CN86102090A - 用重组dna技术制备dna、rna、肽、多肽或蛋白质的方法 - Google Patents

用重组dna技术制备dna、rna、肽、多肽或蛋白质的方法 Download PDF

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CN86102090A
CN86102090A CN198686102090A CN86102090A CN86102090A CN 86102090 A CN86102090 A CN 86102090A CN 198686102090 A CN198686102090 A CN 198686102090A CN 86102090 A CN86102090 A CN 86102090A CN 86102090 A CN86102090 A CN 86102090A
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马克·巴利维特
斯图亚特·阿兰·考夫曼
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Abstract

本发明涉及微生物生产肽或多肽的方法，可以 在一种普通培养基中同时产生出至少有一部分是由 随机合成多聚核甘酸组成的基因，将这样的基因导 入宿主细胞，培养该宿主细胞以便无性繁殖那些随 机基因并导致由其所表达的蛋白质的产生，对所需 的宿主细胞转化体进行鉴别，筛选，分离，然后培养 之，生产出所需的肽或多肽。

Description

本发明的目的是一种获得DNA、RNA、肽、多肽或蛋白质的程序，用含有能够表达这些RNA、肽、多肽或蛋白质的基因的转化的宿主细胞，即利用重组DNA技术实现。
本发明的目的在于以一种可以同时获得的方式产生随机基因或随机基因碎片，这些基因转录和转译之后，大量（至少约为10，000）全新蛋白质或与已知蛋白质的杂化物在含有能够表达这些蛋白质的基因的宿主细胞（细菌或真核的）的存在下产生，并且为了确定它们中的哪一种产生具有新要求特性的蛋白质，随后在上述无性繁殖系中进行筛选，其性质有如结构的，酶的、催化的、抗原的、药理的或配体的特性，以及更一般的化学、生化、生物学等特性。其次本发明的目的在于通过修饰上述随机因或其产物而改进所要求的功能。
同样，本发明的目的表明了获得且随之改进具有可利用的显著的化学、生化或生物学特性的DNA或RNA序列的方法。
因此，本发明显然可供科学、工业和医学上的许多方面的大量应用。
根据本发明，肽或多肽的制备方法的特征是它在同一培养基中同时产生了至少部分是由合成随机多聚核苷酸的构成的基因，它将这样获得的基因导入宿主细胞，同时培养了含有如此基因的转化宿主细胞的独立无性系，因而无性繁殖了随机基因并获得了通过这些随机基因的每一个所表达的蛋白质产物，它以鉴别那些产生了具有至少一种所要求特性的肽或多肽的无性系的方式选择和/或筛选转化的宿主细胞无性系，随之将这样鉴定出的无性系分离出来并培养之，使它们产生至少一种具有所述特性的肽或多肽。当所要求特性需要改进时，该方法包括对产生已确定肽的基因的修饰，继之以上述基因的再次无性繁殖，并选择或筛选转化细胞以确定那些产生至少一种具有改进功能的肽或多肽的细胞。它也包括蛋白质生产物本身的修饰。
利用本技术的首要的方法为，通过四种脱氧磷酸核苷酸A、C、G和T从一个起始线性表达载体的两端的随机共聚产生随机基因，随后以这样的方式合成粘性末端以便形成由一分子具有两个3′末端是互补的随机序列的表达载体所构成的随机起始单链DNA，随之合成随机DNA的副链。
利用本技术的另一方式为，通过没有粘性末端的寡核苷酸的共聚产生随机基因，采用合成随机DNA碎片的方式，然后将这些碎片连到预先线性化的表达载体上。
表达载体可以是一个质粒，显著的是细菌质粒。应用pUC8质粒作为表达载体已经获得了极好的结果。
表达载体也可以是病毒DNA或者质粒与病毒DNA的杂化物。
宿主细胞可以是原核细胞，如HB101和C600，或者是真核细胞。
当应用上述第二种方式的技术时，能够利用形成了一组回文八聚体的寡核苷酸。
利用下述回文八聚体取得了极好的结果：
5′GGAATTCC    3′
5′GGTCGACC    3′
5′CAAGCTTG    3′
5′CCATATGG    3′
5′CATCGATG    3′
还能够应用形成一组回文七聚体的寡核苷酸。
应用下列回文七聚体取得了好结果：
5′XTCGCGA    3′
5′XCTGCAG    3′
5′RGGTACC    3′
此处X＝A、G、C或T、R＝A或T
按照利用这些特别有利的技术的方法，可以从一转化宿主细胞的独立无性系培养物中分离并提纯质粒的转化DNA，转化宿主细胞按上述方法获得，经后用至少一种对应于在回文八聚体或七聚体上所表现出来的而在所用表达载体上所没有的特异限制切割位点的限制性内切酶切割这些质粒;切割后将限制性内切酶钝化，然后同时用T4DNA连接酶处理这样获得的线性随机DNA碎片的群体，以这样的方式得到了含有随机序列的新的一套DNA，由此这个新群体含有比在起始群体中的基因数量更大的随机基因量。然后用这批新的转化DNA转化宿主细胞并无性繁殖这些基因，最后选择和（或）筛选并分离转化宿主细胞的新无性系并培养之以生产至少一种肽或多肽，例如，一种新型的具有所要求特性的蛋白质。
任何其它的用以产生并无性繁殖随机DNA序列以使其表达为新肽、多肽或蛋白质，或表达为融合蛋白质的新部分，继之以筛选或寻找所要求特性并获得至少一种产生具有所要求特性的肽的无性系的方法，都能够根据本发明而应用。
作为选择宿主细胞无性系标准的特性可为由给定无性系所产生的肽或多肽催化给定化学反应的能力。
进而，对于几种肽和（或）多肽的生产来说，上述性质可为催化一系列反应的能力，这些反应将一组起始化合物变成至少一种目标化合物。
为了生产由几种或许多种反身自催化的肽和（或）多肽所构成的群体，上述特性可为在适宜环境中由氨基酸和（或）寡肽催化合成该群体的能力。
上述特性也可以是选择性地修饰给定化合物的生物学或化学性质的能力，例如，选择性修饰一个多肽的催化活性的能力。
上述特性还可以是模拟、抑制、或修饰至少一种生物活性化合物的至少一种生物功能的能力，例如激素、神经递质、附着因子、生长因子以及DNA复制和（或）转录和（或）RNA的转译的特异调节因子。
上述特性亦可为肽或多肽结合于一给定配体上的能力。
本发明的目的还在于，将通过上述技术得到的肽或多肽用于配体的检测和（或）滴定的用法。
