论文部分内容阅读
目的确证 5D4 cDNA是全长 cDNA，并为制备抗 5D4不同表位的单克隆抗体准备抗原。方法构建重组表达质粒 pGEX- 5X- 3- 5D4全长和 pGEX- 5X- 3- 5D4 5′侧 ,并转化大肠杆菌 DH5α ,使其表达 5D4分化抗原，用 Northern印迹分析和 RT- PCR检测 5D4 mRNA在不同组织和免疫细胞株中的表达状况。结果 5D4 mRNA有 5种转录本，它们在不同组织有不同的表达， 1.9 kb RNA在所检测的 8种组织中只见于小肠组织和脾脏。 5D4 mRNA在不同免疫细胞的表达量不同。在大肠杆菌 DH5α中表达出了相对分子质量约为 66 000和 60 000的 5D4- GST融合蛋白。结论 1 846 bp 5D4 cDNA为全长 cDNA，并在大肠杆菌 DH5α中成功表达出了与 GST融合的 5D4分化抗原全长 (66 000)及其跨膜区侧氨基端蛋白质 (60 000)。
Objective To confirm that 5D4 cDNA is a full-length cDNA and to prepare antigens for the preparation of monoclonal antibodies against different epitopes of 5D4. Methods The full length of pGEX-5X-3-D4 and the 5 ’side of pGEX-5X-3-D4 were constructed and transformed into E. coli DH5α to express 5D4 differentiated antigen. Northern blot analysis and RT-PCR were used to detect 5D4 mRNA In different tissues and immune cell lines in the expression status. Results There were 5 transcripts of 5D4 mRNA, which expressed differently in different tissues. The 1.9 kb RNA was found only in small intestine tissue and spleen in the 8 tissues tested. The expression of 5D4 mRNA is different in different immune cells. The 5D4-GST fusion protein with relative molecular masses of about 66 000 and 60 000 was expressed in E. coli DH5α. Conclusion 1 846 bp 5D4 cDNA is a full-length cDNA. The full-length 5D4 antigen (66 000) fused to GST and its transmembrane domain side amino-terminal protein (60 000) were successfully expressed in E. coli DH5α.