A RAPID METHOD OF CLONING FUNCTIONAL VARIABLE-REGION ANTIBODY GENES IN ESCHERICHIA-COLI AS SINGLE-CHAIN IMMUNOTOXINS
A RAPID METHOD OF CLONING FUNCTIONAL VARIABLE-REGION ANTIBODY GENES IN ESCHERICHIA-COLI AS SINGLE-CHAIN IMMUNOTOXINS
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DOI：
10.1073/pnas.87.3.1066
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发表时间：
1990-02-01
影响因子：
11.1
通讯作者：
PASTAN, I
中科院分区：
文献类型：
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作者：
CHAUDHARY, VK;BATRA, JK;PASTAN, I
我们设计了一种基于聚合酶链式反应(PCR)的策略，用于快速克隆功能性抗体基因作为单链免疫毒素。从产生与卵巢癌细胞反应的抗体(OVB3)的杂交瘤中提取的RNA被用作模板来制备第一条cDNA链。然后用两组DNA引物(I)杂交到轻链可变区和重链可变区的末端，(Ii)编码连接肽，(Iii)含有合适的限制性内切酶位点，合成并扩增第二链。经过30个循环的聚合酶链式反应，将含有轻链可变区和重链可变区序列的DNA片段克隆到含有部分假单胞菌外毒素基因的大肠杆菌表达载体中。在大肠杆菌中表达了编码重组单链免疫毒素的克隆，并对其与携带OVB3抗原的细胞结合或杀伤的能力进行了评估。通过使用这种方法，可以从杂交瘤RNA中快速克隆许多不同抗体的功能可变区序列。
We have devised a strategy based on polymerase chain reaction (PCR) for the rapid cloning of functional antibody genes as single-chain immunotoxins. RNA from a hybridoma producing an antibody (OVB3) that reacts with ovarian cancer cells was used as a template to make the first strand of a cDNA. Then a second strand was synthesized and amplified by using two sets of DNA primers that (i) hybridized to the ends of the light- and heavy-chain variable regions, (ii) encoded a linker peptide, and (iii) contained appropriate restriction enzyme sites for cloning. After 30 cycles of PCR, the DNA fragments containing sequences encoding the light- and heavy-chain variable regions were cloned into an Escherichia coli expression vector containing a portion of the Pseudomonas exotoxin gene. Clones encoding recombinant single-chain immunotoxin were expressed in E. coli and the protein product was assessed for its ability to bind to or kill cells bearing the OVB3 antigen. By using this approach it should be possible to rapidly clone the functional variable region sequences of many different antibodies from hybridoma RNA.