兔BK通道β亚基多克隆抗血清的制备

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。

Preparation of polyclonal antiserum against β subunit of rabbit BK channel

AIM: To prepare polyclonal antiserum against beta subunit of rabbit BK channel in mice. METHODS: Gene encoding the intracellular fragment of rabbit BK channel's beta subunit was amplified by RT-PCR. The GST-beta 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 verified to be BK channel beta 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-beta fusion protein was over 95%. The polyclonal antiserum against GST-beta fusion protein could recognize both GST-beta fusion protein and beta 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 beta subunit has been cloned. The polyclonal antiserum against beta subunit of rabbit BK channel with high titer and specificity has been prepared successfully.