兔BK通道β亚基多克隆抗血清的制备

Preparation of polyclonal antiserum (against) β subunit of rabbit BK channel

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。

AIM: To prepare polyclonal antiserum (against) β subunit of rabbit BK channel in mice. METHODS: Gene encoding the intracellular fragment of rabbit BK channel’s β subunit was amplified by RT-PCR. The GST-β fusion protein was expressed in E.coli. The fusion protein from PAGE gel was used to immunize BALB/c mice and prepare polyclonal antiserum. The specificity of antiserum was identified by ELISA and Western blot. RESULTS: A (unique) band about 300 bp was amplified by RT-PCR and was werified to be BK channel β subunit by DNA sequencing. The SDS-PAGE analysis showed that the M_r of the fusion protein was about 37 000. The purity of GST-β fusion protein was over 95%. The polyclonal antiserum against GST-β fusion protein could recognize both GST-β fusion protein and β protein in rabbit tissues. The highest titer of the antiserum was about 1∶128 000, as shown by Western blot and ELISA, respectively. CONCLUSION: The gene encoding the intracellular fragment of rabbit BK channel’s β subunit has been cloned. The polyclonal antiserum against β subunit of rabbit BK channel with high titer and specificity has been prepared successfully.