期刊文献+

用基因组DNA剪接技术克隆SIgA相关基因 被引量:2

Cloning of Genes by Genomic DNA Splicing for Secretory IgA Production
下载PDF
导出
摘要 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.
出处 《中国生物工程杂志》 CAS CSCD 北大核心 2008年第6期1-6,共6页 China Biotechnology
基金 国家科技支撑计划资助项目(2006BAD06A15)
关键词 分泌型IGA 基因组DNA剪接 重叠延伸聚合酶链式反应 Secretory Immunoglobulin A Genomic DNA splicing Overlapping PCR
  • 相关文献

参考文献11

  • 1Snoeck V, Peters I R, Cox E. The IgA system : a comparison of structure and function in different species. Vet Res, 2006, 37 (3) : 455-467
  • 2Woof J M, Kerr M A. The function of immunoglobulin A in immunity. J Pathol, 2006, 208(2) : 270 -282
  • 3Johansen F E, Braathen R, Brandtzaeg P. The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA. J Immunol, 2001, 167(9) : 5185 -5192
  • 4Hanson L A, Korotkova M. The role of breastfeeding in prevention of neonatal infection. Semin Neonatol, 2002, 7 (4) : 275 - 281
  • 5Ma J K, Hiatt A, Hein M, et al. Generation and assembly of secretory antibodies in plants. Science, 1995, 268 (5211): 716 -719
  • 6Johansen F E, Natvig Norderhaug I, Roe M, et al. Recombinant expression of polymeric IgA: incorporation of J chain and secretory component of human origin. Eur J Immunol, 1999, 29(5): 1701-1708
  • 7Berdoz J, Blanc C T, Reinhardt M, et al. In vitro comparison of the antigen-binding and stability properties of the various molecular forms of IgA antibodies assembled and produced in CHO cells. Proc Natl Acad Sci U S A, 1999, 96(6) : 3029-3034
  • 8Chintalacharuvu K R, Morrison S L. Production of secretory immunoglobulin A by a single mammalian cell. Proc Natl Acad Sci U S A, 1997, 94(12) : 6364-6368
  • 9An X, Lu J, Huang J D, et al. Rapid assembly of multipleexon cDNA directly from genomic DNA. PLoS ONE, 2007, 2 (11): e1179
  • 10Wu X, Munroe D J. EasyExonPrimer : automated primer design for exon sequences. Appl Bioinformatics, 2006, 5 ( 2 ) : 119-120

二级参考文献6

  • 1Fan CF,Mei XG.A simple,efficient,and economical method for recovering DNA from agarose gel[J].Prep Biochem Biotechnol,2005,35:71
  • 2Pusch C.A simple and fast procedure for high quality DNA isolation from gels using laundry detergent and inverted columns[J].Electrophoresis,1997,18:1103
  • 3Pramatarova A,Yelle J,D'Amours B,et al.Efficient recovery of cloned human cytomegalovirus DNA fragments from agarose gels[J].J Virol Methods,1994,46:1
  • 4Mukhopadhyay T,Roth JA.A simple and efficient method for isolation of DNA fragments from agarose gel[J].Nucleic Acids Res,1991,19:6656
  • 5Duro G,Izzo V,Barbieri R,et al.A method for eluting DNA in a wide range of molecular weights from agarose gels[J].Anal Biochem,1991,195:111
  • 6Heery DM,Gannon F,Powell R.A simple method for subcloning DNA fragments from gel slices[J].Trends Genet,1990,6:173

共引文献13

同被引文献18

  • 1WHO: Cumulative number of confirmed human cases of Avian influenza A/(H5N1) reported to WHO. 2008. http://www.who.int/csr/disease/avian_influenza/country/c ases_table_2008 02 28/en/index.html.
  • 2Ma JK, Hiatt A, Hein M, et al. Generation and assembly of secretory antibodies in plants. Science, 1995, 268(5211): 716-719.
  • 3Hammarstrom L, Weiner CK. Targeted antibodies in dairy-based products. Adv Exp Med Biol, 2008, 606(12): 321-343.
  • 4Sambrook J, Fritsch EF, Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. New York: Cold Spring Harbor Laboratory Press, 1989: 880-897.
  • 5Ma JK, Hikmat BY, Wycoff K, et al. Characterization of a recombinant plant monoclonal secretory antibody and preventive immunotherapy in humans. Nat Med, 1998, 4(5): 601-606.
  • 6Johansen FE, Natvig I, Norderhaug M, et al. Brandtzaeg: Recombinant expression of polymeric IgA: incorporation of J chain and secretory component of human origin. Eur J Immunol, 1999, 29(2): 1701170-17011708.
  • 7Berdoz.J, Blanc CT, Reinhardt M, et al. In vitro comparison of the antigen-binding and stability properties of the various molecular forms of IgA antibodies assembled and produced in CHO cells. Proc Natl Acad Sci USA, 1999, 96(6): 3029-3034.
  • 8Chintalacharuvu KR, Gurbaxani B, Morrison SL. Incomplete assembly of IgA2m(2) in Chinese hamster ovary cells. Mol Immunol, 2007, 44(13): 3445-3452.
  • 9WHO. Cumulative Number of Confirmed Human Cases of Avian Influenza A/(H5N1) Reported to WHO. http://www.who.int/csr/disease/avian_influenza/country/cases_table_2010_03_29/en/index.html, 2010.
  • 10Corthesy B. Recombinant immunoglobulin A: powerful tools for fundamental and applied research. Trends Biotechnol, 2002, 20(2): 65-71.

引证文献2

二级引证文献2

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部