按照应用的特别有利的方式，选择转化宿主细胞无性系的标准是这些肽或多肽模拟或改变一生物活性分子例如一种蛋白质的作用的能力;产生至少一种具有如此特性的肽或多肽的转化宿主细胞无性系的筛选和（或）选择通过制备或获得对抗活性分子的抗体进行，随之在将它们提纯之后利用这些抗体鉴别含有那些结合有上述对抗活性分子的抗体的肽或多肽的无性系，然后通过培养已经鉴别了的无性系分离并提纯由这些无性系所产生的肽或多肽，最后对肽或多肽进行体外检测，证实它具有模拟或修饰上述分子的作用的能力。随机肽模拟或修饰上述分子作用的能力，能够通过修饰给该肽编码的基因，用修饰了的基因重新转化宿主细胞并对那些改进了所要求功能的修饰进行选择或筛选而得到改善。此外，上述肽可被化学修饰，或诱导从而改善其功能。
根据这种方法应用本发明的此项技术，作为选择标准的特性是具有至少一种与给定抗原的抗原决定部位之一相似的抗原决定基的性质。
本发明继续下去可由前述方法得到多肽并用作化疗活性物质。
尤其在上述抗原是EGF的情况下，本发明可获得用于上皮癌化疗的多肽。
本发明还适用于应用前述技术制备疫苗，其特征为分离抗致病作用的抗体，例如在给一能够产生抗此作用物的抗体的动物体内注射进致病作用物之后产生了抗体，这些抗体可用于鉴别产生至少一种最低限度具有类似于致病作用物抗原决定部位之一的抗原决定基蛋白质的无性系，对应于这些无性系的转化宿主细胞进行培养以产生这些蛋白质，从细胞无性系中分离纯化该蛋白质，然后用此蛋白质制备抗致病作用物的疫苗。如上所述，纯化了的蛋白质可经编码该蛋白质的随机基因修饰（例如，致突变）而改进，将这些基因再次转化进适当的表达修饰蛋白质的宿主细胞中，再次筛选或选择那些将结合于抗起始抗原作用物的抗体的并具有改进功能蛋白质，并利用这些改良蛋白质生产疫苗。
例如，为制备抗HVB疫苗，可提取并纯化HVB病毒的至少一种衣壳蛋白，将该蛋白注射进一种能够产生抗这种蛋白质的抗体的动物体内，回收并纯化这样形成的抗体，利用这些抗体去鉴别产生至少一种蛋白质的无性系，该蛋白质具有至少一个类似于HVB病毒的抗原决定部位之一的抗原决定基，随后培养在某种程度上与这些无性系相称的转化宿主细胞无性系以产生该种蛋白质，从这些细胞的无性系培养基中分离并纯化该蛋白质并用它制备抗HVB疫苗。
根据这项技术的改变，通过用对应于DNA杂化物天然部位所表达的蛋白质抗体的亲和层析可鉴别并分离产生具有所要求特性的肽或多肽的转化宿主细胞无性系。
例如，在杂种DNA的天然部位含有一个表达β-半乳糖苷酶基因的情况下，通过用β半乳糖苷酶抗体的亲和层析技术可有利于鉴别并分离上述转化宿主细胞无性系。
杂种肽或多肽的表达和纯化后即可区分并分离它们的新部位。
根据本发明，应用这项技术的一个手段是筛选新的肽、多肽或催化一给定化学反应的蛋白质。
根据利用本发明的技术的先进手段，宿主细胞是大肠杆菌一类的细菌，其基因组既不含有表达β半乳糖苷酶的天然基因，也没有EBG基因，也就是说是Z-，EBG-Ecoli。转化细胞培养于含有X半乳糖及指示剂IPTG的培养基中。并检测到了对β半乳糖苷酶功能阳性的细胞;随后将转化DNA移植于一适宜的宿主细胞无性系中以大规模培养之，从而产生至少一种具有β半乳糖苷酶活性的肽或多肽。
作为选择转化宿主细胞的标准的特性也可以是通过培养这些无性系而产生的肽或多肽结合给定化合物的能力。
该化合物优先选自肽、多肽和蛋白质中，特别是那些调节DNA转录活性的蛋白质。
本发明的目的还在于，具有那些在作为转化宿主细胞无性系的选择标准的特性是这些蛋白质结合控制DNA转录活性或复制的调节蛋白质的能力的情况下获得的蛋白质。
另一方面，上述被结合的化合物也能从DNA和RNA序列中选出。
此外，本发明的另一个目的在于，获得了一种能结合DNA序列的蛋白质，该序列起控制邻近DNA序列的复制和转录的顺式调节的作用，并通过结合限制邻近DNA序列的转录或复制。同样，本发明的另一个目的是获得一种可结合RNA序列的蛋白质并因此控制了该RNA的转译作用或RNA的稳定性。
本发明的目的包括利用由前述第二种情况下得到的蛋白质修饰含在细胞内的DNA序列的转录或复制特性，并表达该蛋白质。
作为其目的本发明亦表明了一种产生DNA的方法，其特征为在同一培养基中同时产生，该基因至少部分由随机合成多聚核苷酸构成，如此获得的基因被导入宿主细胞以产生转化宿主细胞群体，对该群体进行筛选和（或）选择以鉴别那些含有DNA随机序列的宿主细胞，其DNA具有至少一种所要求的特性，最后从如此鉴别的宿主细胞无性系中分离出DNA。被鉴别的DNA的特性可通过随机基因的修饰而改善，例如通过各种已知的或将下述的致突变技术，再克隆到适宜的载体上，转化适宜的宿主细胞，随即筛选或选择出所要求特性的改进水平。
本发明的目的还在于一种产生RNA的方法，特征是在同一培养基中同时产生至少部分是由随机合成多聚核苷酸所构成的基因，该基因导入宿主细胞产生转化宿主细胞群体，这样产生的宿主细胞同时培养，并筛选和（或）选择这个群体以鉴别那些含有具备至少一种所要求特性的RNA序列，并从这样鉴别了的宿主细胞中分离出RNA。如上所述，这样分离的RNA特性可通过对应随机基团的修饰、再转化及再筛选或再选择而得到改善。
上述特性可以是结合给定化合物的能力，该化合物可为诸如肽或多肽或蛋白质，该特性也可为催化给定化学反应的能力，或作为转移RNA的能力。
首先，我们将描述极其有用的进行随机基因的合成以及将这些基因导入细菌以产生转化细菌的无性繁殖技术。
1）在一表达载体上的直接合成。
a）载体的线性化。
30mg，即大约1013pUC8表达载体分子通过在37℃下用100单位溶于300微升专为该酶的标准缓冲液中的Pst1限制性内切酶处理两个小时而被线性化。线性化的载体用石炭酸-氯仿处理，然后在乙醇中沉淀，在标准TEB缓冲溶液中溶解为30μl体积并吸附于0.8%琼脂糖凝胶上。在3v/cm范围内走柱三个小时后，线性载体被电洗脱，于乙醇中沉淀并溶于30微升水。
b）用末端转移酶（Td    T）的随机合成
30μg线性化载体与30单位的Td    T于300μl适宜的缓冲液中反应，缓冲液中有1mMd    GPT，1mMd    CTP，0.3m    Md    TTP及1mMd    ATP。选择低浓度d    TTP是为了降低在单一的信使RNA中的“终止”密码子的频率。尽管稍微不顺，但利用比其它脱氧三磷酸核苷酸低一些浓度的d    ATP亦能取得类似的效果。在Pst1位点的3′末端上的共聚过程之后，将反应过程中取出的等分试样在凝胶上进行分析。
当反应达到或超过每3′末端所加入的核苷酸平均值为300时停止反应并用差式示沉淀法或者通过象生物凝胶P60一类的分子筛选柱将游离核苷酸与共聚物分离。在乙醇中沉淀浓缩之后，共聚物与Td    T进一步聚合，首先在有d    ATP，随后为d    TTP存在的情况下进行。最后这两个反应通过凝胶过滤分开并留有短的间隔（30秒到3分钟）以连续加入10-30A继之以10-30T于共聚物的3′末端。
c）随机DNA副链的合成
