性。将两种基因混合或者将突变序列插入到基因中的方法可用于任何治疗上使用的蛋白质,例如白介素I,抗体,tPA,生长激素等等。该方法也用于任何核酸例如启动子或者内含子或者基因的3′未转移区或5′未转移区以提高表达或改变所表达蛋白质的特异性。该方法也可用于使核酶或aptamers。
杂合要求存在使多种多样的区域分离开的同源区。类似于支架状的蛋白质结构特别适合于杂合。当显示介导特定结合的相对非限制环时,保守的支架状蛋白确定了由自我联系进行的总体折迭。这样的支架状蛋白的例子是免疫球蛋白β管以及4个螺旋线束(24)。这种杂交方法结合各种组合的用于结合的突变序列可以制备类似支架状的蛋白。
体外杂合
用体外杂合也可以完成等量的一些标准遗传接合。例如,通过将突变体的核酸与野生型核酸重复混合,同时筛选所需的突变就可以完成’分子回交’。象在传统育种中一样,可以用这种方法将来自不同来源的表型结合到所选的背景中。例如,将其用于除去影响非选择特征(即免疫原性)的中性突变。因此,可以用来确定蛋白质中的哪个突变与生物活性的提高有关,而哪一个无关,用易出错误的诱变或盒诱变方法不能得到有利的突变。
可以从小随机片段混合物正确组装大的功能性基因。可以用所述反应对来自化石(25)的许多片段化的DNA进行基因组装。此外,可以将来自化石的随机核酸片段与来自相关种的相似基因的核酸片段结合。
还设想将本发明方法用于体外扩增进行各种研究和诊断应用所需的来自单个细胞的整个基因组。用PCR进行的DNA扩增在实践中限于约40kb的长度。用PCR扩增整个基因组,例如大肠杆菌(5,000kb)的,就需要约250个引物产生125个40kb片段。由于无法得到足够的序列资料,因而该方法不可行。另一方面,用DNAseI随机消化,然后用凝胶纯化小片段会得到许多可能的引物。用随机小片段的混合物在PCR反应中作为引物,单独或与整个基因组一起作为模板,会产生反向链反应,理论上结果产生含许多基因组拷贝的单个连环体。
当只使用随机片段时(见实施例2)可以得到拷贝数扩增100倍,平均片段大小大于50kb的结果。认为较大的连环体是由于许多较小片段的重叠而产生的。用合成引物得到的特异性PCR产物的性质无法与从未扩增的DNA得到的产物区别开。预期可以将所述方法用于基因组作图。
按照实践者的意愿,可以将杂合的多核苷酸制成随机的或非随机片段。
体内杂合
在体内杂合的实施方案中,在至少两种不同核酸序列存在于各宿主细胞的条件下,将特定核酸序列混合物导入到细菌或真核细胞中。可以用各种不同的方法,将所述片段导入到宿主细胞中。可以用本领域已知的方法,例如用氯化钙处理,用所述片段转化宿主细胞。如果所述片段被插入到噬菌体基因组中,则可以用含有特定核酸序列的重组噬菌体基因组转染宿主细胞。另外,可以用电穿孔,转染,脂染(lipofection),biolistics,结合和类似的方法将所述核酸序列导入到宿主细胞中。
总之,在本实施方案中,所述特定的核酸序列将存在于能够在宿主细胞中稳定复制序列的载体中。另外,设想所述载体编码有关标记基因,以便可以筛选含有载体的宿主细胞。这样确保在导入到宿主细胞后,可以回收突变的特定核酸序列。然而,特定核酸序列的全部混合物不必均存在于载体序列上。相反,需要将足够数量的序列克隆到载体中以确保在所述片段导入到宿主细胞中后,各宿主细胞均含有一个携带至少一个存在于其上的特定核酸序列的载体。还设想不光是将特定核酸序列群体的子集克隆到载体中,该子集还已被稳定地整合到宿主细胞中。
已经发现,当将有相同区的两个片段插入到宿主细胞中时,在两个片段之间发生同源重组。在两个突变的特定核酸序列之间的所述重组在某些情况下,将会产生双或三突变体。
还发现如果某些突变的特定核酸序列存在于线性核酸分子上,则重组的频率会提高。因此,在优选的实施方案中,一些特定的核酸序列存在于线性核酸片段上。
转化后,将宿主转化体置于选择条件下,以鉴定含有带有所需性质的特定核酸序列的那些宿主细胞转化体。例如,如果需要对特定药物的增强抗性,则可以使转化宿主细胞经受增加浓度的特定药物,筛选那些产生突变蛋白质的转化体,所述蛋白质能够赋予增加的药物抗性。如果需要提高特定蛋白质与受体结合的能力,则从转化体诱导蛋白质的表达,然后用本领域熟知的方法,经配体结合试验检测所得的蛋白质以鉴定与配体结合能力提高的突变群体的子集。另外,可以在另一系统中表达所述蛋白质以确保适当的加工。
一旦鉴定了携带所需特征的第一重组的特定核酸序列的子集(女儿序列),就可以使其进行第二个循环的重组。
在第二个重组循环中,可以将重组的特定核酸序列与源突变的特定核酸序列(亲本序列)混合,然后,重复上述的循环。用这种方法,可以鉴定一组第二重组的特定核酸序列,它们具有增强的特征或编码有增强的特性的蛋白质。可以按需要将所述循环重复数次。
还设想在第二或随后的重组循环中,可以进行回交。将所需的特定核酸序列与大量野生型序列混合,以便在转化后至少一个野生型核酸序列和突变的核酸序列存在于同一宿主细胞中,以完成分子回交。与野生型特定核酸序列的重组将会消除那些中性突变,所述中性突变会影响非选择特征如免疫原性,而不会影响所选择的特征。
在本发明的另一实施方案中,设想在第一轮中,在导入到宿主细胞之前,可以将特定核酸序列的子集制成片段。片段必须足够大以便含有与其他序列相同的一些区域,从而与所述其他序列进行同源重组。片段的大小为0.03kb到100kb,优选0.2kb到10kb。还设想在随后的数轮中,在导入到宿主细胞之前,将所有特定核酸序列而不是来自前一轮所选的序列裂解成片段。
可以用本领域已知的各种方法完成序列的片段化。可以在核酸序列的特定位点将所述序列制成随机片段或制成片段。通过断裂核酸或使其经严格的物理处理(例如剪切或辐射)或严格化学试剂(例如游离基团;金属离子;酸处理至脱嘌呤和裂解)可以得到随机片段。如果是DNA,也可以用DNase或类似的核酸酶得到随机片段。可以利用限制酶在特定的位点裂解而得到成片段的序列。制成片段的序列可以是单链或双链的。如果所述序列原本是单链的,那么在将其导入到宿主细胞之前可以用热、化学物质或酶将其变性。分开核酸链的合适的反应条件是本领域熟知的。
可以无限地重复本方法的步骤,但只受限于可得到的可能的突变体数目。在一定的循环数后,会得到所有可能的突变体,再循环就是多余的。
在一个实施方案中,重复重组相同的突变的模板核酸,然后筛选有所需特征的重组体。
因此,将突变模板核酸的起始池或群体克隆到能够在细菌,如大肠杆菌中复制的载体中。不需要特定的载体,只要它能够在大肠杆菌中能自我复制。在优选的实施方案中,设计所用的载体以便表达并生产由与载体相连的突变特定核酸编码的任何蛋白质。优选还有含有编码选择性标记的基因的载体。
将含有突变核酸序列池的载体群体导入到大肠杆菌宿主细胞中。可以经转化,转染或用噬菌体感染将载体核酸序列导入。用于转化细菌的载体浓度就是导入到各细胞中的载体量。一旦存在于细胞中后,同源重组的效率就是在不同载体之间发生的同源重组。这样会产生突变体(女儿),它含有不同于源初亲本突变序列的突变的组合。
然后克隆复制宿主细胞并根据在载体上存在的标记基因进行筛选。只有含有质粒的那些细胞才能在选择条件下生长。
接着检测含载体的宿主细胞是否存在有利的突变。例如,如果待选的基因是一种改进的药物抗性基因,所述检测可由在选择的压力下涂布细胞组成。如果载体表达由突变的核酸序列编码的蛋白质,那么所述选择可包括表达所编码的蛋白质,分离所述蛋白质并检测所述蛋白质以确定例如它是否以提高的效率与所需的配体结合。
一旦鉴定了赋予所需特征的特定女儿突变核酸序列,就分离已与载体相连的核酸或从所述载体中分离核酸。然后将所述核酸与第一或亲本群体核酸混合,重复所述循环。
已经表明利用本方法可以筛选具有提高的所需特性的核酸序列。
在另一实施方案中,将第一代突变体保持在细胞中,然后将亲本的突变序列再加到所述细胞中。因此,按上述完成了实施方案I的第一循环。但是,在鉴定女儿核酸序列后,保留含有这些序列的宿主细胞。
将亲本突变的特定核酸序列(不论作为片段还是被克隆到相同的载体中)导入已含有女儿核酸的宿主细胞中。使得在细胞中发生重组,用上述方法筛选下一代重组体或孙女代。
可以将该循环重复许多次直到得到具有所需特征的核酸或肽。设想在随后的循环中,被加到优选突变体中的突变序列群体来自亲本突变体或任何后代。
在另一实施方案中,本发明提供了进行所得重组体的特定核酸的“分子”回交的方法,以便消除任何中性突变。中性突变是未赋予核酸或肽以所需特征的突变。但是,所述突变常常赋予核酸或肽以不需要的特征。因此,需要消除这样的中性突变。本发明的方法就提供了完成此项工作的方法。
在本实施方案中,用本实施方案的方法得到有所需特征的突变核酸后,分离所述核酸,含有所述核酸的载体或含所述载体的细胞。
然后将所述核酸或载体导入带有大量野生型核酸的宿主细胞中。使得突变体的核酸和野生型的核酸序列进行重组。将所得的重组体置于与突变体核酸相同的选择条件下,只有保留了所需特征的那些重组体才会被选择。不提供所需特征的任何不活动突变型通过与野生型DNA重组而被丢失。可以重复所述循环直到消除了所有不活动突变型。
因此可在分子回交中使用本发明的方法,以便消除不必的或不活动的突变。
实用性
可以对未知突变体的池或特定核酸片段或序列的等位基因盲目地使用本发明的体内重组方法。然而不必知道特定核酸片段的实际DNA或RNA序列。
在一个混合的基因群体内使用重组的方法可用于产生任何有用的蛋白质,例如白细胞介素I,抗体,tPA,生长激素等。可以用所述方法生产具有改变的特异性或活性的蛋白质。还可以用所述方法生产突变核酸序列,例如,启动子区,内含子,外显子,增强子序列,基因的3’非翻译区或5’非翻译区。因此可以用该方法产生高表达率的基因。还可用该方法研究重复DNA序列。最后,可以用该方法突变核酶或aptamers。
在蛋白质中分开多样性区的支架状(scaffold)样区特别适用于本发明.1的方法。保守的支架状区通过自我相连而确定所有的折叠,同时显示了介导特异性结合的相对不受限制的环。所述支架状区的实例是免疫球蛋白β管(barrel)和四螺旋束(bundle)。可以用本发明的方法生产具有用于结合的突变序列的不同组合的支架状区样蛋白质。
用本发明方法还可以完成一些标准遗传交配方法。例如通过将突变体核酸与野生型核酸重复混合,同时筛选所需的突变就可以完成“分子”回交。象在传统育种中一样,可以用本方法将不同来源的表型结合到所选的背景中。这对于例如除去影响非选择特征(即免疫原性)的中性突变是有用的。因此,可用其确定在蛋白质中哪个突变与增强的生物学活性有关,哪个无关。
肽展示方法
可以用本发明方法通过公开的体外和/或体内重组方法,并以任何组合杂合用肽展示方法筛选的多核苷酸序列,其中,有关的多核苷酸编码用于表型(如与预定受体(配体)的亲和性)筛选的展示的肽。
生物药物的研究和分子生物学的不断增长的重要性在于鉴定肽结构,包括肽的一级氨基酸序列,或于生物大分子相互作用的肽模拟。鉴定具有所需结构或功能特性,例如与预定生物大分子(如受体)的结合,包括筛选具有由所述肽氨基酸序列赋予的所需结构或功能特性的大文库或个体文库成员的肽。
除用直接的化学合成方法来产生肽文库外,还报道了数种重组DNA方法。一种是在噬菌体颗粒或细胞表面展示肽序列,抗体,或其他蛋白质。通常在这些方法中,各噬菌体颗粒或细胞是作为展示除天然噬菌体或细胞蛋白质序列之外的一种展示肽的个体文库成员。各噬菌体或细胞含有编码特定的展示肽序列的核苷酸序列信息;因此,通过确定所分离文库成员的核苷酸序列可以确定展示肽的序列。
熟知的肽显示方法包括在丝状噬菌体的表面上展示肽序列,通常是与噬菌体被膜蛋白融合。可将噬菌体文库与固定的,预先确定的大分子或小分子(如受体)一起保温,以便将呈现与固定大分子结合的肽序列的噬菌体颗粒与不呈现与预定大分子结合的肽序列的颗粒区别开。然后回收并复制与固定大分子结合的噬菌体颗粒(即文库成员)以扩增所选的噬菌体亚群体以便进行随后一轮的亲和富集及噬菌体复制。亲和富集及噬菌体复制数轮后,分离由此所选的噬菌体文库的成员,然后确定编码展示肽序列的核苷酸序列,从而鉴定与预定大分子(如受体)结合的肽的序列。所述方法在PCT专利公开号91/17271,91/18980,及91/19818和93/08278文本中有进一步的描述。
后一份PCT专利公开文本描述了用于展示肽配体的重组DNA方法,包括用各融合蛋白生产融合蛋白文库,所述融合蛋白由用于与预定大分子进行潜在结合的第一多肽部分,通常含有可变序列,以及结合DNA的第二多肽部分,例如编码单个融合蛋白的DNA载体组成。当在允许表达融合蛋白的条件下培养转化的宿主细胞时,融合蛋白就与编码它的DNA载体结合。将宿主细胞溶解后,用与在基于噬菌体的展示系统中筛选噬菌体颗粒极为相似的方法,对预定的大分子筛选融合蛋白/载体DNA复合物,以DNA载体在所选融合蛋白/载体DNA复合物中的复制和测序作为鉴定所选文库肽序列的基础。
生产肽和类似聚合物文库的其他系统有重组和体外化学合成方法双重功能。在这些杂交方法中,用无细胞的酶促机制完成文库成员(即肽或多核苷酸)的体外合成。在其中的一种方法中,利用改变选择和PCR扩增的轮次来选择具有与预定蛋白质或预定染料分子结合的能力的RNA分子(Tuerk and Gold(1990)Science249:505;Ellington and Szostak(1990)Nature 346:818)。相似的技术用于鉴定与预定的人转录因子结合的DNA序列(Thiesen and Bach(1990)Nucleic Acids Res.18:3203;Beaudry and Joyce(1992)Science 257;635;PCT专利92/05258和92/14843公开文本)。以相似的方式,已经用体外翻译技术合成所需的蛋白质并且作为生产大肽文库的方法。通常依赖于体外翻译,一般包括稳定的多核糖体复合物的这些方法在PCT专利公开88/08453,90/05785,90/07003,91/02076,91/05058和92/02536文本中有进一步的描述。申请人描述了其中文库成员含有融合蛋白的方法,所述融合蛋白含有具有DNA结合活性的第一多肽部分和具文库成员独特肽序列的第二多肽部分。所述方法适用于无细胞体外选择方式。
