论文部分内容阅读
目的:用小鼠制备抗兔BK通道β亚基的抗血清。方法:采用RT-PCR扩增编码兔BK通道β亚基胞内段的基因。在大肠杆菌中表达GST-β亚基融合蛋白。以从PAGE凝胶上切下的融合蛋白免疫BALB/c小鼠制备多克隆抗血清。用ELISA和Westernblot鉴定抗血清的特异性。结果:用RT-PCR扩增出约300bp的兔BK通道基因。序列分析显示,扩增的序列与已发布的新西兰大白兔骨骼肌BK通道的序列完全一致。在E.coliDH5α成功地表达Mr约为37000GST-β亚基融合蛋白。ELISA和Westernblot检测证明,针对GST-β亚基融合蛋白的抗血清,不仅可特异性地识别GST-β,也可识别源于兔组织的β蛋白。抗血清的最高滴度达1∶128000。结论:克隆了编码兔BK通道β亚基胞内段的基因,并成功地获得可特异性识别新西兰大白兔BK通道β亚基的高滴度抗血清,为BK通道的进一步研究提供了物质基础。
OBJECTIVE: To prepare anti-rabbit anti-rabbit BK channel β subunit antiserum. Methods: The gene coding for the intracellular β-subunit of rabbit BK channel was amplified by RT-PCR. The GST-β subunit fusion protein is expressed in E. coli. Polyclonal antiserum was prepared by immunizing BALB / c mice with the fusion protein cut from the PAGE gels. Antisera were identified by ELISA and Western blot. Results: Approximately 300bp rabbit BK channel gene was amplified by RT-PCR. Sequence analysis showed that the amplified sequence was completely consistent with the published sequence of skeletal muscle BK channels in New Zealand rabbits. Mr was successfully expressed at about 37000GST-β subunit fusion protein in E.coli DH5α. ELISA and Western blot showed that the antiserum against GST-β subunit fusion protein can not only recognize GST-β specifically but also recognize β-protein derived from rabbit tissue. The highest titer of antiserum reached 1: 128,000. CONCLUSION: The gene coding for the intracellular β-subunit of rabbit BK channel has been cloned and successfully obtained high titer antiserum that can specifically recognize β subunit of BK channel in New Zealand rabbits, which provides a material basis for further study of BK channel .