在上述反应结束时每分子载体都具有两个随机序列，它们的3′末端是互补的。共聚物的混合体培养在有利于互补末端杂化的条件下，150mM    NaCl，10mM    Tris-HCl，pH7.6，1mM    EDTA，65℃下10分钟，随后以每小时3-4℃的速度降至22℃。杂种聚合物随后与60单位聚合酶Ⅰ大断片（Klenow）在有四种三磷酸核苷酸（200mM）存在的情况下于4℃反应两个小时。这一步骤完成了从杂种共聚物的3′末端合成副链。从线性载体开始的这步直接合成得到的分子随后就用于转化感受态细胞。
d）感受态无性系的转化
100-200ml浓度为1010细胞/ml的C600细胞的感受态HB101与随机DNA制剂（取自上述）共同培养，条件为存在有6mM CaCl2，6mM Tris-HCl，pH8，6mM MgCl2，0℃，.30分钟。对混合物施加一个37℃3分钟的温度冲击，随后加入400-800ml无抗菌素的NZY培养基。转化的培养物于37℃培养60分钟，然后加入含有40μg/ml的氨苄青霉素的NZY培养基稀释至10立升。37℃培养3-5小时之后离心分离稀释培养液，转化细胞的片状沉淀物冻干储存于-70℃。这样的一个培养物含有3×107-108独立转化株，每一个都含有一个独特的嵌入表达载体的随机基因。
Ⅱ）从没有粘性末端的寡核苷酸起始的随机基因合成。
这项技术建立在这样事实的基础上，明智地选择了回文寡核苷酸的共聚化，使在六种可能的解读密码中的任意一个都没有“终止”密码子的随机基因得以构成，同时保证详细说明了氨基酸的三联体密码的平衡表达。此外，为了避免序列主要部分在用少量的起始回文寡核苷酸时所产生的蛋白质中的重复，寡核苷酸可含有若干碱基，但不超过三倍，下述实例描述了达到这些标准的可能组合之一的用法。
a）八聚体组的选择
下列寡核苷酸群：
5′GGAATTCC    3′
5′GGTCGACC    3′
5′CAAGCTTG    3′
5′CCATATGG    3′
5′CATCGATG    3′
由5组回文构成（这样自身互补序列为回文），这里易于证明它们的随机共聚作用没有产生任何“终止”密码子，并表达了所有的氨基酸。
显然可以利用其它回文八聚体群，它不产生任何“终止”密码子并表达了所有在多肽中发现的氨基酸。显然也可能利用非回文八聚体群或其它寡聚物，条件是，也用了它们的形成双链DNA的互补体。而且可能使用多于5种的回文八聚体。
b）从一组八聚体中集合随机基因群体
一种含有前述寡核苷酸每一种各5μg的混合物（先用标准技术在5′位上磷酸化寡核苷酸）在100μl含有1mMATP，10%聚乙二醇，及100单位TDNA连接酶在适宜的缓冲液中于13℃反应6小时。这一步将八聚体随机聚合成双链状态而没有粘性末端。聚合产物过分子筛（生物凝胶P60）分离回收20-100寡聚体。浓缩后该部分在上述条件下再用TDNA连接酶催化聚合。然后如上所述那些至少集合有100寡聚物的共聚体被分离出来。
c）宿主质粒的制备
PUC8表达载体用Smal酶于适宜的缓冲液中线性化，按上述方法进行。SMal线性化的载体没有粘性末端。因此线性载体用牛小肠碱性磷酸酶（CIP）于每毫克载体一单位的水平在适宜的缓冲液中37℃处理30分钟。CIP随后用石炭酸-氯仿连续两次萃取的方法钝化。线性脱磷酸载体在乙醇中沉淀，然后在水中重新溶解为1mg/ml。
d）随机基因与载体的连接
等摩尔量的载体和聚合物混合并在有1000单位T4DNA连接酶，1mMATP、10%聚乙二醇存在的情况下于适当缓冲液中13℃下培养12个小时。这一步骤将随机共聚物连接于表达载体上并形成双链环状分子，因此它可进行转化。
感受态无性系的转化。
感受态无性系的转化按前述方式进行。
Ⅲ）从一组七聚体开始的随机基因的装配。
这步程序与刚讨论过的不同，不同点在于利用具有可变粘性末端的回文七聚体取代了八聚体。这步程序具有能装配含有少量同源主要部分的随机序列的优点。
a）七聚群体的选择
如例，能用下述三种回文七聚体：
5′XTCGCGA    3′
5′XCTGCAG    3′
5′RGGTACC    3′
这里，X＝A、G、C或T，R＝A或T，这里共聚化不能产生任何“终止”密码子并形成代表所有氨基酸的三联体密码。
显然，可用满足这些同样条件的七聚体的其它群体。
b）一组七聚体的聚合
这步骤合作用与前述八聚体的方式完全相同。
c）粘性末端的消除
这样得到的共聚物在其两个5′末端上有一不成对碱基。因此必须在单一的3′末端加上互补碱基。这一步骤如下进行：10mg双链共聚物在有100μl体积的脱氧磷酸核苷酸（200mM）存在的情况下于4℃与10单位Klenow酶反应60分钟。酶用石炭酸氯仿提取法灭活，共聚物用差示沉淀法净化剩余的游离核苷酸。然后将共聚物按前述方法连接于宿主质粒（先已线性化及脱磷酸化过的）。
应该注意的是后两种已描述过了的方法利用了构成某种限制内切酶特异位点的回文八聚体或七聚体。这些位点在很大程度上是pUC8表达载体所没有的。因此，按照下述方法，有可能相当大地增加随基基因起始制备的复杂性：分离由107独立转化株培养中得到的质粒DNA，而该转化株是按上述最后两种方法之一获得的。该DNA纯化后，用Clal限制性内切酶（方法Ⅱ）或Pst1限制性内切酶（方法Ⅲ）部分酶解。酶灭活后，部分酶解DNA用T4DNA连接酶处理，这样产生了极大量的新序列，同时保持了起始序列的基本特性。随后这个新的随机序列的群体就可用于转化感受态细胞。
另外，通过方法Ⅱ和Ⅲ克隆化的随机基因利用属于克隆载体的内切位点从pUC8表达载体上完整地切割下来并且不在随机DNA序列中表现出来。
通过上述两种方法产生的随机基因内的重组，由于其周期发生的分子主体导致了体内同源性，因而，是一种重要的实现编码序列的体内致突变的附加方法。这样就导致了能被检测的新基因数量的扩大。
最后，对于所有产生新合成基因的方法来说，可用一些普通的技术在体内或体外修饰基因，例如解读密码的改变，相对于其启动子的序列的倒位，点突变，或利用宿主细胞表达一种或几种抑制子tRNA。
考虑到上面的描述，显然能在体外合成极大量的（例如多于十亿）不同的基因，通过核苷酸或寡核苷酸的酶促聚合作用实现。这步骤可以随机方式进行，即由反应混合物中的核苷酸或寡核苷酸的各自浓度所决定。
如上所述，可用两种方法克隆化这样的基因（或编码序列）：共聚作用可直接于一克隆表达载体上进行，该载体已预先线性化了;或先连续进行聚合，随后将聚合物与表达载体相连接。
在这两种情况下，下一步骤是感受态细菌细胞（或培养的细胞）的转化或转染。这一步包括在活细胞中克隆繁殖随机基因，并使之非限定性地增殖和表达。
显然，除了上述方法之外，用所有其他适宜的方法合成随机序列是可行的。尤其能用生化的手段将得自化学合成的DNA或RNA单链寡聚物聚合，随后用确定的技术处理DNA或RNA的这些片段产生双链DNA（cDNA），以便克隆这样的基因。
转化宿主细胞无性系的筛选