展示的肽序列其长度可以有所不同,通常为3-5000个氨基酸长或更长,一般为5-100个氨基酸长,且经常为8-15个氨基酸长。文库可以含有具有不同长度的展示肽序列的文库成员,或可含有固定长度的展示肽序列的文库成员。部分或所有展示的肽序列可以是随机的,伪随机的,限定的核心的,固定的等。本发明的展示方法包括用于体外和体内展示单链抗体的方法,例如,在多核糖体上的新单链Fv或在噬菌体上展示的单链Fv,所述方法可以大规模筛选含有较大可变区序列和结合特异性多样性的单链Fv文库。
本发明还提供随机,伪随机和限定的序列骨架肽文库以及生产并筛选这些文库以鉴定有用化合物(如肽,包括单链抗体)的方法,所述化合物以所需模式与受体分子或所需抗原决定簇或修饰肽的基因产物或的RNA结合。从肽文库成员的文库生产随机,伪随机和限定的序列骨架肽,包括肽文库成员附着于展示肽所来自的多核苷酸模板的展示肽或展示的单链抗体。附着模式随本发明的具体实施方案而不同,而且可包括包被在噬菌体颗粒中或掺入到细胞中。
利用亲和富集的方法可以筛选很大的肽和单链抗体文库,并筛选编码所需肽或单链抗体的多核苷酸序列。然后分离多核苷酸序列并杂合以便将所选肽(或其预定部分)或单链抗体(或仅其VH,VL或CDR)的氨基酸序列组合性重组。用这些方法,人们可以鉴定的由于具有对分子的所需的结合亲和性,肽或单链抗体并开发杂合方法以迅速地集中于所需的高亲和肽或scFv(单链Fv)。然后用适用于任何用途(例如治疗或诊断)的常规方法大量合成肽或抗体。
本发明的显著优点是事先不需要考虑预期的配体结构以用来分离所需的肽配体或抗体。所鉴定的肽会有生物学活性,这意味着包括对所选受体分子的特异性结合亲和性,在某些情况下,还包括阻断其他化合物结合、刺激或抑制代谢途径、作为信号或信使、刺激或抑制细胞活性等的能力。
本发明还提供杂合通过亲和筛选展示有新肽(包括单链抗体)的多核糖体的文库而选择的多核苷酸池的方法,所述新肽为结合预定受体(如哺乳动物蛋白类受体,例如peptidergic激素受体,细胞表面受体,与其他蛋白质结合以形成细胞内蛋白质复合物,如异二聚物等的细胞内蛋白质等)或抗原决定簇(例如固定的蛋白质,糖蛋白,寡糖等)的文库成员。
收集用这些方法中的任何方法在第一轮筛选(通常用对受体,如配体结合的亲和选择)中筛选的多核苷酸序列,然后在体外和/或体内重组杂合所述池以生产含有重组的所选多核苷酸序列的群体。使重组的所选多核苷酸序列经至少一次下一轮筛选。在后几轮中筛选的多核苷酸序列可被直接地使用,测序,和/或经一或多轮的杂合和选择。也可以将所选的序列与编码中性序列(即对结合无实质性影响)的多核苷酸序列回交,例如通过与野生型或实质上等同于所选序列的天然产生的序列回交来生产类似天然的,免疫原性较低的功能肽。总之,在回交过程中,用随后的选择保留与预定受体(配体)的结合特性。
在杂合所选的序列之前或同时,可以诱变所述序列。在一个实施方案中,将所选的文库成员克隆在原核载体(如质粒,噬菌粒,或噬菌体)中,其中产生代表不同文库成员的个体菌落(或噬菌斑)的集合。然后对单个的所选的文库成员进行操作(例如经定点诱变,盒诱变,化学诱变,PCR诱变等)以产生代表序列多样性的核心文库成员的集合,所述序列多样性以所选的文库成员为基础。可以对单个所选的文库成员或池的序列进行操作以掺入或者在所述单个所选文库成员序列的片段上,或在其全长上掺入。随机突变,伪随机突变,限定的核心突变(即含有变异体和非变异体残基位置和/或含有可含选自限定氨基酸残基子集的变异体残基位置),密码子为基础的突变等,然后用本文所述的体外和/或他内重组杂合来杂合诱变后的所选文库成员。
本发明还提供含有本发明单个文库成员多样性的肽文库,其中(1)所述多样性的各文库成员含有通过杂合所选序列的池而产生的序列,和(2)各单个文库成员含有在所述多样性中不同于其他单个文库成员的可变肽片段序列或单链抗体序列的可变肽片段序列或单链抗体序列(尽管由于不均匀扩增,随机可能性等,某些文库成员可能以多于1个拷贝/文库存在)。
本发明还提供产物的生产方法,其中具有(或编码的肽具有)预定结合特异性的所选的多核苷酸序列经下列过程形成:(1)筛选抗预定受体(如配体)或抗原决定簇(如抗原大分子)的展示肽或展示的单链抗体文库,然后鉴定和/或富集与所述预定受体或抗原决定簇结合的文库成员以产生所选文库成员的池,(2)通过重组与预定的抗原决定簇结合所选的文库成员(或扩增或克隆其拷贝)并且由此从文库中分离和/或富集而进行杂合以产生杂合的文库,和(3)筛选抗预定受体(如配体)或抗原决定簇(如抗原大分子)的杂合的文库并鉴定和/或富集与预定受体或抗原决定簇结合的文库成员以产生所选杂合文库成员的池。
抗体展示和筛选方法
可以用本发明方法通过任何公开的体外和/或体内重组方法并以任何组合杂合用抗体展示方法筛选的多核苷酸序列,其中有关的多核苷酸编码用于表型(例如用于结合预定抗原(配体)的亲和性)筛选的展示的抗体。
已经设计了各种分子遗传学方法以得到由极大量可存在于免疫球蛋白链上的不同可变区所代表的大量免疫学组分。天然产生的种系免疫球蛋白重链基因座位由位于不同(D)片段基因串联排列上游的可变(V)片段基因的分开的串联排列组成,D片段基因自身位于连接(J)区基因串联排列的上游,J区位于恒定(C)区基因的上游。在B淋巴细胞发育的过程中,发生V-D-J重排,其中通过重排形成融合的D-J片段,然后又与V片段重排形成V-D-J相连的产物基因,从而形成重链可变区基因(VH),如果是生产性重排,V-D-J编码重链的功能可变区(VH)。相似地,轻链基因座位上,数个V片段之一与数个J片段之一重排以形成编码轻链可变区的基因。
免疫球蛋白上许多可能的可变区的所有组成成分部分来自在B细胞发育中重排过程中V和J片段(在重链情况下,和D片段)连接的大量组合性可能性。在重链可变区中其他的序列多样性产生于V-D-J连接过程中非均一地重排和N区的增加。此外,特异性B细胞克隆的抗原选择作用选择在重链和轻链可变区之一或两者中有非种系突变的高亲和突变体;这是一种称为“亲和性成熟”或“亲和性加强”的现象。通常,这些“亲和性加强”集结在可变区的特定区域,常常在互补性决定区(CDRs)。
为了克服通过抗原-刺激的B细胞发育(即免疫)生产和鉴定高亲和性免疫球蛋白的许多限制,已经研究了各种可操作的原核表达系统以生产可用于筛选高亲和性的抗特异性抗原的抗体的组成型抗体文库。在大肠杆菌和噬菌体系统中表达抗体的最近进展(见“Altermative Peptide Display Methods”同下文)已经产生了实际上可通过从表征的杂交瘤克隆抗体基因或用抗体基因文库(如从Ig cDNA)重新筛选得到任何特异性的可能性。
在可筛选为噬菌斑或溶原菌落的噬菌体λ表达系统中已经生产了抗体的组成型文库(Huse et al.(1989)Science 246:1275;Caton and Koprowski(1990)Proc.Natl.Acad.Sci.(U.S.A.)87:6450;Mullinax et al(1990)Proc.Natl.Acad.Sci.(U.S.A.)87:8095;Persson et al.(1991)Proc.Natl.Acad.Sci.(U.SA.)88:2432)。已经描述了噬菌体抗体展示文库和λ噬菌体表达文库的各种实施方案(Kang et al.(1991)Proc.Natl.Acad.Sci.(U.S.A.)88:4363;Clackson et al.(1991)Nature 352:624;McCafferty et al.(1990)Nature 348:552;Burton et al.(1991)Proc.Natl.Acad.Sci.(U.S.A.)88:10134;Hoogenboom et al.(1991)Nucleic Acids Res.19:4133;Chang et al.(1991)J.Immunol.147:3610;Breitling et al(1991)Gene 104:147;Marks et al.(1991)J.Mol.Biol.222:581;Barbas et al.(1992)Proc.Natl.Acad.Sci.(U.S.A.)89:4457;Hawkins and Winter(1 992)J.Immunol.22:867;Marks et al.(1 992)Biotechnology 10:779;Marks et al.(1992)J.Biol.Chem.267:16007;Lowman et al.(1991)Biochemistry 30:10832;Lerner et al.(1992)Science 258:1313;均引入本文作为参考)。通常,用固定(如通过与层析树脂共价结合以便用亲和层析富集反应性噬菌体)和/或标记(如以筛选噬菌斑或挑取菌落)的受体(如多肽,碳水化合物,糖蛋白,核酸)筛选噬菌体抗体展示文库。
一种特别有利的方法是利用所谓单链片段可变(scFv)文库(Marks et al.(1992)Biotechnology 10:779;Winter G and Milstein C(1991)Nature 349:293;Clackson et al.(1991),在所引的书中;Marks et al.(1991)J.Mol.Biol.222:581;Chaudhary et al.(1990)Proc.Natl.Acad.Sci.(U.SA.)87:1066;Chiswell et al.(1992)TIBTECH 10:80;McCafferty et al.(1990)在所引的书中;and Huston et al.(1988)Proc.Natl.Acad.Sci.(U.S.A.)85:5879)。已经描述了各种展示在噬菌体被膜蛋白上的scFv文库的实施方案。
从1988年开始,用抗体基因工程方法就很容易生产Fv片段的单链类似物和其融合蛋白。通常第一步包括得到编码具有所需结合特性的VH和VL区的基因;这些V基因可以从特定的杂交瘤细胞系中分离,或从组成型V-基因文库中筛选或通过V基因合成来制备。通过将成分V基因和编码适当设计的接头肽,例如(Gly-Gly-Gly-Gly-Ser)3或接头肽等同物的寡核苷酸连接起来形成单链Fv。接头桥接第一个V区的C-末端和第二个的N-末端,连接成VH-接头-VL或VL-接头-VH。理论上,scFv结合位点可以精确地复制其亲本抗体结合位点的亲和性和特异性。
因此,scFv片段由通过灵活的接头肽连接到一单链多肽中的VH和VL区组成。组装成scFv基因后,将其克隆到噬菌粒中,然后在M13噬菌体(或相似丝状噬菌体)的顶部表达为与噬菌体pIII(基因3)被膜蛋白融合的融合蛋白。通过培养(panning)重组噬菌体来富集表达所需抗体的噬菌体,所述重组噬菌体带有与预定抗原决定簇(例如靶抗原,受体)结合的scFv群体。
连接的文库成员的多核苷酸提供了在筛选或选择方法后复制文库成员的基础,同时还提供了通过核苷酸测序确定展示的肽序列或VH和VL氨基酸序列相同性的基础。抗原在适宜的表达系统中克隆并表达展示的肽或单链抗体(如scFv)和/或其VH和VL区或其CDRs。经常将编码分离的CDRs的VH和VL区与编码恒定区(CH和CL)的多核苷酸相连以形成编码完整抗体,(如嵌合的或完全的人)抗体片段等。经常将编码分离的CDRs的多核苷酸移植到编码适宜可变区骨架(和可选择的恒定区)多核苷酸中以形成编码完整抗体,(如嵌合的或完全的人的)抗体片段等。可以用抗体通过免疫亲和层析分离制备量的抗原。所述抗体的其他用途是诊断和/或确定疾病的阶段(如瘤形成),用于治疗疾病,例如瘤形成,自体免疫疾病,AIDS,心血管病,感染等。
已经报告了各种方法以增加scFv文库组成型的多样性,从而扩大结合种类(个体基因型谱)的所有组成。用PCR可以迅速从特定杂交瘤或作为基因文库从非固定的细胞中克隆可变区,在可以组合的VH和VL盒的分类中提供组成型多样性。此外,例如通过随机,伪随机或定位诱变可将VH和VL盒自身多样化。通常,在或靠近互补性决定区(CDRs),经常在第三个CDR,CDR3将VH和VL盒多样化。由于有易差错的PCR和化学诱变(Deng et al.(1994)J.Biol.Chem.269:9533),已经表明酶促反向PCR诱变是一种简单可行的构建相对大的scFv定点诱变突变体文库的方法(Stemmer et al.(1993)Biotechniques 14:256)。Riechmann等人((1993)Biochemistry 32:8848)使用通过简并寡核苷酸PCR和所得scFv突变体的噬菌体展示的定点随机化列出了半推理设计的抗体scFv片段。Barbas等人((1992),在所引的书中)试图回避限制所有组分大小的问题,而该问题是由于使用偏移的可变区序列通过随机化在人破伤风类毒素-结合Fab的合成CDR区中的序列而造成的。
