Zhong Xiufeng, Li Yongping, Huang Shuqi, Ning Bo, Zhang Chunyan, Zheng Jianliang, Feng Guanguang
Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou 510060, China.
Yan Ke Xue Bao. 2002 Sep;18(3):185-9.
To clone the variable region gene of light chain of monoclonal antibody against human retinoblastoma and to analyze the characterization of its nucleotide sequence as well as amino acid sequence.
Total RNA was extracted from 3C6 hybridoma cells secreting specific monoclonal antibody (McAb) against human retinoblastoma (RB), then transcripted reversely into cDNA with olig-dT primers. The variable region of the light chain (VL) gene fragments was amplified using polymerase chain reaction(PCR) and further cloned into pGEM -T Easy vector. Then, 3C6 VL cDNA was sequenced by Sanger's method. Homologous analysis was done by NCBI BLAST.
The complete nucleotide sequence of 3C6 VL cDNA consisted of 321 bp encoding 107 amino acid residues, containing four workframe regions (FRs) and three complementarity-determining regions(CDRs) as well as the typical structure of two cys residues. The sequence is most homological to a member of the Vk9 gene family, and its chain utilizes the Jkl gene segment.
The light chain variable region gene of the McAb against human RB was amplified successfully, which belongs to the Vk9 gene family and utilizes Vk-Jkl gene rearrangement. This study lays a good basis for constructing a recombinant antibody and for making a new targeted therapeutic agents against retinoblastoma.
克隆抗人视网膜母细胞瘤单克隆抗体轻链可变区基因，并分析其核苷酸序列及氨基酸序列特征。
从分泌抗人视网膜母细胞瘤（RB）特异性单克隆抗体（McAb）的3C6杂交瘤细胞中提取总RNA，然后用寡聚dT引物反转录成cDNA。采用聚合酶链反应（PCR）扩增轻链可变区（VL）基因片段，并进一步克隆到pGEM -T Easy载体中。然后，用桑格法对3C6 VL cDNA进行测序。通过NCBI BLAST进行同源性分析。
3C6 VL cDNA的完整核苷酸序列由321 bp组成，编码107个氨基酸残基，包含四个框架区（FRs）、三个互补决定区（CDRs）以及两个半胱氨酸残基的典型结构。该序列与Vk9基因家族的一个成员同源性最高，其轻链利用Jkl基因片段。
成功扩增了抗人RB McAb的轻链可变区基因，该基因属于Vk9基因家族，利用Vk-Jkl基因重排。本研究为构建重组抗体及制备抗视网膜母细胞瘤新型靶向治疗药物奠定了良好基础。
