Amplification and sequence analysis of anti-D variable region gene with leader peptide sequence |
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Authors: | CAO Kai-yuan FU Yong-shui XU Lin YUAN Guang-qing HUANG Xiao-rong DAI Shu-qin TANG Yong-ping |
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Affiliation: | 1. Department of Immunology, Zhongshan Medical College, Sun Yat-sen University, Guangzhou 510089, China;2. Guangzhou Blood Center, Guangzhou 510095, China |
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Abstract: | AIM:To amplify from leader peptide region and obtain human monoclonal anti-D variable region gene with high specificity and affinity, and analyze the nucleotide and deduced amino acid sequences.METHODS:The total RNA was extracted from an Epstein-Barr-virus-transformed cell line secreting monoclonal anti- (rhesus D) antibody. The leader region primers containing a ribosome recognition site were designed. By using PCR method, the cDNA of human anti-(rhesus D) antibody (IgM κ) variable region gene was amplified. Cloning and subsequent sequence analysis of the variable region gene was performed. The deduced amino acid sequence was also compared and analyzed with previously published sequences.RESULTS:A band of approximate 440 and 410 base pairs were amplified using heavy chain primers and light chain primers, respectively. Sequence analysis indicated that the deduced amino acid sequence was in agreement with the characterization of the amino acid present in the human Ig variable region. CONCLUSION:The cloning and sequencing of a human anti- (Rhesus D) antibody variable region cDNA will make benefits for production of recombinant anti-(Rhesus D) antibody and prevention of Rh haemolytic disease in newborns. |
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Keywords: | Antibodies monoclonal Sequence analysis Polymerase chain reaction Variable region gene |
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