CDR随机化有可能仅对重链CDR3的约1×1020就产生CDRs,以及大约相似量的重链CDR1和CDR2突变体和轻链CDR1-3突变体。单独或一起考虑,重链和/或轻链CDR随机化的组成要产生抑制量的噬菌体克隆以生产单独代表所有可能组合的一个克隆文库,其中大多数是非结合的。用目前的转化技术和噬菌体展示系统要生产这样大量的一级转化体并不容易。例如Barbas等人(1992,在所引的书中)只产生了5×107个转化体,这只代表了所有随机化CDRs文库潜在多样性的很小的部分。
尽管有这些实质性的限制,但scFv的噬菌体展示还是产生了各种有用的抗体和抗体融合蛋白。已经表明双特异性单链抗体介导有效的肿瘤细胞溶解(Gruber et al;(1994)J.Immunol.152:5368)。已表明抗-Rev scFv的细胞内表达抑制HIV-1的体外复制(Duan et al.(1994),Proc.Natl.Acad.Sci.(USA)91:5075),抗-p21rasscFv的细胞内表达抑制Xenopus卵母细胞的减数分裂成熟(Biocca et al.(1993)Biochem.Biophys.Res.Commun.197:422)。还报告了用于诊断HIV感染的重组scFv,表明了scFv的诊断实用性(Lilley et al.(1994)J.Immunol.Meth.171:211)。也已经报告了scFv与第二多肽,如毒素或血纤维蛋白激活蛋白相连的融合蛋白(Holvost et al.(1992)Eur.J.Biochem.210:945;Nicholls et al.(1993)J.Biol.chem.268:5302)。
如果可能生产具有更多抗体多样性并克服常规CDR诱变和随机化方法的许多限制的scFv文库,就可以使适用于治疗和诊断用途的scFv抗体的数量和质量得到很大的提高,所述的常规CDR诱变和随机化方法只能覆盖潜在序列组合中很小的部分。为了达到这一目的,用本发明的体外和体内杂合方法重组已经从核酸得到(通常经PCR扩增或克隆)的CDRs,所述核酸是从筛选的展示抗体得到的。可以在细胞上,噬菌体颗粒上,多核糖体或其中有抗体与编码它的核酸相连的适宜的抗体展示系统上展示所述的展示抗体。在改变中,CDRs开始是从生产抗体的细胞(例如来自免疫的野生型小鼠,人或能够生产WO92/03918,WO93/12227和WO94/25585中的人抗体的转基因小鼠的浆细胞/脾细胞),包括从其得到的杂交瘤的mRNA(或cDNA)得到的。
收集用任何这些方法在第一轮筛选(通常通过亲和性筛选与抗原(例如,配体)结合的展示的抗体)中筛选的多核苷酸序列,然后经体外和/或体内重组进行杂合,特别是杂合CDRs(通常杂合重链CDRs和其他重链CDRs以及轻链CDRs和其他轻链CDRs)以得到杂合的池,所述池含有重组的筛选过的多核苷酸序列群体。将重组的选择的多核苷酸序列在选择方式中表达随后再经至少一轮筛选。直接使用,测序在随后的选择中筛选的多核苷酸序列,和/或使其经一次或多次的杂合和筛选过程,直到得到具有所需结合亲和性的抗体。也可以将所选的序列与编码中性抗骨架序列的多核苷酸序列(即对结合抗原没有实质性的功能影响)回交,例如通过与人可变区骨架回交以得到类似人的抗体序列。总而言之,在回交过程中,用随后的选择作用来保留与预定抗原结合的特性。
另外,或与注明的改变相结合,可以改变靶抗原决定簇的效价以控制所选scFv文库成员的平均结合亲和性。可以将靶抗原决定簇以不同的密度,通过例如包括竞争抗原决定簇,通过稀释或通过本领域已知的其他方法,与表面或底物结合。可以用高密度(效价)的预定抗原决定簇富集具有相对低亲和性的scFv文库成员,而低密度(效价)可优选地富集较高亲和性的scFv文库成员。
为了产生不同的可变片段,通过连接到预定位点(如CDR)插入编码随机,伪随机,或限定的核心序列组肽序列的合成寡核苷酸插入集合。相似地,用定点诱变,CDR-取代等通过突变CDR来扩展单链抗体盒的一个或多个CDRs的序列多样性。在杂合前,可以在克隆和扩增的宿主中增殖所得的DNA分子,或将其直接使用(即可以避免在宿主细胞中增殖时可能发生的丢失),然后杂合所选的文库成员。
通过亲和富集技术从所述文库中筛选编码所需可变片段肽序列或所需单链抗体的肽/多核苷酸复合物(文库成员)。利用固定的大分子或对所需肽序列特异性的抗原决定簇,例如受体,其他大分子,或其他抗原决定簇达到所述目的。重复亲和筛选方法可以富集编码所需序列的文库成员,然后将其分离以进行收集和杂合,测序和/或进行进一步增殖和亲和富集。
通过洗涤除去没有所需特异性的文库成员。确定对于所需的各肽序列或单链抗体及固定的预定大分子或抗原决定簇所需的洗涤程度和严谨度。通过调节结合温育和随后洗涤的条件,可以在一定程度上控制回收的新肽/DNA复合物的结合特征。在固定的大分子亲和性的特定范围内,将选择用于新肽/DNA复合物的温度,pH,离子强度,二价阳离子浓度以及洗涤体积和持续的时间。以慢解离速率为基础的选择(通常预测到高亲和性)是最常用的实施途径。通过在存在饱和量的游离预定大分子条件下继续保温或通过增加洗涤体积,次数和时间长度来达到所述目的。在各情况下,要预防解离的新肽/DNA或肽/RNA复合物的再结合,而且随着时间的增加,回收亲和性越来越高的新肽/DNA或肽/RNA复合物。
也可以使用对结合和洗涤过程有其他改进的方法以发现具有特殊特征的肽。某些肽的亲和性依赖于离子强度或阳离子浓度。当需要从肽中除去蛋白质的条件柔和时,这对于在各种蛋白质的亲和纯化中使用的肽来说是有用的特征。
一种改变是利用多种结合靶(多种抗原决定簇,多种受体)以便同时筛选scFv文库,筛选具有不同结合特异性的多重性的scFv。由于scFv文库的大小经常限制潜在scFv序列的多样性,因此,一般我们希望scFv文库尽可能地大。出于时间和经济上的考虑,产生许多很大的多核糖体scFv-展示文库会受到限制。为了避免这个实际问题,在单个文库中可以同时筛选多种预定的抗原决定簇(受体),或对大量抗原决定簇进行依次筛选。在一种改变中,可以混合在不同珠(或珠组)上编码的多种靶抗原决定簇,然后在适宜的结合条件下与多核糖体-展示scFv文库保温。然后通过亲和筛选,收集的含有多种抗原决定簇的珠来分离scFv文库成员。通常,随后的亲和筛选过程包括混合相同的小珠,其子集或含有仅一种或两种抗原决定簇的小珠。所述方法提供了有效的筛选,而且与实验室自动化,批量加工,筛选方法的高处理量相协调。
在本发明中可以使用各种技术以便使肽文库或单链抗体文库多样化,或在杂合前或同时,使在较早的panning过程中发现的可变片段肽或VH,VL或CDRs周围多样化使其对预定大分子或抗原决定簇具有足够的结合活性。在一种方法中,将筛选的阳性肽/多核苷酸复合物(在较早的亲和富集过程中鉴定的那些)测序以确定活性肽的一致性。然后在这些活性肽的基础上合成寡核苷酸,在各步骤中通过低水平的所有碱基掺入以得到有轻微改变的一级寡核苷酸序列。将所述(轻微)简并寡核苷酸的混合物在适当的位置克隆到可变片段序列中。这种方法对起始肽序列产生了系统的控制的改变,然后可用于杂合。然而,它要求在诱变前对单个阳性新肽/多核苷酸复合物测序,因此对于扩大少量所回收的复合物的多样性并筛选具有更高结合亲和性和/或更高结合特异性的变异体来说是有用的。在一种改变中,经诱变PCR扩增所选的阳性肽/多核苷酸复合物(特别是可变区序列,在体外和/或体内杂合的扩增产物)并在测序前再进行一轮或多轮的筛选。对于单链抗体可以使用大致相同的方法以便通过在杂合前或同时将CDRs或相邻的骨架区多样化而扩大多样性并提高结合亲和性/特异性。如果需要,用能够与包括所选文库成员进行体外重组的诱变寡核苷酸抑制杂合反应。因此,可以将合成的寡核苷酸和PCR片段(用易于差错或高精确度方法合成的)混合物加到体外杂合混合物中,然后掺入到所得的杂合文库成员(杂合体)中。
本发明的杂合能够产生大的CDR-变异体单链抗体文库。产生所述抗体的一种方法是在杂合前或同时将合成的CDRs插入到单链抗体和/或随机化的CDR中。通过参考已知的人CDR的序列资料来筛选合成的CDR盒序列并且根据下列原则由实施慎重选择:合成的CDRs与已知CDR序列至少有40%的序列一致性,而且优选的与已知序列至少有50-70%的一致性。例如,在Kabat等人(1991,在所引的书中)所列的天然产生的人CDR序列的基础上,通过合成一批寡核苷酸序列来产生一批合成的CDR序列;计算合成CDR序列的池以使其编码与至少一种已知的天然产生的CDR序列有至少40%一致性的CDR肽序列。另外,可以比较一批天然产生的CDR序列以得到一致序列以便将在频率高的残基位置(即占已知CDR序列至少5%)所用的氨基酸掺入到合成CDRs的相应位置。通常比较并观察一些(如3到约50个)一致的CDR序列,将一致CDRs之间的天然序列变异列成表,然后合成一批编码CDR肽序列的寡核苷酸,所述CDR肽序列包括所有或大多数观察到的天然序列变异的置换。例如并不是为了限制,如果一批人VHCDR序列含有以Tyr,Val,Phe,或Asp为C-末端氨基酸,那么就可以设计合成CDR寡核苷酸序列的池以使所述C-末端CDR残基是这些氨基酸中的任何一种。在一些实施方案中,掺入除在CDR序列中的残基位置上的天然残基之外的残基:经常插入保守的氨基酸取代物,而且可以改变高达5个残基位置以掺入与天然CDR序列相比是非保守的氨基酸取代。可以将所述CDR序列用于一级文库成员(在第一轮筛选前)和/或可用其抑制所选文库成员序列的体外杂合反应。本领域专业人员很容易构建限定的和/或简并序列的所述池。
所述合成的CDR序列含有至少一个不是已知天然CDR序列的成员。实施者可任选包括或不包括相对于重链CDR N区的随机或伪随机序列部分;在V-D和D-J的连接中N区序列的范围是1个核苷酸至约4个核苷酸。一批合成的重链CDR序列含有至少约100个特有的CDR序列,通常至少为1,000个特有的CDR序列,优选地为至少约10,000个特有的CDR序列,经常多于50,000个特有的CDR序列;但是在一批中通常不超过1×106个特有的CDR序列,尽管有时存在1×107-1×108个特有CDR序列,特别是如果在天然人CDRs中,在没有保守氨基酸取代物的位置或在该位置保守氨基酸取代物稀少(即少于0.1%)的情况下,允许保守氨基酸取代。总之,在文库中所包括的特有CDR序列的数量不能超过所述文库中一级转化体预期数量的10倍以上。所述单链抗体通常可以以约至少1×107M-1的亲和性,优选有约至少5×107M-1的亲和性,更优选的约至少1×108M-1到约至少1×109M-1的亲和性或更高,有时高达1×1010M-1或更高的亲和性与预定抗原(如免疫原)结合。通常预定抗原是人蛋白质,例如人细胞表面抗原(如CD4,CD8,IL-2受体,EGF受体,PDGF受体),其他人生物大分子(如血栓调节蛋白,蛋白质C,碳水化合物抗原,sialyl Lewis抗原,L-选择素(selectin)),或与人疾病无关的大分子(如细菌LPS,病毒颗粒衣壳蛋白或被膜糖蛋白)等。
抗原在各种系统中加工并表达具有所需特异性的高亲和单链抗体。例如已经在植物中生产了scFv(Firek et al.(1993)Plant Mol.Biol.23:861),而且很容易在原核系统中制备(Owens RJ(1994)J.Immunol.Meth.168:149;Johnson S and Bird RE(1991)Methods Enzymol.203:88)。此外,可以用单链抗体作为构建完全抗体或其各种片段(Kettleborough et al.(1994)Eur.J.Immunol.24:952)。可以分离(如经PCR扩增或亚克隆)可变区编码序列并将其与编码所需人恒定区的序列相拼接以编码人抗体序列,所述抗体更适于免疫原性最小的的人治疗应用。在宿主细胞中表达含有所得的人编码序列的多核苷酸(例如在哺乳动物细胞中从表达载体开始),然后将其纯化用于药物配方。
DNA表达构建体通常包括与编码序列可操作相连的表达控制DNA序列,包括天然相关的或异源的启动子区。优选的,表达控制序列是在能够转化或转染真核宿主细胞的载体中的真核启动子系统。一旦所述载体被插入到适宜的宿主中,就在适于高水平表达核苷酸序列的条件下保持所述宿主,然后收集纯化突变的“工程”抗体。
正如上文所述,在将DNA序列与表达控制序列可操作相连(即定位以确保结构基因的转录和翻译)后,在宿主中表达所述序列。这些载体通常作为附加体或宿主染色体DNA的整合部分在宿主生物体中是可复制的。总之,表达载体将含有选择性标记如,四环素或新霉素以便检测带有所需的DNA序列的那些转化细胞(见,如美国专利4,704,362,该文献引入本文作为参考)。
除象酵母这样的转化微生物外,也可以用哺乳动物组织细胞生产本发明的多肽(见Winnacker,“From Genes to Clones,”VCH Publishers,N.Y.,N.Y.(1987),该文献引入本文作为参考)。实际上真核细胞是优选的,原因在于在本领域中已经研究了许多能够分泌完整免疫球蛋白的适宜的宿主细胞系,而且包括CHO细胞系,各种COS细胞系,Hela细胞系,骨髓瘤细胞系等,但最好是转化的B-细胞或杂交瘤。用于这些细胞的表达载体包括表达控制序列,如复制源点,启动子,增强子(Queen et al.(1986)Immunol.Rev.89:49),和必需的加工信息位点,如核糖体结合位点,RNA拼接位点,多腺苷酸化位点以及转录终止序列。优选的表达控制序列源于免疫球蛋白基因,细胞肥大病毒,SV40,腺病毒,牛乳头状瘤病毒等的的启动子。
通过将增强子序列插入到载体中来提高真核DNA的转录。增强子是10-300bp的提高启动子转录的顺式作用序列。当在转录单位5′或3′时,增强子可以有效地提高转录。如果在内含子内或在其自身编码序列内,它们也是有效的。通常使用的表达增强子包括SV40增强子,细胞肥大病毒增强子,多形瘤增强子和腺病毒增强子。来自哺乳动物系统的增强子序列也是常用的,例如小鼠免疫球蛋白重链增强子。
