SINGLE CHAIN ANTIBODY (SCA) ENCODING GENES - ONE-STEP CONSTRUCTION AND EXPRESSION IN EUKARYOTIC CELLS
SINGLE CHAIN ANTIBODY (SCA) ENCODING GENES - ONE-STEP CONSTRUCTION AND EXPRESSION IN EUKARYOTIC CELLS
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DOI：
10.1038/nbt0291-165
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发表时间：
1991-02-01
期刊：
影响因子：
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通讯作者：
JACOBS, TW
中科院分区：
文献类型：
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作者：
DAVIS, GT;BEDZYK, WD;JACOBS, TW
我们报告的表达，在真核细胞中，编码单链抗体（SCA）的基因和这种基因的快速构建方法。 针对芳香族染料荧光素的SCA由通过同时使用四种PCR引物和轻链和重链免疫球蛋白cDNA的模板以质粒克隆或逆转录的杂交瘤RNA的形式构建的基因合成。 两个引物彼此部分互补，并编码连接SCA多肽的免疫球蛋白轻链和重链可变结构域的多肽接头。 在两个大肠杆菌中，从这样构建的基因合成了功能性的半抗原结合产物。大肠杆菌和裂殖酵母，粟酒裂殖酵母。 我们的研究结果表明，编码单链抗原结合蛋白的基因构建体可以非常快速地合成，仅需要关于相关轻链和重链免疫球蛋白基因的有限序列信息，并且，无论是鼠密码子使用偏好、水生栖热菌DNA聚合酶不忠实性，还是真核细胞环境，都不排除在非淋巴细胞中合成功能性单链抗原结合蛋白，非鼠真核细胞。
We report the expression, in eukaryotic cells, of a gene encoding a single chain antibody (SCA) and a rapid method for the construction of such genes. A SCA directed against the aromatic dye fluorescein was synthesized from a gene constructed by means of the simultaneous use of four PCR primers and templates of both light and heavy chain immunoglobulin cDNAs in the form of either plasmid clones or reverse transcribed hybridoma RNA. Two of the primers were partially complementary to one another and encoded the polypeptide linker which joins the immunoglobulin light and heavy chain variable domains of the SCA polypeptide. A functional, hapten-binding product was synthesized from the gene thus constructed in both E. coli and the fission yeast, Schizosaccharomyces pombe. Our results demonstrate that gene constructs encoding single chain antigen binding proteins can be synthesized very rapidly with only limited sequence information about the pertinent light and heavy chain immunoglobulin genes, and, that neither murine codon usage bias, Thermus aquaticus DNA polymerase infidelity, nor the eukaryotic cellular environment preclude the synthesis of functional single chain antigen binding proteins in non-lymphatic, non-murine eukaryotic cells.