本发明方法的最后一步是通过选择或筛选检测转化或转染细胞，其目的为分离一至数种这样的细胞，即其转化的或转染的DNA导致具有所要求特性的转录产物（RNA）或转译产物（蛋白质）的合成。这些特性可为诸如酶促的、功能的，或结构的特性。
根据本发明，该技术的最重要的方面之一是能同时筛选或选择一种可利用的产物（RNA或蛋白质）及产生该产物的基因。此外，前述合成并克隆化的DNA可为完全分离具有可利用生化特性的形成产物的DNA序列而被选择或筛选。
现在我们将举非限定性的实例说明对于转化细胞无性系选择或筛选的最佳方法，因而从工业或医学应用的角度看，新型蛋白质是有意义的。
这些方法之一建立在这样的想法上，通过已确定的技术产生或获得的多克隆或单克隆抗体直接抗蛋白质或其它类型的生化或医学分子，此处该分子是或已经是提取的、致免疫的，随后用这些抗体作为探针从大量的用随机基因转化的无性系中鉴别那些其蛋白与这些抗体反应的无性系。这步反应是存在于随机基因合成的多肽与起始分子之间的结构（抗原决定部位）同源的结果。用这种方法能够分离若干具有类似于起始分子上的抗原决定部位或抗原决定因子的分子特征的新型蛋白质。这样的新型蛋白质是模拟、刺激、调节、或限定起始分子作用的侯补者。显然这种选择或筛选手段具有非常多的制药和生物医学用途。如上所述，按具有类似于起始分子的抗原决定部位鉴定的肽可通过修饰为上述鉴定了的蛋白质或肽编码的随机基因，继之以用已知方法再克隆和再筛选或诱导那些肽本身而被改良。
下面我们在具体情况下举非限定性实例说明这项操作的第一种方式：
EGF，（表皮生长因子）是存在于血液中的小蛋白质，其作用为刺激表皮细胞的生长。这个效果是由EGF与位于表皮细胞膜上的特异受体的相互作用而得到的。通过给动物注射结合于以增强EGF的免疫原性的KLH（铜孔
Figure 86102090_IMG1
血青蛋白）的EGF，制备直接抗EGF的抗体。纯化免疫动物的抗-EGF抗体，用诸如过亲和层析柱的方法，这里的配体是EGF或相当于EGF碎片的合成肽。纯化了的抗EGF抗体随之作为探针筛选大量的溶于氯仿的细菌无性系，在固体支持器上进行。抗EGF抗体结合那些类似起始抗原的抗原决定部位的随机肽或蛋白质。含有这种肽或蛋白质的无性系在与放射性蛋白质A固体支柱培养或与一放射性抗抗体抗体培养之后用射线自显影术显示出来。
这些步骤鉴别那些每一种都含有一种与筛选抗体反应的蛋白质（或其基因）的无性系。在大量细菌细胞或病毒噬菌斑（例如总数约为1，000，000）菌落中进行筛选是可行的且可检测出数量约为1毫微克的极少量的蛋白产物。随后培养已经鉴定了的无性系，这样检出的蛋白质用常规方法纯化。这些蛋白质在上皮细胞培养中进行体外检验以确定它们是否抑制、模拟或调节这些培养中的EGF的作用。在这样获得的蛋白质中，有些可被用于上皮细胞癌的化疗中。这样获得的蛋白质的活性能够通过用类似上述方法或将所述蛋白质衍生的方法，使为蛋白质编码的DNA突变而得到改善。
本方法的改进型是纯化这些随机肽、多肽或蛋白质，它们具有抗原决定部位或使它们被抗某些抗原例如一种病原体抗原的抗体所结合的分子特征，随后用已鉴定的肽作为疫苗或更一般地利用它们在免疫系统产生抗致病作用物的免疫性或产生其他作用，例如产生耐受性或特别是由于这些肽、多肽或蛋白质与直接抗该抗原的抗体结合而降低对应于给定抗原的过敏性。显然可在体外及体内使用该肽、多肽或蛋白质。
更准确地说，在与抗给定抗原X的抗体反应的新型蛋白质的群体中，每一个都具有至少一个与X相同的抗原决定基，所以该群体具有一个与X相同的抗原决定基群体。它使该群体或亚群可用作疫苗产生抗X的免疫性。例如易于纯化一或几种乙型肝炎病毒的衣壳蛋白。将这些蛋白注射进兔子一类的动物体内，可用亲和柱纯化法回收对于起始抗原的抗体。如上所述，这些抗体可用于鉴定产生至少一种蛋白质的无性系，该蛋白质具有类似于起始抗原的至少一种抗原决定部位的抗原决定基。纯化后，这些蛋白可（单独或联合）作为抗原以产生抗乙型肝炎的保护作用。疫苗的最终产物不需要进一步接触起始致病作用物。它不要求由乙肝病毒得到的起始抗原是蛋白质抗原，而宁愿它们能包括多糖或其他抗原决定因子。此外，在有利的情况下，不需要接触起始病原体，而将抗由与病原体接触的动物或人体产生的病原体的高效价循环抗体作为起始步骤筛选被那些高效价抗体所结合的肽。这样的高效价循环抗体可达到所有循环抗体的1%-10%，因此，已确定的随机肽的1%-10%作为交叉反应将具有类似（可能未知的）病原体的抗原决定因子。该确定了的交叉反应肽混合物可作疫苗。与高效价抗体分子交叉反应的肽的亚型或可通过筛选与血清交叉反应肽的集合体以及鉴别那些对于它们在血清中存在高水平循环抗体的肽而被选择出来。这个筛选出的交叉反应肽的集合体可随之用于生产免疫性的疫苗，或作为具有其他免疫修饰作用的物质。
在上述方法的描述中注意到，已经描述了一些选择和筛选的方法。所有这些方法可要求从一转化无性系中提纯一种特定的蛋白质。这些蛋白质的提纯可通过已知方法以及利用特别是凝胶层析，离子交换，以及亲和层析技术而进行。另外，由随机基因产生的蛋白质已能以杂种蛋白质的形式无性繁殖，该蛋白质具有象β半乳糖苷酶的序列，该酶允许抗抗β半乳糖苷酶抗体的亲和层析，以及随后的杂种部位的分解（即允许杂种蛋白的新部位与细菌部位的分离）。
下面我们叙述根据建立在检测这些肽或多肽催化特定反应的能力的基础上的辅助筛选方法筛选肽或多肽及相应的基因的原理和技术。
作为一个具体的和非限定性的例子，我们将描述在一特定的能够催化乳糖分解，通常为β半乳糖苷酶进行的功能的蛋白质的情况下的筛选。
如上所述，程序的第一步是产生表达载体的大群体，其中每个载体均能表达一个独特的新型的蛋白质。例如，具体地说，选择pUC8表达载体于Pst1限制优点克隆DNA的随机序列。这样获得的质粒随之导入-E.coli无性系中，其基因组中的β半乳糖苷酶、Z的天然基因以及与前者无关但能突变成β-半乳糖苷酶的辅助基因EBG均已被消除。这样的宿主细胞（Z-，EBG-）不能自己水解乳糖。并因此利用乳糖作为生长的碳源。这样就可用这个宿主无性系筛选β-半乳糖苷酶功能。
一种简便的用于分析那些具有新的表达β半乳糖苷酶功能基因的转化E.coli的生物学检验法是将上述转化的细菌培养在含有X-半乳糖的培养基中。在这种情况下，所有表达了β半乳糖苷酶功能的细菌菌落都可见到是呈蓝色的菌落。用这样一种生物检验法能测得甚弱的催化活性。这种酶的特异活性范围在每秒钟10-10，000个产物分子。