哺乳动物表达载体系统通常还包括可选择的标记基因。适宜标记基因的实例包括二氢叶酸还原酶(DHFR),胸苷激酶基因(TK)或赋予药物抗性的原核基因。前两种标记倾向于使用在未将胸苷加到生长培养基中就不能生长的突变体细胞系。根据其在非补充培养基中生长的能力来鉴定转化的细胞。作为标记的有用的原核药物抗性基因的实例包括赋予G418,霉酚酸和潮霉素抗性的基因。
根据细胞宿主类型,用熟知的方法,可以将含所需的DNA片段的载体转移到宿主细胞中。例如常用于原核细胞的氯化钙转染,而磷酸钙处理。脂染或电穿孔用于其他细胞宿主。用于转化哺乳动物细胞的其他方法包括使用聚凝胺,原生质体融合,脂质体,电穿孔和微注射(见,Sambrook et al.同上文)。
一旦表达后,可以用本领域的标准方法,包括硫酸铵沉淀,分离柱层析,凝胶电泳等纯化本发明的抗体,个体突变的免疫球蛋白链,突变的抗体片段和其他免疫球蛋白多肽(见Scopes R.,Protein Purification,Springer-Verlag,N.Y.(1982))。纯化后,按照部分或均一性的要求,将多肽用于治疗或研究和完成检测方法,没有荧光染色等(见,Immunological Methods,Vols.I and II,Eds.Lefkovits and Pernis,Academic Press,New York,N.Y.(1979 and 1981))。
用本发明方法生产的抗体可用于诊断和治疗。用以说明而不是限制为目的,可以用它们治疗癌,自体免疫疾病或病毒感染。为了治疗癌,通常抗体将与常在癌细胞上表达的抗原,例如erbB-2,CEA,CD33以及许多其他抗原和本领域专业人员熟知的结合成员结合。
酵母两杂交筛选试验
可以用杂合重组对通过筛选两杂交筛选系统来鉴定结合预定多肽序列的文库成员而得到的所选文库成员池多样化。收集所选的文库成员,然后进行体外和/或体内重组。然后在酵母两杂交系统中筛选杂合的池以筛选结合所述预定多肽序列(如SH2区)或结合其他预定多肽序列(如来自另一蛋白质的SH2区)的文库成员。
鉴定结合预定多肽序列的多肽序列鉴定方法已经使用了所谓“两杂交”系统,其中预定多肽序列存在于融合蛋白中(Chien et al.(1991)Proc.Natl.Acad.Sci.(USA)88:9578)。所述方法通过重建转录活化因子(Fields S and Song O(1989)Nature 340:245),酵母Ga14转录蛋白来鉴定蛋白质-蛋白质之间的相互作用。通常,该方法是以酵母Ga14蛋白质的特性为基础的,所述蛋白质由导致DNA结合和转录活化的可分开的区组成。构建编码两种杂交蛋白质的多核苷酸并将其导入到酵母宿主细胞中,所述两种杂交蛋白,一种由与已知蛋白质的多肽序列融合的酵母Ga14 DNA结合区组成,另一个由与第二蛋白质的多肽序列融合的Ga14活化区组成。两种融合蛋白之间的细胞间结合重建了Ga14 DNA结合区和Ga14活化区,这样导致与Ga14结合位点可操作相连的报告基因(如lacZ,HIS3)的转录活化。通常用两杂交方法鉴定与已知蛋白质相互作用的新多肽(Silver SC and Hunt SW(1993)Mol.Biol.Rep.17:155;Durfee et al.(1993)Genes Devel.7:555;Yang et al.(1992)Science 257:680;Luban et al.(1993)Cell 73:1067;Hardy et al.(1992)GenesDevel.6;801;Bartel et al.(1993)Biotechniques 14:920;and Vojtek et al.(1993)Cell74:205)。但是,已经用改进的两杂交方法鉴定影响其与第二已知蛋白质结合的已知蛋白质的突变体(Li B and Fields S(1993)FASEB J.7:957;Lalo et al.(1993)Proc.Natl.Acad.Sci.(USA)90:5524;Jackson et al.(1993)Mol.Cell.Biol.13:2899;andMadura et al.(1993)J.Biol.Chem.268:12046)。还已经用两杂交系统鉴定了两个已知蛋白质结构区(Bardwell et al.(1 993)med.Microbiol.8:1177;Chakraborty et al.(1992)J.Bio.Chem.267:17498;Staudinger et al.(1993)J.Biol.Chem.268:4608;andMilne GT and Weaver DT(1993)Genes Devel.7:1755)或引起单个蛋白质寡聚的区(Iwabuchi et al.(1993)Oncogene 8:1693;Bogerd et al.(1993)J.Virol.67:5030)之间的相互作用。还用改进的两杂交系统研究蛋白水解酶的体内或(Dasmahapatra etal.(1992)Proc.Natl.Acad Sci.(USA)89:4159)。另外,可以用大肠杆菌/BCCP相互作用筛选系统(Germino et al.(1993)Proc.Natl.Acad Sci.(USA)90:933;GuarenteL(1993)Proc.Natl.Acad Sci.(USA)90:1639)鉴定相互作用的蛋白质序列(即异二聚的或形成更高级异多聚体的蛋白质序列)。收集用两杂交系统选择的序列,将其杂合,然后导入到用于随后一或多轮筛选以鉴定与含预定结合序列结合的多肽序列的两杂交系统中。将由此鉴定的序列进行比较以鉴定一致序列或一致序列核心。
从上述公开内容可以理解,本发明有广泛的应用。因此提供下列实施例是为了说明而不是限制。
在下列实施例中,下列缩写有下列含义。如果在下文未说明,那么所述缩写含有本领域已知的含义。
ml=毫升
μl=微升
μM=微摩尔
nM=毫微摩尔
PBS=磷酸缓冲的盐
ng=毫微克
μg=微克
IPTG=异丙基硫代-β-D-半乳糖苷
bp=碱基对
kb=千碱基对
dNTP=三磷酸脱氧核苷酸
PCR=聚合酶链反应
X-gal=5-溴-4-氯-3-吲哚基-β-D-半乳糖苷
DNAseI=脱氧核糖核酸酶
PBS=磷酸缓冲的盐
CDR=互补性决定区
MIC=最小抑制浓度
scFv=抗体的单链Fv片段
总之,在各种文献中描述了标准的重组DNA技术,例如Sambrook et al.,1989,Molecular Cloning:A Laboratory Manual,Cold Spring Harbor Laboratry;Ausubel et al.1987,Current Protocols in Moleculat Biology,Vols 1 and 2和补充,and Berger andKimmel,Methods in Enzymology,Volume 152,Guide to Molecular ClongingTechniques(1987),Academic Press,Inc.Sam Diego,Ca,以上文献均全部引入本文作为参考。按照制造商的说明使用限制酶和多核苷酸修饰酶。使用ABI混合物,在Applied Biosystems Inc.Model 394 DNA合成仪上合成寡核苷酸。如果需要,根据实践者的需要选择扩增预定DNA序列的PCR引物。
实施例
实施例1.LacZα基因重装配
1)底物制备
重组反应的底物是来自pUC18(图2)(28;Gene BankNo.XO2514)的野生型LacZα基因的dsDNA聚合酶链反应(“PCR”)产物。引物序列是5′AAAGCGTCGATTTTTGTGAT3′(SEQ ID NO:1)和5′ATGGGGTTCCGCGCACATTT3′(SEQ ID NO:2)。根据厂家说明书以Wizard PCR制品(Promega,Madison WI)从PCR产物中去掉游离的引物。发现游离引物的清除是重要的。
2)DNAseI消化:
用溶于100μl[50mM Tris-HCl pH7.4,1mM MgCl2]中的0.15单位的DNAseI(Sigma,St.Louis MO)在室温下消化大约5μg DNA底物10-20分钟。消化的DNA在2%低熔点琼脂糖凝胶上电泳。经过电泳到DE81离子交换纸(Whatman,Hillsborough OR)上从2%低熔点琼脂糖凝胶上纯化10-70碱基对(bp)的片段。用1M NaCl从纸上洗脱出DNA片段并用乙醇沉淀。
3)DNA重装配:
将纯化的片段以10-30ng/μl的浓度重悬于PCR混合物(各0.2mM dNTP,2.2mM MgCl2,50mM KCl,10mM Tris-HCl pH9.0,0.1%Triton X-100,0.3μl Taq DNA聚合酶,总体积50μl)。在此时不加引物。在MJ Research(Watertown MA)PTC-150热循环器中使用的重组程序是94℃ 60秒,30-45个循环的[94℃ 30秒,50-55℃ 30秒,72℃ 30秒]及72℃ 5分钟。小片段重组进较大序列的PCR后在重组25,30,35,40和45个循环后取反应样品(图2)。
由于100-200bp片段的重组可产生正确大小的单个PCR产物,10-50碱基片段典型地产生正确大小的一些产物以及不均一分子量的产物。大多数这种大小不均一性似乎是由于产物末端是单链序列,因为限制性酶消化后获得正确大小的单一带。
4)有引物的PCR
将重组产物稀释进含上述引物(SEQ ID NO:1和2)各0.8μM的PCR混合物中并进行由[94℃ 30秒,50℃ 30秒,72℃ 30秒]组成的循环大约15次PCR循环后,获得正确大小的单一产物(图2)。
5)克隆和分析:
用末端限制性酶BamHI和EcoO109消化上面第4步的PCR产物并按上面第2步所述进行凝胶纯化。将重组的片段连接进用BamHI和EcoO109消化的pUC18上。按厂家建议(Stratagene,San Diego CA)在标准条件下用连接混合物转化大肠杆菌并涂布到含100μg/ml氨苄青霉素,0.004%X-gal和2mM IPTG的琼脂板上。鉴别出所得的鉴定为++重组子的具有HinD III-NheI片段的菌落,因为它们是蓝色的。
本实施例说明了携带LacZα基因的1.0kb序列可消化成10-70bp的片段且这些凝胶纯化的10-70bp片段可重组进正确大小的单一产物中以便84%(N=377)的所得的菌落是LacZ+(相对于无交换的94%;图2)。
编码来自所得LacZ-菌落的LacZ基因的DNA根据厂家说明书用测序试剂盒(美国生化公司,Cleveland OH)进行测序,发现该基因由于重组过程而具有点突变(表1)。发现11/12类型的取代且无阅读框。
表1
经诱变交换导入突变
转变 频率 易位 频率
G-A 6 A-T 1
A-G 4 A-C 2
C-T 7 C-A 1
T-C 3 C-G 0
G-C 3
G-T 2
T-A 1
T-G 2
测序了总共4,437碱基的交换LacZ DNA
从DNA测序测定10-70bp片段的DNA重组期间点诱变率为0.7%(N=4,473),这与PCR误差倾向相似。由于不受任何理论的限制,据信如果较大片段用于重组或如果加入防止阅读聚合酶,点诱变率可能较低。
当组合14个这些点突变的LacZ-菌落的质粒DNA并以上述方法再重组/交换时,34%(N=291)的所得菌落是LacZ+,这些菌落推测由不同菌落的DNA重组产生。
假定诱变率为0.7%(10),PCR误差引起的单一点突变的预期回复率可能是<1%。
因此,可从小片段的随机混合物经过效率惊人且简单的反应重组大DNA序列。本技术的一个应用是基于同源性的相关序列的重组或交换。
实施例2.LacZ基因和完整质粒DNA杂合
1)LacZ基因的杂合
使用2个LacZ基因构建体测量以75个碱基分开两个标记之间的交换。在LacZα基因的两个分隔区插入终止编码子用作阴性标记。各标记是具有4个终止密码子的25bp非同源序列,其中两个在LacZ基因阅读框中。25bp非同源序列在图3中以大盒表示。终止密码子以盒表示或划线。含LacZα基因+-和-+型(图3)的2个1.0kb LacZ模板的1∶1的混合物用DNAseI消化并按实施例1所述纯化出100-200bp的片段。除了加入0.5μl聚合酶且总体积为100μl外在与实施例1的重组中所述相似的条件下进行交换程序。
克隆后,获得的蓝色菌落数是24%(N=386);它与兰色菌落的理论最大数(即,25%)相近,表明在两个标记之间完成了重组。所有10个蓝色菌落含预期的HindIII-NheI限制性片段。
2)完整质粒DNA的杂合
也试验了完整2.7kb质粒(pUC18-+和pUC18+-)。用DNA seI消化含LacZα的+-和-+型(图3)的两个2.9kb质粒的1∶1混合物并按实施例1所述纯化100-200bp的片段。除了进行[94℃ 30秒,55℃ 30秒,72℃ 30秒]的60次循环外在与上面步骤(1)重组所述相似的条件下进行交换程序。凝胶分析表明:经过交换程序后,大多数产物大于20kb。因此,完整2.7kb质粒(pUC18-+和pUC18+-)从不加引物的随机100-200bp片段中高效重组。
用在质粒上具有单一位点的限制性酶(EcoO109)消化后,大多数产物由预期大小的单一带组成。将该带凝胶纯化,再连接并将DNA用于转化大肠杆菌。转化子按实施例1所述涂布在0.004%X-gal板上。所得的质粒11%(N=328)是兰色,因而是++重组子。
