Cloning and sequencing of the VL and VH genes from the anti-hCD154 McAb hybridoma cell line / 中国病理生理杂志

ABSTRACT

AIM:To obtain the light chain region(VL) and heavy chain region(VH) genes from the hybridoma cell line and analyse their sequence for construction of the engineering antibody against hCD154. METHODS: In this research ,total RNA was extracted from the hybridoma cell line 7E8, which secretes McAb against hCD154, and subjected to reverse transcription. The VL gene and VH gene were amplified by PCR, cloned into puc18 vector and sequenced by Sanger's dideoxymediated chain-termination method. RESULTS: The VL cDNA of 7E8 McAb consists of 341 bp encoding 113 amino acid residues. Compared with mouse Ig database, the VL region is in accord with the characterization of DNA sequence present in the mouse Ig Vk region , it belong to mouse V?2 light chain. The VH cDNA of 7E8 McAb consists of 354 bp encoding 118 amino acid residues. Compared with mouse Ig database, the VH region is in accord with the characterization of DNA sequence present in the mouse Ig VH region. CONCLUSION: The DNA squence analysis showed that the cloned genes code the light and heavy chain variable region of mouse respectively.