假设一个由一个随机基因合成的蛋白质有微弱的特异活性，水平约为每100秒1分子，这样的催化活性仍能被检出。培养基中含有X-半乳糖的培养皿中，在有不能产生代谢变化的诱导物IPTG（异丙基硫代半乳糖苷）存在的情况下，出现一个蓝色区域要求大约每平方毫米分解1010-1011个X半乳糖分子。一个表达了一个弱酶且占据了1mm2的表面区域的细菌菌落有约107-108个细胞。如果每个细胞只有一个弱酶复制品，每个细胞应需要催化分解10，000-100个X半乳糖去被检测，这就要求2.7-270个小时。因为筛选能将每个细胞的每一质粒的复制品的数量增加到每个细胞有5-20个复制品，甚至达到100或1000个，而且因为相当于10%的细胞蛋白质可被新基因表达，在每个细胞有100个酶分子的微弱活性的情况下检验菌落变蓝所需的时间约为0.27-2.7个小时。
作为这些事实的结果，筛选大量独立的每一个都表达不同的新基因的细菌菌落，并利用表达β半乳糖苷酶作用的能力作为选择的标准是完全可行的。在一个直径为10cm的培养皿中进行大约2000个菌落的筛选是可能的。因此，可在一大片1平方米的X半乳糖琼脂培养基上筛选大约20，000，000个菌落。
需要严重关切的是在X半乳糖培养皿上显示蓝色的细菌落可能是假的阳性，因为在细菌产生代谢乳糖能力的基因组中发生了突变，或者只是因为那些起因于菌落细胞所表达的新蛋白质的催化活性。这样的假阳性可直接用从阳性菌落上纯化表达载体DNA并重新转化Z-，EBG-E.coli宿主细胞的方法除去。如果β半乳糖苷酶活性是由于被表达载体中的新基因编码的新蛋白质，则所有那些被该载体转化的细胞都将表现β半乳糖苷酶作用。相反，如果起始的蓝菌落是由于宿主细胞基因组中的突变，则为罕有的事件并与转化无关，这样能够表现β半乳糖苷酶功能的转化Ecoli新无性系细胞的数量将很小或零。
应该强调从所有阳性无性系（蓝色的）中批量同时纯化所有表达载体随后重新转化天然细菌的能力。假设目的是进行筛选以选出具有催化功能的蛋白质，以及一种新的肽或多肽至少微弱地表现出这个功能的概率是10-6，同时Ecoli细菌宿主无性系发生突变，使它能表现出同样功能的概率是10-5，随后预测出从筛选出的20，000，000转化细菌中，20个阳性无性系可归因于表达载体所携带的新基因，另200个阳性无性系将是基因组突变的结果。从所有220个阳性细菌无性系中大量纯化表达载体继之以用这些表达载体混合物重转化天然细菌将产生大量的阳性无性系，该无性系由所有那些用20个为具有所要求功能的新蛋白质编码的表达载体所转化的细菌，以及极少量的由基因组突变所引起的且含有200个无意义的表达载体的细菌无性系所组成。从阳性细菌菌落中纯化表达载体的几个周期，继之以这样的重转化，使得非常少的对于所要求的催化活性为真正阳性的表达载体得以检出，尽管对于同样的功能在宿主细胞中有高背景比例的突变。
在这类筛选操作之后，可用已确立的技术纯化新蛋白质。大量的这种蛋白质的生产由实用蛋白质的鉴别以及同时的为同样的蛋白质编码的基因的鉴别一起进行的事实所构成。结果，或者使用相同的表达载体，或者将新基因移植于一个更适宜的表达载体中以大量合成和分离。
将这种筛选的方法用于任意酶促的作用是可行的，对于该作用有一适宜的生物学检验法。对于这种筛选，所要求的酶促功能对宿主细胞不必是实用的。不但对于酶促功能，而且对于其它任何所要求的能建立一种适宜的生物学检验法的特性都能进行筛选。此外，甚至在于-X-gal培养皿上所显示的β半乳糖苷酶功能这样简单的情况下，在100，000，000甚或1，000，000，000新基因的数量级上对其催化活性或其它所要求特性进行筛选是可行的。
转化宿主细胞的选择。
另一方面，选择技术能够用于任意特性，催化的或其它的，这里特性的存在与否可理解为含有为新基团编码的表达载体的宿主细胞生存的要素，或亦可用于选择那些编码和表达所要求新基因的病毒。作为一个非限制性的但具体的例子，β半乳糖苷酶功能的选择将被描述。一个适宜的Z-，EBG-E.coli无性系不能以乳糖为唯一碳源而生长。因此，在进行了上述第一步骤之后，能够在选择的情况下，或者逐渐减少其它碳源，或者从一开始就只用乳酸培养极大量的被为新基因编码的表达载体所转化的宿主细胞。在这样选择的过程中，通过重组或直接回收表达载体在体内致突变，以及在体外用各种诱变剂或其它任何常见技术使其新基因致突变，在执行所要求的催化功能的能力上有适当的改进。象在这种情况下，当选择技术和简便的生物检测技术同时存在时，开始可用选择技术丰富显示β半乳糖苷酶作用的宿主细菌的表现，随后在培养皿上对X-gal培养基进行筛选从而高效率地证实哪些是阳性细胞。在没有方便的生物检测的情况下，应用逐渐严谨的选择是最容易的纯化一种或少量独特的宿主细胞的方法，这些细胞的表达载体为催化所要求反应的蛋白质编码。
应用这些技术能够发现具有多种多样结构及除催化一种特定反应的能力以外的功能特征的新型蛋白质。例如，能够对在DNA上结合顺式调节位点并因此抑制宿主细胞功能之一的表达或阻断DNA的转录，刺激转录等的新型蛋白质进行筛选。
例如，就E.coli而言，一个乳糖操纵子（i-）阻遏物中的无性突变体显示了β半乳糖苷酶功能，基本上是由于乳糖操纵基因不被抑制。这个类型的所有细胞在含有X-gal培养皿上产生蓝色无性系。能够用合成新型蛋白质的表达载体转化这类宿主细胞，并在X-gal培养皿上进行筛选以检测那些不是蓝色的无性系。其中的一些表现了新蛋白质结合了乳糖操纵基因并表达了β半乳糖苷酶的合成的情况。随后进行大量分离这样的质粒，再转化，i-宿主分离那些不产生β半乳糖苷酶的无性系，然后进行详细的鉴定。
如上所述，这种方法可用于产生然后完全分离可利用蛋白质，而且包括RNA和DNA作为具有可利用特性的产物。这一方面起因于该方法是产生可与其它分子或生化组分直接作用的DNA随机序列，另一方面，这些在表达载体中克隆化的序列被转录成它们本身能进行多重生化作用的RNA。
本方法用于产生和选择实用DNA的实例。
本实例说明了对实用DNA的选择，以及结合于DNA的调节蛋白的纯化及其作用机制的研究。考虑雌二醇受体，一种用标准技术所获得的蛋白质的制备。在有雌二醇，一种固醇类性激素，存在的情况下，受体改变构象并紧紧地结合在基因组DNA中某些特异序列上，从而影响与性别分化有关的并控制生育力的基因的转录。通过培养含有雌二醇，它的受体以及大量不同的嵌入其载体的随机DNA序列的混合物，随后在硝化纤维膜上过滤混合物，直接选择那些结合在雌激素受体复合物上的随机DNA序列，这里，只有那些被蛋白结合的DNA才能被膜挡住。冲洗和洗脱之后，从膜上释放出的DNA被用于转化细菌。转化细菌培养后，它们所含有的载体被再次纯化并培养几个周期，按上述方法进行过滤和转化。这些方法分离了对雌二醇受体复合物具有更高亲和性的DNA随机序列。这样的序列对许多诊断学和药物学的应用，特别是用于检验用于繁殖力的控制和不育的治疗上的合成雌激素而开放。
实用RNA的产生和选择
假设有大量的按已述方法产生并于一表达载体内并克隆化的随机DNA序列。结果是从这些序列在转化宿主细胞中转录的RNA可为完全实用的产物。作为一个非限制性例子，能够按下述方法选择一个为抑制基因转移RNA（tRNA）编码的随机基因：