3)峰DNA杂合
混合进交换混合物中的寡核苷酸可基于该寡核苷酸侧翼序列与模板DNA的同源性掺入终产物中(图4)。上面描述的LacZ-终止密码突变子(pUC18-+)用作DNAseI消化的模板。将在两端包括与野生型LacZ基因同源的18个碱基的66聚体寡核苷酸以超出原始基因中存在的正确终止密码突变4倍的摩尔数加入到反应中。在与上面步骤2相似的条件下进行交换反应。消化所得的产物,连接后按上面所述插入大肠杆菌中。
表2
兰菌落%
对照 0.0(N>1000)
上链峰 8.0(N=855)
下链峰 9.3(N=620)
上、下链峰 2.1(N=537)
假定由于竞争性杂交,ssDNA似乎比dsDNA更有效。经过调节超出的摩尔数,退火温度,或同源性长度可大范围地改变插入程度。
实施例3.在完全缺乏引物的DNA重装配
用限制性酶EcoRI,EcoO109,XmnI和AlwNI消化质粒pUC18,产生大约370,460,770和1080bp的片段。电泳并从2%低熔点琼脂糖凝胶中分别纯化这些片段(370和460碱基的带不能分离)。产生在3个单独的试管中的大型片段,中型片段和2个小片段的混合物。
按实施例1所述用DNAseI消化各片段,对于各原始片段从2%低熔点琼脂糖凝胶中纯化50-130bp的片段。
自PCR混合物(如上面实施例1所述)中加入纯化的消化片段至片段终浓度为10ng/μl。不加入引物。分别对三个消化的DNA片段混合物进行重组反应75次循环[94℃ 30秒,60℃ 30秒]以琼脂糖电泳分析产物。
结果清楚地显示从纯化片段中有效地重新形成的1080,770和370及460bp的带,证实交换根本不需使用任何引物。
实施例4.IL-1β基因杂合
本实施例描述了可获得基于同源性不超过15个碱基的交换。作为一个例子,交换人和鼠IL-1β基因。
具有大肠杆菌密码子用法的鼠IL1-β基因(BBG49)和人IL1-β基因(BBG2;R&D Systems,Inc.Minneapolis MN)用作交换反应中的模板。人和鼠IL-1β序列的全部同源区平均仅4.1碱基长(图5,同源区用盒表示)。
各基因dsDNA PCR产物的制备,引物的清除,DNAseI消化和10-50bp片段的纯化与上面实施例1所述相似。用于PCR反应的引物序列是:5′TTAGGCACCCCAGGCTT3′(SEQ ID NO:3)和5′ATGTGCTGCAAGGCGATT3′(SEQ ID NO:4)。
用DNA聚合酶I的Klenow片段进行交换反应的前15个循环,在每次循环中加入1单位的新鲜酶。将DNA加入实施例1的PCR混合物中,该混合物没有聚合酶。操作方法是94℃ 1分钟,然后进行15次[95℃ 1分钟,干冰/乙醇上10秒(直到冷冻),25℃保温大约20秒,加入1U的Klenow片段并在25℃保温2分钟]的循环。在每次循环中经变性步骤后,在干冰/乙醇中将试管迅速冷却并预热到退火温度。然后加入热变性聚合酶。在每次循环中都需加入该酶。使用本方法,获得了高水平的变换,其基础仅是未间断的具同源性的少数碱基(图5,交换位置以
表示)。
经过这15次循环操作后,加入Taq聚合酶,进行无引物的另外22次交换反应循环[94℃30秒,35℃30秒]。
然后将反应物稀释20倍。加入下列引物至终浓度为0.8μM:5′AACGCCGCATGCAAGCTTGGATCCTTATT3′(SEQ ID NO:5)和5′AAAGCCCTCTAGATGATTACGAATTCATAT3′(SEQ ID NO:6),按上面实施例1所述进行PCR应。第二引物对与第一对不同,仅仅因为据信限制性位点的改变是必需的。
用XbaI和SphI消化PCR产物后,将片段连接进XbaI-SphI-消化的pUC18。用双脱氧DNA测序试剂盒(美国生化公司,Cleveland OH)根据厂家说明书测定一些菌落的插入序列。
经过对9个菌落的DNA测序发现总共17个交换。一些交换仅以未间断同源的1-2个碱基为基础。
发现为了加强以短同源性为基础的有效交换,需要非常低的有效退火温度。使用任何热稳定性聚合酶,PCR机器的冷却时间(以1-2度/秒从94℃到25℃)引起的有效退火温度比系统的退火温度更高。因此,以Taq聚合酶为基础的方法都不能产生交换,即使所使用的超出IL1-β基因的10倍。相反,热变性聚合酶,如DNA聚合酶I的Klenow片段可用于精确地获得低退火温度。
实施例5.TEM-1β内酰胺酶基因的DNA杂合
在β内酰胺酶模型系统上试验了直接分子进化的诱变DNA变换的实用性。TEM-1β内酰胺酶是一种非常有效的酶,主要以扩散限制其反应速率。本实施例确定了改变其反应特异性和获得对药品头胞噻肟的抗性是否可能,正常情况下它不水解头胞噻肟。
经过在具有不同水平的头胞噻肟(Sigma,St.Louis MO)平板上涂布10μl 10-2稀释的大肠杆菌XL1-blue细胞(Stratagene,San Diego CA)的过夜细菌培养物(大约1000cfu)确定头胞噻肟对缺乏质粒的细菌细胞的最低抑制浓度(MIC),接着在37℃下培养24小时。
在头胞噻肟上生长对稠密的细胞具有敏感性,因此需要将相似的细胞数涂布到各板上(经过在简单LB板上涂布获得)。均匀地进行1000个细胞的涂布。
1)起始质粒构建
使用携带细菌性TEM-1β-内酰胺酶基因的pUC18衍生物(28)。TEM-1β-内酰胺酶基因赋予细菌对大约0.02μg/ml的头胞噻肟产生抗性。用下列两个引物对载体序列进行PCR:Primer A(SEQ ID NO:7):5′TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGATTT3′andPrimer B(SEQ ID NO:8):5′TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCACATTT3′以及用其它如下两个引物对β内酰胺酶基因序列进行PCR:Primer C(SEQ ID NO:9):5′AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT,和Primer D(SEQ ID NO:10):5′AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT。
将Sfi1限制性位点加到启动子5′端和该基因3′端。
两个反应产物用SfiI消化,混合,连接并用于转化细菌。
所得的质粒是pUC182Sfi。该质粒含有携带TEM-1曲因和P-3启动子的Sfi1片段。
37℃培养24小时后头胞噻肟对携带该质粒的大肠杆菌XL1-blue(Stratagene,San Diego CA)最低抑制浓度是0.02μg/ml。
经过逐步在2倍增加的药物水平上再涂布稀释库的细胞(大约107cfu)确定无交换时β内酰胺酶基因对头胞噻肟抗性增加的能力。无交换时可获得高达1.28μG/ml的抗性。这代表抗性增加64倍。
2)DNAseI消化
第一次交换反应的底物是经过用引物C和D(两者都含SfiI位点)对pUC182Sfi进行PCR获得的0.9kb的dsDNA。
以Wizard PCR制品(Promega,Madison WI)在每次循环时去掉PCR产物的游离引物。
用在100μl 50mM Tris-HCl pH7.4,1mM MgCl2中的0.15单位的DNAseI(Sigma,St.Louis MO)在室温下消化大约5μg的DNA底物10分钟,经电泳到DE81离子交换纸(Whatman,Hillsborough OR)上从2%低熔点琼脂凝胶上纯化100-300bp的片段,以实施例1所述的方法用1M NaCl洗脱和乙醇沉淀。
3)基因杂合
将纯化片段以10-30ng/μl的浓度重悬于PCR混合物(各0.2mM的dNTP,2.2mMMgCl2,50mM KCl,10mM Tris-HCl pH9.0,0.1%Triton X-100)。此时不加引物。在MJ Research(Watertown MA)PTC-150热循环器中使用的重组方法是94℃ 60秒,然后进行40次[94℃ 30秒,50-55℃ 30秒,72℃ 30秒]的循环,再72℃ 5分钟。
4)用引物扩增重装配产物:
将重组产物稀释进含引物(C和D)各0.8μM的PCR混合物并进行20次[94℃ 30秒,50℃ 30秒,72℃ 30秒]的PCR循环后,获得900bp大小的单一产物。
5)克隆和分析:
用末端限制性酶SfiI消化900bp的产物并进行琼脂糖凝胶纯化后,用T4 DNA连接酶(BRL,Gaithersburg MD)将900bp产物连接进载体pUC182Sfi的单一SfiI位点。混合物电穿孔进大肠杆菌XL1-蓝细胞并涂布到含0.32-0.64μg/ml头胞噻肟(Sigma,St.Louis MO)的LB板上。将细胞在37℃生长24小时,从板上刮下所得的菌落作为细胞库并用作下一轮交换的PCR模板。
6)随后的重装配步骤
将3中每轮交换后获得的转化子涂布到水平增加的头胞噻肟上。合并含有最高水平头胞噻肟平板上的菌落(>100,保持多样性)并用作下一轮PCR反应的模板。
在步骤(5)中以0.32-0.64μg/ml获得的头胞噻肟菌落的混合物用作下一轮交换的模板。10μl在LB培养基中的细胞用作重组过程的模板,重组方法是99℃ 10分钟,然后进行35次[94℃ 30秒,52℃ 30秒,72℃ 30秒]循环,再按上面所述在72℃进行5分钟。
按上面步骤(5)所述消化重组产物并连接进pUC182Sfi。混合物电穿孔进大肠杆菌XL1-blue细胞并涂布到含5-10μg/ml头胞噻肟的LB平板上。
除了细胞涂布到含80-160μg/ml头胞噻肟的LB板上外以5-10μg/ml获得的菌落用于与第一、和第二轮相似的第三轮。第三轮后,以80-160μg/ml获得菌落,在浓度增加的头胞噻肟上重新涂布后,37℃培养24小时后可获得高达320μg/ml的菌落(MIC=320μg/ml)。
在头胞噻肟上的生长取决于细胞密度,需要所有的MICs标准化(在本例中大约1,000细胞/板)。在更高的细胞密度,获得在高达1280μg/ml时生长。在1,280μg/ml上生长的5个最大菌落涂布2次以产生单个菌落,以菌落PCR产物的限制性图谱分析Sfil插入片段。
获得了-个对头胞噻肟抗性增加16,000倍(MIC=0.02μg/ml到MIC=320μg/ml)的突变子。
选择后,将选定克隆的质粒转移回到野生型大肠杆菌XL1-blue细胞(Stratagene,San Diego CA)中以确保测定药物抗性的菌落不是由染色体突变引起。
三轮交换和选择产物对TEM-1β内酰胺酶扩大的广谱抗菌素头胞噻肟的最小抑制浓度增加1.6×104倍。相反,无交换的重复涂布导致抗性仅增加16倍(PCR误差或序列盒突变)。
7)序列分析
生长于1,280μg/ml的所有5个最大菌落具有与野生型TEM-1酶相同的限制性图谱。从一个这些菌落中获得的质粒的sfiI插入片段以双脱氧DNA测序(美国甘化公司,Cleveland OH)根据厂家说明书测序。所有的碱基数与修改的pBR322序列相对应(29),氨基酸序数与ABL标准编序方法相对应(30)。以Z字母编码代表氨基酸,以一字母码表示核苷酸。术语G4205A指4205核苷酸从鸟嘌呤变成腺嘌呤。
发现9个单一碱基取代。G4205A位于β内酰胺酶P3启动子(31)-35和-10位之间。Chen和Clowes(31)获得的启动子上游突变位于这里所用的Sfil片段外,因而不能检测出来。四个突变是沉默突变(A3689G,G3713A,G3934A和T3959A),四个导致氨基酸改变(C3448T导致Gly238Ser,A3615G产生Met182Thr,C3850T产生Glu104Lys,G4107产生Ala18Val)。
8)分子回交
然后使用与额外的野生型DNA发生的分子回交,以便去掉非义必需突变。
以上面所述的正常交换相同的方法对来自第三轮DNA交换的选定质粒进行分子回交,除了DNAseI消化和交换反应在超出40倍的野生型TEM-1基因片段存在的条件下进行外。为了使回交更有效,在交换反应中使用非常小的DNA片段(30至100bp)。回交突变子再次在含80-160μg/ml头胞噻肟(Sigma,St.Louis MO)的LB板上选择。
在超出40倍的野生型TEM-1 DNA存在的条件下用第一轮回交菌落的DNA重复该回交交换。使用小DNA片段(30-100bp)增加回交的效率。再次在含80-160μg/ml头胞噻肟的LB平板上选择第二轮回交的突变子。
将所得的转化子涂布在160μg/ml头胞噻肟上,将合并的菌落再次涂布到水平增加到1,280μg/ml的头胞噻肟上。重新涂布在1,280μg/ml获得的最大菌落以得到单一菌落。
该回交突变子抗生比野生型高32,000倍(MIC=640μg/ml)。该突变菌株对头胞噻肟抗性比以前报道的临床或工程TEM-1来源的菌株高64倍。因此,似乎DNA交换对直接分子进化的至少一些循环而是快速且有效的工具。
使用双脱氧DNA测序试剂盒(美国生化公司,Cleveland OH)根据厂家说明书测定回交突变子SfiI插入片段的DNA序列(表3)。该突变子有9个单碱基对突变。与预期一样,所有4年以前鉴定的沉默突变丢失,回复到野生型基因的序列。在回交克隆中保留了启动子突变(G4205A)以及四个氨基酸突变中的3个(Glu104Lys,Met182Thr,和Gly238Ser),表明它们对高水平的头胞噻肟抗性是必需的。然而,发现了2个新沉默突变(T3842C和A3767G)以及3个导致氨基酸改变的新突变(C3441T导致Arg241 His,C3886T导致Gly92Ser,G4035C导致Ala42Gly)。尽管这两个沉默突变不影响蛋白质的一级序列,但它们可能影响蛋白质的表达水平(例如以mRNA结构)和甚至可能影响蛋白质的折叠(经过改变密码子应用并从而改变中止位点,它可能涉及蛋白质的折叠)。
