用基因组DNA剪接技术克隆SIgA相关基因

Cloning of Genes by Genomic DNA Splicing for Secretory IgA Production

目的:克隆分泌型IgA(SIgA)相关基因——J链基因(IgJ)、多聚免疫球蛋白受体基因(pIgR)和IgA重链恒定区基因(IGHA),为进一步构建SIgA真核表达质粒奠定基础。方法:采用"基因组DNA剪接"技术,根据已发表的IgJ、pIgR和IGHA的核苷酸序列,通过计算机软件分别设计各个基因片段外显子的优化引物,从人外周血基因组DNA中直接扩增各基因的外显子序列;然后人工设计融合相邻外显子的融合引物,采用重叠PCR技术,把各基因片段的外显子串联起来形成全长编码序列,完成基因组DNA的体外剪接。扩增的PCR产物纯化后克隆到pGEM-T Easy Vector中,通过DNA测序对阳性克隆进行分析鉴定。结果:PCR扩增的IgJ、pIgR和IGHA基因与预期大小一致;测序结果表明实验获得的上述基因与GenBank中的目标基因序列完全一致。结论:通过基因组DNA剪接技术成功克隆人类SIgA3个相关基因,提示此技术是合成多外显子cDNA的有效手段。

Objectives： To clone immunoglobulin J chain （IgJ） gene, polymeric （pIgR） gene and human immunoglobulin A heavy chain constant region coding construction of secretory immunoglobulin A expressing plasmids. Methods： According immunoglobulin receptor sequence （IGHA） for 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 muhiple-exon cDNAs.