大量（＞108）的随机序列转化到在精氨酸E.基因（arg E.gene）上有一“nonsense”突变的感受态细菌宿主。这些转化了的细菌平板培养在没有精氨酸而有对该质粒选择性的抗菌素（如果载体是pUC8为氨苄青霉素）的基本培养基上。只有那些已经变成能合成精氨酸的转化的细菌才能够生长。这个表现型既可由一宿主基因组的回复突变，又可由于细胞中的抑制基因tRNA的存在而引起。易于检测每一转化菌落以确定arg+表现是否由于在其载体中存在随机基因;它满足了纯化该菌落质粒并证实它在所有它所转化的arg E细胞上产生了Arg+表现型的需要。
能够催化一系列反应的蛋白质的选择。
下面我们介绍另一种对独立应用开放，建立在同时平行选择一定量能够催化一系列相关反应的新蛋白质的原理基础上的选择方法。
本方法的基本设想如下：给定一起始系列的化合物作为建筑积木或建筑成分，由此有望通过一系列催化的化学反应合成一或数种所要求的化合物，有极多的反应途径可以部分或全部互相替换，并且在热力学上全都是可能的，它们将建筑积木构成所要求的目标化合物。如果至少一个将建筑积木化合物构成目标化合物的反应途径的每一步由每个都是催化的反应所组成的话，高效合成目标化合物是有利的。另一方面，在许多独立的或部分独立的反应途径中它是否被催化相对来说不是很重要的。在前述方法中，我们已经说明了如何能得到大量的每一个都表达一个独特的新型蛋白质的宿主细胞。
这些新型蛋白质的每一个都是催化所有可能将建筑积木群体构成目标化合物的反应集合体中的某个可能反应的候补者。如果足够大量的随机蛋白质存在于含有建筑积木化合物的反应混合物中，以致于足够大量的可能的反应被催化，则一系列关联的将建筑积木化合物的集合体构成目标化合物的反应将被新蛋白质的亚群所催化就是高概率的了。显然这种方法可被扩展到同时催化不只是一种，而是几种目标化合物。
根据这个原理，为了同时选择一套催化一系列所要求的化学反应，如下所述那样进行是可能的。
1.选定构成建筑积木化合物所要求的集合体，优先利用适当量的独特化学物质以增加有可能同时发生的导向所要求的目标化合物的反应数量。
2.用适当体积的反应介质，加入大量的从合成它们的转化或转染细胞中分离出的新的随机蛋白质。进行检验以确定是否已形成了目标化合物。如果是的话，进一步确定这步形成要求有新蛋白质的混合物参与。如果是这样的话，则混合物应该含有一个催化一个或几个将建筑积木集合体构成目标化合物的反应途经的蛋白质亚群。纯化分离合成一套新型随机蛋白质的无性系的起始群体。随后重新检验亚群看它能否催化与产生目标化合物关联的反应。
更确切地说，作为一个非限制性的例子，下面我们介绍一套能够催化一个特异小肽，特别是一个五肽，从由更小的肽和氨基酸所构成的建筑积木集合体开始的合成的新型蛋白质的选择。所有的肽都是由20种不同类型的氨基酸线性序列所组成，方向为从氨基末端到羧基末端。任何肽能以专一的方法通过两种较小的肽（或两种氨基酸）的末端缩合或一较大的肽的水解而形成。具有M个残基的肽可因此通过M-1步缩合反应而形成。反应的数目R，通过它一个具有1，2，3......M个残基长度的肽链可被互相转换，比可能的分子种类的数目T要大。这可表示为R/T≌M-2。因此，从肽的给定群体开始，大量的独立或部分独立的反应导致了特异目标肽的合成。选择了一个其存在可通过一些常见检测技术，例如HPLC（液相高压色谱），纸层析等，而被方便地测得的五肽。在一稀释的水介质中形成一条肽键需要能量，但如果参与缩合反应的肽充分浓缩的话，则在热力学上肽键的形成比水解有利，而且在有一适宜的酶催化剂，例如胃蛋白酶或胰蛋白酶，存在的情况下，不需要ATP或其它高能化合物的参与就能高效地进行。这样一个其氨基酸用3H，14C，35S作为示踪物而被放射性标记的，由建筑积木集合体所构成的小肽反应混合物可在足够高的浓度下用于缩合反应。
例如，可按下述那样进行：每种氨基酸和有2～4个氨基酸的小肽各15mg，用作建筑积木集合体，溶于0.25ml每毫升0.1M    pH7.6的磷酸缓冲液中。大量按上述方法繁殖和分离的新蛋白质从它们的细菌宿主细胞中被纯化出来。新蛋白质混合物在同样的缓冲液中溶解至最终浓度约为0.8～1.0mg/ml。0.25～0.5ml的蛋白质混合液加入于建筑积木混合物中。在25～40℃下保温1～40个小时。每隔一定时间取出8ml等分试样，第一份作为“空白”（blank）在新蛋白质混合物加入之前取出。这些等分试样通过层析法用正丁醇-乙酸-吡啶-水（30∶6∶20∶24）作为溶剂进行分析。层析分离谱干燥后用茚三酮或放射自显影术（用不用增感屏均可）分析。因为构成建筑积木集合体的化合物被放射标记，所以目标化合物是放射性的，并具有足够高的可在1～10ng级别测得的特异活性。代替标准的层析分析法，可用HPLC（高压液相层析法），它可更快并更简单地进行。更一般地说，所有常用分析技术都可应用。结果能测得相对于作为起始建筑积木的化合物重量计不到百万分之一的目标化合物的产率。
如果在上述条件下合成五肽，而不是用一从不含随机基因的表达载体转化的宿主细胞得到的提取物，五肽的形成不是细菌杂质的结果，因此在反应混合物中要求新蛋白质亚群的存在。
下一步骤是细胞特定亚群的分离，这种细胞含有具有催化产生目标五肽的系列反应的新型蛋白质的表达载体。举例说明，如果形成该序列的反应数目是5，则约有5种新蛋白质催化必须的反应。如果含有为新基因编码的表达载体的细菌克隆库具有若干总数约为1，000，000的性质截然不同的新基因，则所有这些表面载体全部被分离并再转化到108个细菌的100个不同的集合体中，载体对细菌的比例足够低，即平均转化了的集合体中细菌的数量约为起始基因的半数，即大约500，000。因此，任何已知细菌的100个集合体之一含有5种临界的新蛋白质的全部集合体的概率是（1/2）5＝1/32。在100个起始细菌集合体中，大约3个含有5个临界转化株。在这些集合体的每一个当中，新基因的总数只有500，000而不是1，000，000。在目前状况下，经连续重复20次，本方法分离了5种临界新基因。随之对5种随机基因的这个集合体致突变及选择，从而改善了必要的催化功能。在必须催化20个反应并同时分离编码新蛋白质的20个基因的情况下，它满足了调节转化的多重性的要求，因而108个细菌的每一个集合体得到了10e6个随机基因的80%，并使用了200个这样的细菌集合体。在一个集合体中发现所有20种新蛋白质的几率是0.820≌0.015。因此，在200个集合体中大约有2个将具有所需催化目标化合物形成的20个新基因。分离20种新基因所需要的周期数约为30。
上述原理和技术从肽对化学的许多方面作用的角度归纳出，在这些方面中化学反应在水介质中，温度，pH和浓度等条件允许一般的酶促作用发生的条件下进行。在每一种情况下必须用一种检验方法检测要求的目标化合物的形成。也必须选择足够量的建筑积木化合物扩大产生目标化合物的反应序列的数量。