表3
β内酰胺酶的突变
突变类型 非回交 回交
氨基酸改变 Ala18Lys -
Glu104Lys Glu104Lys
Met182Thr Met182Thr
Gly238Ser Gly238Ser
- Ala42Gly
- Gly92Ser
沉默 T3959A -
G3934A -
G3713A -
A3689G -
- T3842C
- A3767G
启动子 G4205A G4205A
回交和非回交突变具有一个启动子突变(以其本身或组合导致表达水平增加2-3倍)以及3个普通氨基酸改变(Glu104Lys,Met182Thr和Gly238Ser)。Glu104Lys和Gly238Ser是存在于一些头胞噻肟抗性或其它TEM-1衍生物中的突变(表4)。
9)表达水平比较
经过对根据Witholt,B.(32)的方法以渗压休克制备的外周胞质提取物进行SDS-聚丙烯酰胺凝胶电泳(4-20%;Novex,San Diego CA)比较了β内酰胺酶基因在野生型质粒,非回交突变子和回交突变子中的表达水平。
纯化的TEM-1β内酰胺酶(Sigma,St.Louis MO)用作分子量标记,缺乏质粒的大肠杆菌XL1-blue细胞用作阴性对照。
突变子和回交突变子与野生型基因相比似乎产生的β内酰胺酶蛋白质率高2-3倍。启动子突变似乎导致β-内酰胺酶水平增加2-3倍。
实施例6.TEM-1β内酰胺酶基因的突变组合的构建
为了确定不同突变组合的抗生以及为了比较新突变子和公开的突变子,将一些突变子构建进相同质粒背景中。两个突变Glu104Lys和Gly238Ser称为头胞噻肟突变子。构建的所有突变子组合具有启动子突变,允许比较选定的突变子。结果如表4所示。
经PCR将特定的突变组合导入野生型pUC182Sfi中,每一突变使用2个寡核苷酸。
获得下列突变的寡核苷酸是:Ala42Gly(SEQ ID NO:11)AGTTGGGTGGACGAGTGGGTTACATCGAACT和(SEQ IDNO:12)AACCCACTCGTCCACCCAACTGATCTTCAGCAT;Gln39Lys:(SEQ ID NO:13)AGTAAAAGATGCTGAAGATAAGTTGGGTGCAC GAGTGGGTT和(SEQ ID NO:14)ACTTATCTTCAGCATCTTTTACTT;Gly92Ser:(SEQ ID NO:15)AAGAGCAACTCAGTCGCCGCATACACTATTCT和(SEQ ID NO:16)ATGGCGGCGACTGAGTTGCTCTTGCCCGGCGTCAAT;Glu104Lys:(SEQ ID NO:17)TATTCTCAGAATGACTTGGTTAAGTACTCACCAGT CACAGAA和(SEQ ID NO:18)TTAACCAAGTCATTCTGAGAAT;Met182Thr:(SEQ ID NO:19)AACGACGAGCGTGACACCACGACGCCTGTAGCAATG和(SEQ ID NO:20)TCGTGGTGTCACGCTCGTCGTT;Gly238Ser单独:(SEQ ID NO:21)TTGCTGATAAATCTGGAGCCAGTGAGCGTGGGTCTCGCGGTA和(SEQ ID NO:22)TGGCTCCAGATTTATCAGCAA;Gly238Ser and Arg241His(组合):(SEQ ID NO:23)ATGCTCACTGGCTCCAGATTTATCAGCAAT和(SEQ ID NO:24)TCTGGAGCCAGTGAGCATGGGTCTCGCGGTATCATT;G4205A:(SEQ ID NO:25)AACCTGTCCAGGCCACCATGGCCTAAATACAATCAAATATGTATCCGCTTATGAGACAATAACCCTGATA。
从合成的寡核苷酸中凝胶纯化出这些分离的PCR片段。组合各片段10ng,重组反应在94℃进行1分钟,然后经过25次[94℃ 30秒,50℃ 30秒和72℃ 45秒]循环。在含SifI的外侧引物(来自实施例5的引物C和D)存在的条件下对重组产物进行PCR 25个循环。用SfiI消化DNA并插入野生型pUC182Sfi载体中,获得下列突变子组合(表4)。
表4
名称 | 基因型 | MIC | MIC来源 |
TEM-1 | 野生型 | 0.02 | |
Glu104Lys | 0.08 | 10 | |
Gly238Ser | 016 | 10 | |
TEM-15 | Glu104Lys/Gly238Ser* | 10 | |
TEM-3 | Glu104Lys/Gly238Ser/Gln39Lys | 102-32 | 37,15 |
ST-4 | Glu104Lys/Gly238Ser/Met182Thr* | 10 | |
ST-1 | Glu104Lys/Gly238Ser/Met182Thr/Ala18Val/T3959A/G3713A/G3934A/A3689G* | 320 | |
ST-2 | Glu104Lys/Gly238Ser/Met182Thr/Ala42Gly/Gly92Ser/Arq241His/T3842C/A3767G* | 640 | |
ST-3 | Glu104Lys/Gly238Ser/Met182Thr/Ala42Gly/Gly92Ser/Arq241His* | 640 |
*所有这些突变子另外还含有G4205A启动子突变。
推断保守的突变在MIC的15个倍增体中占9个。
Glu104Lys单独显示出仅导致倍增体的MIC达0.08μg/ml,且Gly238Ser(在一些情况下具有另一个氨基酸改变)仅导致MIC为0.16μg/ml(26)。双突变子Glu104Lys/Gly238Ser具有MIC为10μg/ml。这一突变子与TE-15相对应。
这些相同的Glu104Lys和Gly238Ser突变与Glu39Lys(TEM-3)或Thr263Met(TEM-4)组合导致高水平的抗性(TEM-3为2-32μg/ml,TEM-4为8-32μg/ml)(34,35)。
含有在回交后保守的3个氨基酸改变的突变(Glu104Lys/Met182Thr/Gly238.Ser)也具有MIC为10μg/ml。这意味着各新选择的突变子加上3个已知的突变后产生的突变体导致基因对cefotaxime的抗性进一步增加32到64倍。
天然存在的,衍生自临床TEM-1的酶(TEM-1-19)各含有仅5-7个相同突变(前述)的不同组合。由于这些突变在基因中的位置完全分离,所以经过单个区域的序列盒诱变不能获得具有高cefotaxime抗性的突变子。这可以解释为什么经过标准序列盒诱变研究获得的最大MIC仅为0.64μg/ml(26)。例如,在这一研究中分别发现G104Lys和Gly238Ser突变MIC低于0.16μg/ml。使用DNA交换允许发生组合,从而发现Glu104Lys/Gly238Ser组合的MIC为10μg/ml。
本实施例的重要局限是使用单个基因作为直始点。预期如果大量相关的,天然存在的基因发生交换会发现更好的组合。存在于该混合物中的多样性比经过诱变交换产生的随机突变更有意义。例如,预期可使用来自单一物种相关基因的所有组成成份,例如预先存在的免疫系统的多样性,或从许多不同种类获得的相关基因。
实施例7.经过所有6个突变子CDRs文库的DNA杂合改进抗体A10B
A10B scFv抗体,小鼠抗-兔IgG由Pharmacia(Milwaukee WI)赠送。使用商业上可获得的Pharmacia噬菌体演示系统,它使用pCANTAB5噬菌体演示载体。
原始A10B抗体仅具有能重复的低亲合力,因为获得的克隆仅微弱地结合固相抗原(兔IgG),(以噬菌体ELISA(Pharmacia试验试剂盒)或噬菌体滴定测量)。在竞争试验中对A10B抗体结合产生50%抑制的兔IgG浓度是13微微摩尔。观察到的低亲合力可能是由于A10B克隆的不稳定性。
根据厂家说明书测序A10B scFv DNA(美国生化公司,Cleveland OH)。根据与Kabat(33)的比较,该序列与现存抗体相似。
1)噬菌体DNA的制备:
具有A10B野生型抗体基因(10μl)的噬菌体DNA在99℃保温10分钟;然后在72℃ 2分钟。向噬菌体DNA中加入PCR混合物(50mM KCl,10mM Tris-HClpH9.0,0.1%Triton X-100,各200μm的dNTP,1.9mM MgCl2),各0.6μm的引物和0.5μm Taq DNA聚合酶(Promega,Madison WI)。PCR程序进行了35个[94℃30秒,45℃ 30秒,72℃ 45秒]循环。使用的引物是:5′ATGATTACGCCAAGCTTT3′(SEQ ID NO:26)和5′TTGTCGTCTTTCCAGACGTT3′(SEQ ID NO:27)。
然后电泳850bp PCR产物并从2%低熔点琼脂糖凝胶中纯化。
2)片段化
将300ng凝胶纯化的850bp带用存在于50mM Tris-HCl pH7.5,10mM MgCl2中的0.18单位的DNAseI(Sigma,St.Louis MO)室温消化20分钟。消化的DNA在2%低熔点琼脂凝胶上分离并从凝胶中纯化出50至200bp之间的带。
3)试验文库的构建
本实验的目的是试验CDRs的插入是否有效。
合成下列具有内部限制性酶位点的CDR序列。“CDR H”指重链中的CDR,“CDR L”指抗体中轻链上的CDR。
具有限制性位点的CDR寡聚体:CDR H1(SEQ ID NO:34)5′TTCTGGCTACATCTTCACAGAATTCATCTAGATTTGGGTGAGGCAGACGCCTGAA3′CDR H2(SEQ ID NO:35)5′ACAGGGACTTGAGTGGATTGGAATCACAGTCAAGCTTATCCTTTATCTCAGGTCTCGAGTTCCAAGTACTTAAAGGGCCACACTGAGTGTA3′CDR H3(SEQ ID NO:36)5′TGTCTATTTCTGTGCTAGATCTTGACTGCAGTCTTATACGAGGATCCATTGGGGCCAAGGGACCAGGTCA3′CDR L1(SEQ ID NO:37)5′AGAGGGTCACCATGACCTGCGGACGTCTTTAAGCGATCGGGCTGATGGCCTGGTACCAACAGAAGCCTGGAT3′CDR L2(SEQ ID NO:38)5′TCCCCCAGACTCCTGATTTATTAAGGGAGATCTAAACAGCTGTTGGTCCCTTTTCGCTTCAGT3′CDR L3(SEQ ID NO:39)5′ATGCTGCCACTTATTACTGCTTCTGCGCGCTTAAAGGATATCTTCATTTCGGAGGGGGGACCAAGCT3′
将CDR寡聚体以超出10倍的摩尔数加入上面步骤(2)50至200bp之间的纯化A10B抗体DNA片段中。加入PCR混合物(50mM KCl,10mM Tris-HCl pH9.0,0.1%Triton X-100,1.0mM MgCl2各200μm的dNTP,0.3μl Taq DNA聚合酶(Promega,Madison WI),总体积为50μl),交换程序是在94℃进行1分钟,在72℃进行1分钟,然后进行35次循环:94℃ 30秒,55℃ 30秒,72℃ 30秒。
将1μl交换混合物加入100μl PCR混合物(50mM KCl,10mM Tris-HCl pH9.0,0.1%Triton X-100,各200μm的dNTP,1.9mM MgCl2,各0.6μm的两种外侧引物(SEQ ID NO:26和27,见下面),0.5μl Taq DNA聚合酶)中,PCR程序进行30个[94℃ 30秒,45℃ 30秒,72℃ 45秒]循环。所得的850碱基对大小的DNA片段混合物用酚/氯仿提取并用乙醇沉淀。
外侧引物是:
外侧引物1:SEQ ID NO:27
5′TTGTCGTCTTTCCAGACGTT3′
外侧引物2:SEQ ID NO:26
5′ATGATTACGCCAAGCTTT3′
将850bp PCR产物用限制性酶SfiI和NotI消化,从低熔点琼脂糖凝胶中纯化并连接进从Pharmacia,Milwankee WI获得的pCANTAB5表达载体中。根据Invitrogen(San Diego CA)阐述的方法将连接的载体电穿孔进TG1细胞(Pharmacia,MilwallKee WI)并涂布成单个菌落。
将所得菌落的DNA加入100μl PCR混合物(50mM KCl,10mM Tris-HCl,pH9.0,0.1%Triton X-100,各200μm的dNTP,1.9mM MgCl2,0.6μm的外侧引物1(SEQ ID NO:27;见下面),6个内部引物(SEQ ID NO:40-45;见下面),和0.5μl Taq DNA聚合酶)中,PCR过程进行35次[94℃ 30秒,45℃ 30秒,72℃45秒]循环。以琼脂糖凝胶电泳测定PCR产物的大小并用于确定哪一个具有限制性位点的CDRs被插入。CDR内部引物:H1(SEQ ID NO:40)5′AGAATTCATCTAGATTTG3′H2(SEQ ID NO:41)5′GCTTATCCTTTATCTCAGGTC3′H3(SEQ ID NO:42)5′ACTGCAGTCTTATACGAGGAT3′L1(SEQ ID NO:43)5′GACGTCTTTAAGCGATCG3′,L2(SEQ ID NO:44)5′TAAGGGAGATCTAAACAG3′,L3(SEQ ID NO:45)5′TCTGCGCGCTTAAAGGAT3′
