RESUMO
AIM: To investigate the effects on protein expression of big-conductance Ca(2+)-sensitive K(+) channel (BKca) beta1 subunit caused by high cholesterol in Rabbit Oddi's sphincter (SO) cells. METHODS: The rat-anti-rabbit polyclonal antiserum against beta1 subunits of BKca channel of SO cell was prepared. And the protein expression of BKca channel beta1 subunit of SO tissue was detected by semi-quantitative immunohistochemical staining. RESULTS: The protein expression of BKca channels beta1 subunit of SO tissue in HC group was reduced, and there's statistically significant difference between the HC group and the control group. CONCLUSION: High cholesterol can reduce the protein expression of BK Channel's beta1 subunit in Rabbits' SO which suggests high cholesterol can affect the function of BKca channel.
Assuntos
Colesterol/metabolismo , Colesterol/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Esfíncter da Ampola Hepatopancreática/metabolismo , Animais , Colesterol/administração & dosagem , Ensaio de Imunoadsorção Enzimática , Feminino , Soros Imunes/análise , Soros Imunes/imunologia , Subunidades beta do Canal de Potássio Ativado por Cálcio de Condutância Alta/imunologia , Camundongos , Plasmídeos/genética , Plasmídeos/metabolismo , Coelhos , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/metabolismo , Esfíncter da Ampola Hepatopancreática/citologiaRESUMO
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.
