Cloning of genes by genomic DNA Splicing for secretory IgA production
用基因组DNA剪接技术克隆SIgA相关基因

Objectives: To clone immunoglobulin J chain (IgJ) gene, polymeric immunoglobulin receptor (pIgR) gene and human immunoglobulin A heavy chain constant region coding sequence (IGHA) for construction of secretory immunoglobulin A expressing plasmids. Methods: According to the "Genomic DNA Splicing" technique established in our lab, highly efficient exon primers were designed by software to amplify the exons directly from genomic DNA extract. An overlapping PCR was then performed with manually designed overlapping primers to join adjacent exons together to form a full-length coding sequence. The full-length PCR products were purified and ligated with pGEM-T Easy Vector. After transformation, clones were screened and positive cloned were subjected to sequencing. Results: The full-length PCR products had the expected molecular weight and sequence analysis confirmed that the cloned sequences were identical to the relative entries of the GenBank database. Conclusion: IgJ, pIgR and IGHA genes are successfully assembled by the "Genomic DNA Splicing" technique, which suggests that this technique could be a reliable strategy for cloning of multiple-exon cDNAs.