在野生型A10B抗体DNA的预期位置插入6个合成的CDRs(图7)。这些研究表明:尽管特定克隆的6个CDRs中的每一个成为具有限制性位点的CDR的机会较小,但大多数克隆携带至少一个具有限制性位点的CDR,并产生了具有限制性位点的CDR的任意可能的组合。
4)突变的互补决定区(“CDRs”)的构建:
根据我们的序列资料,制备了与6个CDRs相应的6个寡核苷酸。CDR(Kabat定义)在合成时突变的比率为70(现存碱基):10∶10∶10,以大约20碱基的侧翼序列在5′和3′两侧,当以摩尔数超出的量混合进未诱变的抗体基因片段混合物中时它为CDR的插入提供了同源性。所得的突变序列在下面给出:
CDR文库的寡核苷酸:CDR H1(SEQ ID NO:28)5′TTCTGGCTACATCTTCACAACTTATGATATAGACTGGGTGAGGCAGACGCCTGAA3′CDR H2(SEQ ID NO:29)5′ACAGGGACTTGAGTGGATTGGATGGATTTTTCCTGGAGAGGGTGGTACTGAATACAATGAGAAGTTCAAGGGCAGGGCCACACTGAGTGTA3′CDR H3(SEQ ID NO:30)5′TGTCTATTTCTGTGCTAGAGGGGACTACTATAGGCGCTACTTTGACTTGTGGGGCCAAGGGACCACGGTCA3′CDR L1(SEQ ID NO:31)5′AGAGGGTCACCATGACCTGCAGTGCCAGCTCAGGTATACGTTACATATATTGGTACCAACAGAAGCCTGGAT3′CDR L2(SEQ ID NO:32)5′TCCCCCAGACTCCTGATTTATGACACATCCAACGTGGCTCCTGGAGTCCCTTTTCGCTTCAGT3′CDR L3(SEQ ID NO:33)5′ATGCTGCCACTTATTACTTGCC AGGAGTGGAGTGGTTATCCGTACACGTTCGGAGGGGGGACCAAGC T3′
黑体字及划线序列是使用核苷酸70∶10∶10∶10的混合物合成的突变序列,其中70%是野生型核苷。
将摩尔数超出10倍的CDR突变寡核苷酸加入以上面步骤(2)得到的长度在50至200bp之间的纯化A10B抗体DNA片段中。加入PCR混合物(50mM KCl,10mMTris-HCl pH9.0,0.1%Triton X-100,1.9mM MgCl2,各200μm dNTP,0.3μl TaqDNA聚合酶(Promega,Madison WI),总体积为50μl),交换程序在94℃进行1分钟,72℃ 1分钟,然后进行35个[94℃ 30秒,55℃ 30秒,72℃ 30秒]循环。
将1μl交换混合物加入100μl PCR混合物(50mM KCl,10mM Tris-HClpH9.0,0.1%Triton X-100,各200μm的dNTP,1.9mM MgCl2,各0.6μm的两个外侧引物(SEQ ID NO:26和27,见下面),0.5μl Taq DNA聚合酶)中,PCR程序进行30个[94℃ 30秒,45℃ 30秒,72℃ 45秒]循环,所得的850碱基对大小的DNA片段混合物用酚/氯仿提取并用乙醇沉淀。
外侧引物是:外侧引物1:SEQ ID NO:27 5′TTGTCGTCTTTCCAGACGTT3′外侧引物2:SEQ DI NO:26 5′ATGATTACGCCAAGCTTT3′
5)将scFv抗体DNA克隆进pCANTAB5
用限制性酶SfiI和NotI消化850bp PCR产物。从低熔点琼脂糖凝胶中纯化并连接进从Pharmacia,Milwaukee WI获得的pCANTAB5表达载体中。根据Invitrogen(San Diego CA)阐述的方法将连接的载体电穿孔进TG1细胞(Pharmacia,Milwaukee WI),按照厂家建议的说明书使用辅助噬菌体生长噬菌体文库。
使用6轮选择筛选在该方法产生的文库中改进抗体的存在。
6)高亲合力克隆的选择
用兔IgG(Jackson Immunoresearch,Bar Harbor ME)以10μg/孔在37℃覆盖96孔微8滴板的15个孔1小时,然后用2%的溶于PBS的脱脂半乳在37℃封闭1小时。
用100μl 2%乳液在室温下封闭100μl的噬菌体文库(1×1010cfu)30分钟,然后加到15个孔的各孔中并在37℃保温1小时。
然后用含0.5%Tween-20的PBS洗涤各孔3次,每次10分钟。结合的噬菌体用100μl洗脱缓冲液(甘氨酸-HCl,pH2.2)洗脱,接着用2M Tris pH7.4立即中和并转染用于噬菌体生产。重复该选择循环6次。
第6个循环后,挑出单个噬菌体克隆并以噬菌体ELISA比较相对亲合力,用Pharmacia(Milwaukee WI)的试剂盒根据厂家建议的方法试验兔IgG的特异性。
当以Western分析试验时,与野生型A10B比较,最好的克隆表达水平提高大约100倍。与最佳克隆在竞争试验中产生50%抑制的兔IgG浓度是1微微摩尔。最佳克隆对兔抗原具有可重复的特异性。以噬菌体显示的抗体拷贝数似乎增加。
实施例8.经部分基因的直接重复进行体内重组
以两部分灭活的相同基因(β-内酰胺酶)拷贝构建质粒以证实这两个直接重复的共同区之间的重组产生全长的,有活性的重组基因。
使用携带细菌TEM-1β内酰胺酶基因的pUC18衍生物(Yanish-Perron et al.,1985,Gene 33:103-119)。TEM-1β内酰胺酶基因(“Bla”)赋予细菌对大约0.02μg/ml的cefotaxime产生抗性。用2个引物对载体序列进行PCR:Primer A(SEQ ID NO:46)5′TTCTATTGACGGCCTGTCAGGCCTCATATATACTTTAGATTGTTTT3′Primer B (SEQ ID NO:47)5′TTGACGCACTGGCCATGGTGGCCAAAAATAAACAAATAGGGGTTCCGCGCACATTT3′
及用另外两个引物对β-内酰胺酶基因序列进行PCR:Primer C(SEQ ID NO:48)5′AACTGACCACGGCCTGACAGGCCGGTCTGACAGTTACCAATGCTT3′Primer D(SEQ ID NO:49)5′AACCTGTCCTGGCCACCATGGCCTAAATACATTCAAATATGTAT3′
将Sfil限制性位点加到启动子5′端和β内酰胺酶基因3′端。
用Sfil消化两个反应产物,混合,连接并用于以下述程序转化受体大肠杆菌。所得的质粒是pUC182Sfi-Bla-Sfi。该质粒含有携带Bla基因和P-3启动子的Sfil片段。
携带pUC182Sfi-Bla-Sfi的大肠杆菌XL1-blue(Stratagene,San Diego CA)在37℃培养24小时后的cefotaxime最低抑制浓度为0.02μg/ml。
使用同源区域将pBR322的四环素基因克隆区pUC18Sif-Bla-Sfi,产生pBR322TetSfi-Bla-Sfi。然后用SspI和FspI限制性消化pBR322 TetSfi-Bla-Sfi去掉TEM-1基因并连接末端,产生pUC322 TetSfi-Sfi。
使用标准PCR技术和下列引物扩增TEM-1基因的重叠区:Primer 2650(SEQ ID NO:50)5′TTCTTAGACGTCAGGTGGCACTT3′Primer 2493(SEQ ID NO:51)5′TTT TAA ATC AAT CTA AAG TAT3′Primer 2651(SEQ ID NO:52)5′TGCTCATCCACGAGTGTGGAGAAGTGGTCCTGCAACTTTAT3′,以及rimer 2652(SEQ ID NO:53)ACCACTTCTCCACACTCGTGGATGAGCACTTTTAAAGTT
用Sfil和BstX1消化2个所得的DNA片段并连接进pBR322 Tet Sfi-Sfi的Sfi位点。所得的质粒称为pBR322Sfi-BL-LA-Sfi。质粒的图谱以及功能性β-内酰胺酶的质粒内重组和再构建示意图在图9中表示。
将质粒电穿孔进TG-1或JC8679大肠杆菌细胞。大肠杆菌JC8679是RecBC sbcA(Dliner et al.,1993,NAR 21:5192)。将细胞涂布到含四环素的琼脂平板上。然后将生长的那些克隆涂布到含100μg/ml氨苄青霉素的固体琼脂板上并计数活菌落数。将表现出氨苄青霉素抗性的那些转化子中的β内酰胺酶基因插入片段以标准PCR技术扩增,使用的引物是:Primer 2650(SEQ ID NO:50)5′TTCTTAGACGTCAGGTGGCACTT3′和Primer 2493(SEQ ID NO:51)5′TTTTAAATCAATCTAAAGTAT3′
测量插入片段的长度。1kb插入片段的存在表明该基因已成功地重组,如图9和表5所示。
表5
细胞 | 菌落 | 菌落 | 菌落 |
TG-1 | 131 | 21 | 3/3 at 1 kb |
JC8679 | 123 | 31 | 4/4 at 1 kb |
载体对照 | 51 | 0 |
大约17-25%的四环素抗性菌落也是氨苄青霉素抗性,经菌落PCR测定全部氨苄青霉素抗性菌落已正确重组。因此,位于相同质粒的部分基因将成功地重组以产生功能基因。
实施例9.经全长基因的直接重复进行体内重组
构建具有β内酰胺酶基因不同等位基因的两个全长拷贝的质粒。两个基因的同源重组产生该基因的单个重组全长拷贝。
pBR322Tet Sfi-Sfi和pBR322 Tet Sfi-Bla-Sfi的构建描述如上。
按下面构建β内酰胺酶基因的两上等位基因。用pUC18Sfi-Bla-Sfi作模板进行两个PCR反应。一个反应用下列引物进行:Primer 2650(SEQ ID NO:50)5′TTCTTAGACGTCAGGTGGCACTT3′Primer 2649(SEQ ID NO:51)5′ATGGTAGTCCACGAGTGTGGTAGTGACAGGCCGGTCTGACAGTTACCAATGCTT3′
用下列引物进行第二个PCR反应:Primer 2648(SEQ ID NO:54)5′TGTCACTACCACACTCGTGGACTACCATGGCCTAAATACATTCAAATATGTAT3′Primer 2493(SEQ ID NO:51)5′TTT TAA ATC AAT CTA AAG TAT 3′
这样产生两个Bla基因,一个具有5′Sfi1位点和3′BstX1位点,另一个具有5′Bst X1位点和3′Sfi1位点。
用BstX1和Sfi1消化这两个基因后,连接进Sfi1消化的质粒pBR322Tet Sfi-Sfi中,获得具有Bla基因串联重复的质粒(pBR322-Sfi-2BLA-Sfi),(见图10)。将该质粒电穿孔进大肠杆菌细胞中。将细胞涂布到含15μg/ml四环素的固态琼脂板上。然后将那些生长的菌落涂布到含100μg/ml氨苄青霉素的固态琼脂板上并计数活菌落数。将表现出氨苄青霉素抗性的转化子中的Bla插入片段以标准PCR技术使用实施例8描述的方法和引物扩增。1kb插入片段的存在表明:复制基因已重组如表6所示。
表6
细胞 | 菌落 | 菌落 | 菌落 |
TG-1 | 28 | 54 | 7/7 at 1kb |
JC8679 | 149 | 117 | 3/3 at 1kb |
载体对照 | 51 | 0 |
菌落PCR证实串联重复有效重组形成了单个重组基因。
实施例10.多轮直接重复重组-质粒内
为了确定多轮重组是否可用于更快地生产抗性细胞,进行实施例9所述方法的多个循环。
负重组控制由单拷贝的β内酰胺酶基因组成,而正重组实验由插入的两个拷贝的β内酰胺酶直接重复组成。四环素标记用于平衡在每轮中选择cefotaxime抗性的菌落数以补偿连接效率。
在第一轮中,用EcrI消化pBR322TetSfi-Bla-Sfi并用正常和Cadwell PCR混合物(Cadwell and Joyce(1992)PCR Methods and Applications 2:28-33)的1∶1混合物(1ml)进行PCR以获得发生误差的PCR。PCR程序是开始在70℃ 2分钟。然后进行30次循环,每循环在94℃ 30秒;52℃ 30秒和72℃ 3分钟6秒,接着在72℃ 10分钟。
在PCR反应中用于产生一个Bla基因控制质粒的引物是引物2650(SEQ IDNO:5))和引物2719(SEQ ID NO:55)5′TTAAGGGATTTTGGTCATGAGATT3′。这产生扩增DNA片段的混合群体,集中命名为片段#59。这些片段有不同突变数目。
用于两个不同PCR反应以产生两个Bla基因质粒的引物对于第一基因是引物2650(SEQ ID NO:50)和引物2649(SEQ ID NO:51),对于第二基因是引物2719(SEQID NO:55)。这产生两个扩增DNA片段中每一个的混合群体:片段#89(用引物2648和2719扩增)和片段#90(用引物2650和2649扩增)。在每种情况下将不同突变数导入各片段的混合群体中。