为目标五肽的合成所给出的具体实例也可按下述那样被推广：
上述技术从其它产物中产生随机肽和蛋白质。这些肽或蛋白质能对其它化合物起催化的或其它方式的作用。它们同样能构成它们为之起作用的底物。因此，对于这样的随机肽或蛋白质在它们本身中相互作用并因此修饰其中一些的构象，结构或功能的能力的选择（或筛选）是可能的。相似地，能够对这些肽或蛋白质对它们本身催化水解，缩合，转肽或其它修饰肽的反应的能力进行选择（筛选）。例如一个给定的随机肽被随机肽或蛋白质集合体的至少一个成员催化的水解可通过给定蛋白质的放射标记继之以和随机蛋白质混合物在有诸如Mg，Ca，Zn，Fe和ATP或GTP存在的情况下一起培养而被跟踪和测量。标记蛋白质的放射性碎片的出现按前述方法测得。催化该反应的随机蛋白质可按上述方法，通过作为结果出现的转化无性系库的缩减与产生它们的基因一起被分离。
本技术的扩展在于一个能够催化一组将起始建筑积木（氨基酸和小肽）构成一些本组的肽或多肽的反应的随机肽和多肽群体的选择。因此也能选择一个能催化它自己合成的群体;这样的一个反身自催化集合体可在一反应产物被连续稀释而建筑积木的浓度保持恒定的恒化器中建立起来。选择性地，这样一个集合体的合成通过在脂质体中用标准的技术关闭肽的复合物集合体而得到帮助。在一包围该脂质体的高渗水环境中，形成较大肽的缩合反应降低了脂质体内的渗透压，将因为缩合反应而产生的水分子排出了脂质体，因而有利于更大的聚合物的合成。自催化群体的产生可通过两次凝胶电泳和HPLC显示肽和多肽稳定分布的合成而证实。合适的反应体积取决于所用分子种类的数量，以及有利于肽建的形成超过它们的水解所必需的浓度。自催化群体的分子种类的分布能够因为不同的自催化群体的出现而自由变化。构成一个自催化集合体的肽和多肽可以具有与大的起始群体相同的某些成份（由本方法所给出的编码的肽和多肽构成的）而且还能含有不是由为起始群体的肽和蛋白质编码的随机基因群体所编码的肽和蛋白质。
其产物是建立这样一个自催化集合体所必需的随机基因集合体，可按已知方法通过连续缩小转化无性系库而完成分离。此外，一个自催化集合体可以包括由随机基因起始编码的并在自催化集合体内连续合成的编码肽。为了分离这个编码的肽和蛋白质的亚群，自催化集合体可用于通过在动物体内的免疫接种作用得到识别大量的自催化集合体组分的多克隆血清。
该血清可用于筛选随机基因库从而发现那些表达能够与血清中存在的抗体结合的蛋白质的基因。
这个随机基因的集合体表达了大量的存留在自催化集合体内的编码随机蛋白质。这样一个自催化集合体的编码组分存留物可通过随机基因库的连续缩减而被分离，由此，用免疫法检出的亚群首先被除去。
按所述方法获得的这样的肽和蛋白质的自催化集合体可以发现若干实际的应用。

Claims (55)

1、通过微生物法产生肽或多肽的方法，其特征在于至少有一部分是由随机的合成多聚核苷酸组成的基因可在一种普通培养基中同时产生，如此获得的基因被导入宿主细胞中，含有这些基因的转化宿主细胞的独立无性繁殖系同时被培养以便无性繁殖那些随机基因并导致由这些随机基因各自所表达的蛋白质的产生，对这样的转化宿主细胞的无性繁殖系以鉴定那些产生至少具有一种专一性的肽或多肽的无性繁殖系的方式进行选择或筛选，分离那些如此鉴定出的无性繁殖系，然后以产生至少一种具有上述特性的肽或多肽的方式培养之。
2、按照权项1的方法，其特征在于这些基因是由四种类型的脱氧磷酸核苷酸A，C，G，和T的随机共聚作用而产生的，随机共聚作用是从一个预先线性化的表达载体的两个末端开始的，然后通过这样的方式形成粘性末端，即产生包含具有两个随机序列的一个表达载体分子的随机DNA的第一股，所述两个随机序列的3′末端是互补的，随后合成随机DNA的第二股。
3、按照权项1的方法，其特征在于基因是由那些没有粘性末端的双股寡核苷酸的随机共聚作用产生的，其方式为形成随机DNA的碎片，然后在一个预先线性化的表达载体中连接这些碎片。
4、按照权项2或3的方法，基特征在于表达载体是一个质粘。
5、按照权项4的方法，其特征在于表达载体是pUC8。
6、按照权项2或3的方法，其特征在于表达载体是病毒DNA的碎片。
7、按照权项2或3的方法，其特征在于表达载体是质粘和病毒DNA的一种杂化物。
8、按照权项1到7的方法，其特征在于宿主细胞是原核细胞。
9、按照权项1到7的方法，其特征在于宿主细胞是真核细胞。
10、按照权项8的方法，其特征在于，细胞选自HB101和C600中。
11、按照权项3的方法，其特征在于寡核苷酸形成一组四文八聚体。
12、按照权项11的方法，其殊征在于，四文八聚体是下列一组：
5′GGAATTCC  3′
5′GGTCGACC  3′
5′CAAGCTTG  3′
5′CCATATGG  3′
5′CATCGATG  3′。
13、按照权项3的方法，其特征在于寡核苷酸形成一组四文七聚体。
14、按照权项13的方法，其特征在于，四文七聚体是下列一组：
5′XTCGCGA  3′
5′XCTGCAG  3′
5′RGGTACC  3′
其中X＝A，G，C，或T，R＝A或T。
15、按照权项4和权项12或14之一的方法，其特征在于，首先分离和纯化由培养转化宿主细胞的独立无性繁殖系得到的转化DNA，转化的宿主细胞是以在权项11或权项13中限定的方式获得的，然后至少用一个对应着存在于这些四文八聚体和七聚体中的特异限制位占的限制性内切酶切割纯化的DNA，但所用的表达载体上没有该特异限制性位点，此后用DNA连接酶同时处理如此得到的线性化随机DNA碎片的整体，以这样的方法产生一种含有新的随机序列的新的DNA整体，利用这种新的转化DNA整体转化宿主细胞并无性繁殖这样的基因，最后对新的转化细胞无性繁殖系进行选择或筛选和分离，并培育这些无性繁殖系以便产生至少一种具有所需特性的肽或多肽。
16、按照权项1的方法，其特征在于所述的特性为催化一种给定化学反应的能力。
17、按照权项1产生几种肽和/或多肽的方法，其特征在于，所述的特性为催化由给定的起始化合物直至形成至少一个目标化合物的一系列反应的能力。
18、按照权项16产生由一个以上反身自催化的肽和/或多肽组成的整体的方法，其特征在于所述的特性为催化由氨基酸和/或寡肽开始的整体本身的合成的能力。
19、按照权项1的方法，其特征在于，所述的特性为选样修饰一种给定化合物的化学性质或/和生物学性质的能力。
20、按照权项19的方法，其特征在于，所述的特性为选择修饰一种多肽的催化活性的能力。
21、按照权项19的方法，其特征在于，所述的特性为模拟或修饰至少一种生物活性化合物的至少一个生物学功能的能力。
22、按照权项21的方法，其特征在于，所述的生物活性化合物选自激素，神经递质，粘连或生长因素，以及DNA的复制和/或转录，和/或RNA的转解的特异调节剂中。
23、按照权项1的方法，其特征在于，所述的特性为与一种给定配位体结合的能力。
24、用按照权项23所获得的肽或多肽检测和/或滴定配位体。
25、按照权项1的方法，其特征在于，所述的特性为至少有一个与给定抗原的抗原决定部位相似的抗原决定部位。
26、按照权项19和25的方法，其特征在于所述的性质为模拟或修饰一种生物活性分子的作用的能力，对产生具有该特性的至少一种的肽或多肽的转化宿主细胞无性系的筛选和（或）选择，是这样完成的，即制备或获取抗（给定）分子的抗体，并且利用如此获得的这些抗体鉴定那些含有通过抗体而结合的那些肽或多肽的无性繁殖系，然后通过培育这样鉴定的无性繁殖系并分离和纯化由这些无性繁殖系所产生的肽或多肽，最后通过提供这些肽或多肽在体外测定证实它（它们）实际上有模拟或修饰所述分子的作用的能力，模拟或修饰所述分子作用的能力方面的改进可通过对所鉴定的肽或多肽编码的随机基因（不论突变或不突变）的修饰，然后重新筛选和重新试验，或者由已知方法对所鉴定的肽或多肽本身的衍生作用而获得。
27、通过按照权项1或权项26的方法所获得的可作为具有药理学和/或化学治疗作用的活性物质而利用的肽或多肽。
28、通过按照权项25的方法获得的肽或多肽，在体外或体内可用来减小对所述抗原有特异性的游离抗体的浓度，这是通过这些肽或多肽与这些抗体之间形成键而实现的。
29、按照权项27或28的可作为免疫过敏性抑制剂而利用的肽或多肽。
30、通过按照权项25的方法获得的可用作对所述抗原产生忍耐力试剂的肽或多肽。
31、按照权项25的方法，其特征在于抗原是EGF。
32、通过根据权项31的方法获得的肽或多肽，可用于上皮细胞癌的化疗。
33、按照权项1的方法，其特征在于，产生具有所述特性的肽或多肽的转化宿主细胞无性繁殖系通过在相应于由DNA杂种的天然碎片表达的蛋白质的抗体上的亲和层析得到鉴定和分离。
34、按照权项33的方法，其特征在于DNA杂种的天然碎片含有表达β半乳糖苷酶的基因，通过抗β半乳糖苷酶抗体的亲和层析鉴定和分离所述转化细胞宿主的无性繁殖系。
35、按照权项1或34的方法，其特征在于，在杂种肽或多肽的表达和纯化之后，新的碎片得到分离。
36、将按照权项25或权项26的方法用于疫苗的制备，其特征在于，抗病原作用物的抗体被获得并被用于鉴定那些至少产生一个具有至少一个与病原作用物的抗原决定部位之一类似的抗原决定部位的蛋白质的无性繁殖系，相应的转化宿主细胞的无性繁殖系以这样一种方式培育以便产生这种蛋白质，从这种细胞无性繁殖系的培养物中分离和纯化蛋白质并且利用这种蛋白质生产抗病原作用物的疫苗。
37、按照权项36的应用，制备抗HVB疫苗，其特征在于，至少一种HUB病毒的衣壳蛋白质被提取和纯化，这种蛋白质被注入能够形成抗此蛋白质的抗体的动物体内，这些抗体被回收和纯化，用这些抗体鉴定那些至少产生一种具有至少一个与HBV病毒的抗原决定部位之一类似的抗原决定部位的蛋白质的无性繁殖系，以产生这种蛋白质的方式培育相应于这些无性繁殖系的转化宿主细胞的无性繁殖系，从这些宿主细胞的培养物中分离和纯化这种蛋白质，并且用这种蛋白质生产抗HVB疫苗。
38、按照权项1的方法，其特征在于，宿主细胞是大肠杆菌类型的细菌，其基因组既不含天然的β半乳糖苷酶基因，也不含EBG基因，是Z-EBG-大肠杆菌，在也含有诱导物IPTG的X-gel培养基中培养这些转化宿主细胞，对β半乳糖苷酶的功能是阳性的无性繁殖系在这种培养基中被检测，此后，（相应的）DNA被移植到宿主细胞的无性繁殖系中，这种宿主细胞的无性繁殖系适于至少一种具有β半乳糖苷酶功能的肽、多肽或蛋白质的工业生产。
39、按照权项1的方法，其特征在于，所述的特性为与一种给定化合物结合的能力。
40、按照权项39的方法，其特征在于，在肽、多肽和蛋白质中选择所述的化合物。
41、按照权项40的方法，其特征在于，所述的蛋白质是调节DNA的转录活性或复制的蛋白质。
42、按照权项39的方法，其特征在于在DNA和RNA序列中选择所述的化合物。
43、通过按照权项40或42的方法得到的蛋白质。
44、一种产生DNA的方法，其特征在于，在同样的培养基中，至少有一部分是由随机的合成多核苷酸组成的基因被生产出来，如此产生的基因以产生一个转化宿主细胞的整体的方式，被导入宿主细胞中，培育这些基因以便产生如此产生的宿主细胞的独立无性繁殖系，在这个整体上进行筛选和（或）选择以鉴定那些含有至少具有一种所需特性的DNA的那些随机序列的宿主细胞，这样的DNA与所鉴定的宿主细胞培养物分离开。
45、按照权项44的方法，其特征在于所述的特性为与一种给定化合物结合的能力。
46、按照权项45的方法，其特征在于，在肽、多肽和蛋白质中选择所述的化合物。
47、按照权项45的方法，其特征在于，所述的化合物是一种调节DNA转录活性或复制的化合物。
48、按照权项47的方法，其特征在于，所述的化合物是一种控制DNA转录或复制的调节蛋白。
49、通过按照权项46，或权项47的方法获得的DNA序列用作为一个DNA邻近序列的复制或转录的调节序列。
50、按照权项42的方法，其特征在于，所获得的蛋白质具有修饰DNA的转录活性、复制，或稳定性的能力。
51、利用通过按照权项48的方法获得的蛋白质在含有该序列的DNA和表达该蛋白质的细胞中修饰DNA序列的转录，复制或稳定性。
52、生产RNA的方法，其特征在于，在同样的培养基中，同时产生至少有一部分是由合成的随机多核苷酸组成的基因，如此得到的基因以产生一个转化宿主细胞的整体的方式导入宿主细胞，同时培养如此产生的转化宿主细胞的独立无性繁殖系，在这个整体上进行的筛选和（或）选择是以鉴定那些含有至少具有一种所需特性的RNA随机序列的宿主细胞的方式进行，这种RNA从如此鉴定的宿主细胞的培养物中分离出来。
53、按照权项52的方法，其特征在于所述的特性为与一种给定化合物结合的能力。
54、按照权项52的方法，其特征在于所述的特性为催化一种给定化学反应的能力。
55、按照权项52的方法，其特征在于所述的特性应为一种转移RNA。
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US5976862A (en) 1999-11-02
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US5824514A (en) 1998-10-20
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DE3590766C2 (zh) 1991-01-10
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DE229046T1 (de) 1987-12-17
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