经过有误差的PCR后,用Sfil消化扩增DNA片段#59的群体,然后克隆进pBR322TetSfi-Sfi以产生质粒pBR322Sfi-Bla-Sfi1的混合群体。
经过有误差的PCR后,用SfiI和BstXI在50℃消化扩增DNA片段#90和#89的群体并连接进pBR322TetSfi-Sfi以产生质粒pBR322TetSfi-2Bla-Sfi1的混合群体(图10)。
将质粒pBR322Sfi-Bla-Sfi1和pBR322Sfi-2Bla-Sfi1电穿孔进大肠杆菌JC8679并涂布在具有不同cefotaxime浓度的琼脂板上以选择抗性菌株并涂布在四环素板上滴定。
挑出等数目的菌落(根据在四环素上生长的菌落数),在LB-tet上生长并从菌落中提取DNA。这是一轮重组。用EcrI消化该DNA并按上面所述用于第二轮有误差的PCR。
经过5轮后,一个片段质粒对cefotaxime的MIC(最低抑制浓度)是0.32,而两个片段质粒的MIC是1.28。结果表明,经过5次循环后,重组获得的抗性比体内重组存在的抗性高4倍。
实施例11.经片段的电穿孔进行体内重组
按所述制备含pUC18Sfi-Bla-Sfi的感受态大肠杆菌。如上所述质粒pUC18Sfi-Bla-Sfi含标准TEM-1β-内酰胺酶基因。
获得来自pUC18Sfi-cef-Sfi(克隆ST2)(Stemmer WPC(1994))Nature370:389-91,本文引用作为参考)的TE-1衍生的cefotaxime抗性基因,它赋予携带该质粒的大肠杆菌对cefotaxime的MIC为640μg/ml。在一个实验中,将完整的质粒pUC18Sfi-cef-Sfi DNA电穿孔进具有质粒pUC18Sfi-Bla-Sfi的大肠杆菌细胞中。
在另一实验中,将含来自pUC18Sfi-cef-Sfi的cefotaxime基因的DNA片段使用引物2650(SEQ ID NO:50)和2719(SEQ ID NO:55)经PCR扩增。所得的1kb PCR产物以DNase消化成<100bp的DNA片段,将这些片段电穿孔进已含有pUC18Sfi-Bla-Sfi的感受态大肠杆菌细胞中。
然后经过将转化细胞涂布到具有不同cefotaxime浓度的琼脂板上试验来自两个实验中的转化细胞对cefotaxime的抗性。结果在表7中表示。
表7 菌落/cefotaxime浓度
无DNA对照 | 0.16 | 0.32 | 1.28 | 5.0 | 10.0 |
ST-2突变子,整体 | 14 | ||||
ST-2突变子,片段 | 4000 | 2000 | 800 | 400 | |
野生型,整体 | 1000 | 120 | 22 | 7 | |
野生型,片段 | 27 | ||||
18 |
该结果表明电穿孔后完整ST-2 Cef基因插入了细菌基因组成质粒中。由于大多数插入片段是同源的,预期该基因插入质粒中,取代野生型基因。St-2的Cef基因片段也有效地插入质粒的野生型基因中。对导入野生型基因(整体或片段)和无DNA观察不到cefotaxime抗性的急剧增加。因此,St-2片段显示出比野生型片段产生更大的cefotaxime抗性。期望从增加抗性基因库中制备的重复的插入片段会导致抗性增加。
因此,分离具有St-2基因片段,产生增加的cefotaxime抗性的那些菌落并提取质粒DNA。使用上面描述的PCR方法扩增该DNA。用DNase将扩增DNA消化成片段(<100bp),将2-4μg该片段电穿孔进已含有pUC322Sfi-Bla-Sfi的上面所述的感受态大肠杆菌细胞中。将转化的细胞涂布在含有不同cefotaxime浓度的琼脂上。
作为对照,也使用具有质粒pUC18Sfi-Kan-Sfi的感受态大肠杆菌。将pUC18Sfi-cef-Sfi PCR产物消化的DNA片段电穿孔进这些细胞中。在卡那霉素基因和β内酰胺酶基因间无同源性,因此不会发生重组。
本实验重复2轮,结果在表8中显示。
表8
次数 | 浓度 | 对照 | 抗性菌落 |
1重复涂布 | 0.16-0.640.32 | 菌落10小 | 菌落1000 |
2重复涂布 | 10 | 10100sm@2.5 | 40050@10 |
3 | 401280 | 100sm | 100sm |
实施例12.重组形式的确定
设计本实验以确定哪一种重组形式在每个循环中产生最多重组子。
在第一种方法中,用PCR引物扩增载体pUC18Sfi-Bla-Sfi以产生大型和小型片段。大片段具有质粒和部分Bla基因的末端,小片段编码中央Bla基因。使用实施例8的PCR方法产生具有完整Bla基因的第三个片段。以上面描述的方法将大质粒片段和含完整Bla基因的片段同时电穿孔进大肠杆菌JC8679细胞中,将转化子涂布在不同浓度的cefotaxime上。
在第二种方法中,扩增载体pUC18Sfi-Bla-Sfi以产生如上面第一种方法中分离的大质粒片段。经PCR获得各包含部分完整Bla基因的2个片段,使两个片段一起覆盖完整的Bla基因。将大质粒片段和2个Bla基因片段都电穿孔进感受态的大肠杆菌JC8679细胞中,将转化子涂布在不同浓度的cefotaxime上。
在第三种方法中,将载体和质粒都电穿孔进大肠杆菌JC8679细胞中并将转化子涂布在不同浓度的cefotaxime上。
在第四种方法中,将完整Bla基因电穿孔进已含有载体pUCSfi-Sfi的大肠杆菌细胞保并将转化子涂布在不同浓度的cefotaxime上。作为对照用完整Bla基因或仅用载体电穿孔大肠杆菌JC8671细胞。
结果在图11中表示,与使用具有完整Bla基因的一个片段相比,两个片段插入载体的效率低100倍。第3步研究表明:插入效率取决于自由DNA末端的存在,因为在这一步研究中设有获得重组子。然而,方法3中的结果也是由于载体电穿孔的效率低,当表达载体已存在于感受态细胞中时,载体电穿孔的效率不再是因素,甚至用未切割的载体可获得有效的同源重组。
实施例13.优化载体效果的序列盒杂合试剂盒
为了提供能赋予优化表型的载体(例如,载体编码序列的最大表达,如克隆的基因),提供了一套试剂盒,包含各种能被交换的序列盒,并选择优化的交换子。图12是一个实施方案的图示,每个基因座具有许多序列盒。例如,在细菌表达系统中,图13显示了在各自基因座使用的序列盒例子。给字基因座的各序列盒(例如,本实施例中的所有启动子)两侧是能重叠邻近基因座序列盒侧翼序列且优选也能参与同源重组或非同源重组(例如,lox/cre或flp/frt系统)的基本相同的序列,以便实现在一个基因座内而基本上不是基因座之间的序列盒的交换。
在试剂盒中以PCR片段的形式提供序列盒,其中各序列盒类型或单个序列盒种类包装在分离的试管中,经过合并试管的内容物以装配完整质粒或其主要部分构建载体文库,其中经过使每个基因座序列盒重叠的侧翼序列与邻近基因座的序列盒杂交来实现。装配的载体连接到预定的目的基因上以形成载体文库,其中各文库成员包含预定的目的基因和序列盒的组合,这经过序列盒的联接来确定。将载体转移进合适的宿主细胞中,在适合于表达的条件下培养细胞并选择所需的表型。
尽管使用认为是优选的实施例对本发明进行了描述,但应清楚本发明不受所公开实施例的限制。相反,本发明试图覆盖包括在所附权利要求的实质和范围内的各种修改和等价方法。
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Claims (32)
1.用于将一个或多个突变导入模板双链多核苷酸的方法,其中模板双链多核苷酸已裂解成所需大小的双链随机片段,该方法包括:
a)将一种或多种单链或双链寡核苷酸加入所得的双链片段群体中,其中所说的寡核苷酸包含与模板多核苷酸相同的区域和异源区域;
b)将所得的双链随机片段与寡核苷酸的混合物变性成单链片段;
c)在导致所说单链片段在单链片段之间相同区域上退火的条件下将所得的单链片段群体与聚合酶保温并形成诱变的双链多核苷酸;以及
d)重复步骤(b)和(c)。
2.权利要求1的方法,其中在双链片段混合物中特定双链片段的浓度不超过总DNA重量的1%。
3.权利要求1的方法,其中不同特定双链片段的数目包括至少大约100个。
4.权利要求1的方法,其中双链片段的大小从56bp到5kb。
5.权利要求1的方法,其中诱变的双链多核苷酸的大小包含50bp到100kb。
6.生产具有生物活性重组蛋白质的方法,包括:
a)在提供将所说模板多核苷酸裂解成具有所需大小的随机双链片段的条件下处理包含编码野生型蛋白质的双链模板多核苷酸的样品;
b)向所得的随机片段的群体中加入一种或多种单链或双链寡核苷酸,其中所说的寡核苷酸包含模板多核苷酸的相同区和异源区;
c)将所得的双链随机片段和寡核苷酸的混合物变性成单链片段;
d)在导致所说的单链片段在相同区域退火的条件下将所得的单链片段群体与聚合酶保温并形成诱变的双链多核苷酸;
e)重复步骤(c)和(d);
f)从诱变的双链多核苷酸表达重组蛋白质。
7.权利要求6的方法,其中在步骤(a)中双链片段混合物中的特定双链片段的浓度不到总DNA重量的1%。
8.权利要求6的方法,其中步骤(a)中不同的特定双链片段的数目包含至少大约100个。
9.权利要求6的方法,其中双链片段的大小是从大约5bp到5kb。
10.权利要求6的方法,其中诱变的双链多核苷酸大小包括从50bp到100kb。
11.权利要求6的方法,进一步包含从重组蛋白质群体中选择所需的重组蛋白质。
12.获得嵌合多核苷酸的方法,包含:
a)在提供将所说的模板多核苷酸裂解成所需大小的随机双链片段的条件下处理包含不同双链模板多核苷酸的样品,其中所说的不同模板多核苷酸含有相同区和异源区;
b)将包含在步骤(a)产生的处理样品中的所得随机双链模板片段变性成单链片段;
c)在提供靶单链片段在相同区域退火的条件下将所得的单链片段与聚合酶保温并形成包含模板多核苷酸序列的嵌合双链多核苷酸序列;
d)按需要重复步骤(b)和(c)。
13.权利要求12的方法,其中在步骤(a)中双链片段混合物中的特定双链片段浓度不超过总DNA重量的1%。
14.权利要求12的方法,其中步骤(a)不同的特定双链片段的数目包含至少大约100个。
15.权利要求12的方法,其中双链片段的大小是从大约5bp到5kb。
16.权利要求12的方法,其中诱变的双链多核苷酸的大小包含从50bp到100kb。
17.复制模板多核苷酸的方法,该方法包括:体外结合单链模板多核苷酸与来自模板多核苷酸裂解和变性的小随机单链片段,在形成双链模板多核苷酸群体的条件下,在核酸聚合酶存在的条件下保温所说的核酸片段混合物。
18.一种产生适于亲和作用筛选或表型筛选的演示肽或演示抗体文库的方法,该方法包括:
(1)获得包含演示肽或演示抗体和编码所说演示肽或演示抗体的相关多核苷酸的第一群选定的文库成员,并获得所说相关多核苷酸或其拷贝,其中所说的相关多核苷酸包含基本相同序列的区域,以及
(2)在适于PCR扩增的条件下合并且片段化所说的相关多核苷酸或拷贝,进行PCR扩增,且由此同源重组所说的片段使形成重组多核苷酸的杂合库,其中所说的杂合库的重组多核苷酸的主要部分不存在于第一群选定的文库成员中。
19.权利要求18的方法,进一步包含将突变导入所说的多核苷酸或拷贝中。
20.权利要求19的方法,其中经过进行PCR扩增导入突变。
21.权利要求20的方法,其中PCR扩增是有误差的PCR。
22.权利要求18的方法,包含另一步筛选杂合库的文库成员以鉴定具有结合预定大分子之能力的单个杂合文库成员。
23.权利要求18的方法,其中经过选择不同于对预定分子的结合亲和力的表型特征来获得第一群选定的文库成员。
24.权利要求18的方法,其中合并第一群选定的文库成员并片段化,经体外PCR同源重组。
25.权利要求18的方法,其中合并第一群选定的文库成员并体外片段化,所得的片段转移进宿主细胞或有机体并同源重组形成体内杂合文库成员。
26.权利要求18的方法,其中第一群选定的文库成员在游离的可复制载体上克隆或扩增,所说的多重性载体转移进细胞和同源重组以形成体内杂合文库成员。
27.产生适于亲和作用筛选或表型筛选的演示肽或演示抗体的文库的方法,该方法包括:
(1)获得包含演示肽或演示抗体和编码所说演示肽或演示抗体的相关多核苷酸的第一群选定的文库成员,获得所说相关多核苷酸或其拷贝,其中所说的相关多核苷酸包含基本相同序列的区域;且
(2)在游离可复制载体上克隆或扩增所说相关多核苷酸或拷贝并将所说的多重性载体转移进细胞和同源重组以形成体内杂合文库成员。
28.权利要求27的方法,进一步包含将突变导入所说的多核苷酸或其拷贝中。
29.权利要求27的方法,其中所说的游离可复制载体包含许多相关多核苷酸或其拷贝的直接重复。
30.一种产生适于亲和作用筛选的演示抗体的文库的方法,该方法包含:
(1)获得包含演示抗体和编码所说的演示抗体的相关多核苷酸的第一群选定的文库成员,获得所说的相关多核苷酸或其拷贝,其中所说的相关多核苷酸包含基本相同的可变区框架序列的区域;以及
(2)在适于PCR扩增的条件下合并和片段化所说的相关多核苷酸或拷贝以形成其片段,进行PCR扩增,并由此同源重组所说的片段以形成包含新CDR组合的重组多核苷酸的杂合库,其中包含CDR组合的所说杂合库的重组多核苷酸主要部分不存在于第一群选定的文库成员中。
31.权利要求30的方法,包含另一个步骤,其中对杂合库进行亲和筛选以选择结合预定抗原决定基的杂合文库成员,并由此选择许多选定的杂合文库成员。
32.权利要求31的方法,包含另一步杂合许多选定的杂合文库成员并从1到大约1000次循环进行筛选。
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