pICln毕赤酵母表达载体的构建及转染
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摘要
pICln(p-protein,I-currents,Cl-chloride,n-nucleotide sensitive)是1992年发现的一种蛋白质,推测它与细胞体积的调节有关。目前认为,pICln与ClC-2、P-糖蛋白是阴离子通道的三种“候选”蛋白。在增强低渗环境下细胞的存活率、参与剪接体的剪接组装、作用于细胞骨架参与细胞体积调节等多方面发挥作用。了解pICln的结构,对于研究其生物学活性非常重要。因此克隆了pICln基因序列,构建了真核表达载体并转染毕赤酵母细胞,以期获得较纯的蛋白质,为其结构和功能的研究做准备。
本试验从大鼠肾脏中提取总RNA,利用特异性引物扩增出目的片段,将目的基因序列与克隆载体pMD18-T Simple连接,然后再连接于表达载体pGAPZ a A上。重组质粒经BlnI线性化后转染毕赤酵母细胞SMD1168中表达,取上清液作SDS-PAGE和Western Blot检测。
本试验成功地克隆出了pICln基因序列,其大小约为750bp。经测序、PCR和酶切鉴定证实pICln准确地插入毕赤酵母表达载体pGAPZ a A中,氯化锂转染后,重组载体通过同源重组整合到酵母基因组中,为深入研究pICln奠定了基础。
pICln(p-protein,I-currents,Cl-chloride,n-nucleotide sensitive) is the cDNA of a new protein that was cloned From pig kidney epithelial cell(MDCK cell)by Paulmichl et al.in 1992.Because it caused a significant changes in chloride ion current when it was expressed in Xenopus oocytes,so it is possible that pICln is an anion channel.But there are somebody think that it is a regulatory protein ion channel.
pICln are mainly distributed in the cytoplasmic components in rat heart, Xenopus oocytes and MDCK cells,and in the cell membrane in human reticulocyte. The position of pICln in cells has been controverted.Strange et al.proposed a model in 1996:When the cells volume changed with the external environment changing,pICln transfer between the membrane and cytoplasm.That is,when cells are put in a hypotonic environment,pICln transfer to the cytoplasmic membrane which constitute ion channels,then mediate ion flow;when the external environment of the cells tend to isotonic and the cells volume restore normally, pICln leaves the cell membrane and re-position in cytoplasm.
There is evidence that at hypertonic conditions pICln will also play a regulatory role.The proposed model of this theory seems to solve the position of pICln in the cells,but we still lack of strong evidence to support this theoretical model.
Analyzing of the pICln Sequence,p107-152 sequence is considered the basic function region where pICln can play the action of anti-swelling.The bacteria with this sequence can survive under hypotonic environment.This means that this sequence has the activity of anti-swelling and it is the active region of pICln.
pICln is associated with Sm that prevent Sm associating with SMN.So the Assembly,processing and modification of the spliceosome is affected,pICln can also affect the cytoskeletal proteins.It regulates the cells volume by associating with actin and myosin.
pICln is found in many kinds of tissues and cells,and pICln of different species are highly homologous.This points that pICln has very important function in vivo.At present,we only know that pICln can regulate the cells volume,but in the process of regulation of cells volume,the pICln how to play a role remains unclear.
We know little about the function of pICln,we need to understand the basic chemical structure so that we can learn about the biological function of this protein.The levels of pICln in cells are very low,so it is very difficult for us to know and research much about it.The purpose of this study is that pICln will be built into the eukaryotic expression system and to obtain the high-level expression of fusion protein.The fusion protein was released into the extracellular,and this is very beneficial to recover and purify protein.The advantage of eukaryotic expression system is that this system can modify and process gene and protein,and also can avoid the new synthetic protein degrading. For example the synthetic protein glycosylation.
primers was designed by Primer 5.0 software and was made from the Shanghai Public Health Synthesis Ltd.Upstream primer:5' TGA ATT CTT GGT TCC TGT GGA GCA ATG C 3',The introduction of restriction sites EcoRI:Downstream primer:5 ' ACT CGA GAT CCT CAG TGG TCA ACG TCT G 3',The introduction of restriction sites XbaI。Extract the total RNA from the rat kidney,reverse transcriptase fragment synthesis.1%agarose gel electrophoresis examination,it has the obvious specificity banding about 750bp,match to the theoretical value。After purified by agarose gel extraction kit,and connected with clone pMD18-T simple vector, transformed into theE.coli competent cells of JM109,selected positive clones by blue-white screening,named after the sequencing pMD18-T(+)。Extracted positive colonies from the recombinant plasmid,double digested with EcoRI andXbaI。The fragments were separate by 1%agarose gel electrophoresis,in 2700bp and 750bp place we get the specific band,they consistent with the known fragment size.Expression vector pGAPZαA was digested With EcoRI and XbaI,separately recycle the vector and fragment which are all double-digestion products by gel Recycling Kit,UV detection of sub-ray spectrometer on the target gene fragment and double-digestion products.Mixed by the molar ratio of 1:10,added T4 ligase and reacted overnight in 16℃,the connected products were transformed into E.coli cells,cultivated overnight in 37℃on selection medium containing the Zeocin,picked the positive colonies and Amplified,restriction identified by enzyme digestion,after 1%agarose gel electrophoresis,there are two specific bands at 3100bp and 750bp place under the UV lamp.Name after sequencing pGAPZαA-pICln.Recombinant plasmid linearization with BlnI,then transfected into Pichia pastoris cells SMD1168 with lithium chloride method.Paved on the YPD medium containing Zeocin,cultivate two days in 30℃.The screening of recombinant clones are inoculated into YPD liquid medium,Cultivated 72 hours in 30℃.Removed the part of the culture every 36 hour,after high-speed centrifugation,extract supernatant,stored in 4℃for SDS-PAGE and Western Blot testing.SDS-PAGE detected the expression of pICln shows that the molecular weight of 29KDa to 44 KDa,match with known molecular weight with us.Western Blot results showed that the obtained protein can be combined with the specific polyclonal antibodies.Proved that this protein is the protein we need.
本试验从大鼠肾脏中提取总RNA,利用特异性引物扩增出目的片段,将目的基因序列与克隆载体pMD18-T Simple连接,然后再连接于表达载体pGAPZ a A上。重组质粒经BlnI线性化后转染毕赤酵母细胞SMD1168中表达,取上清液作SDS-PAGE和Western Blot检测。
本试验成功地克隆出了pICln基因序列,其大小约为750bp。经测序、PCR和酶切鉴定证实pICln准确地插入毕赤酵母表达载体pGAPZ a A中,氯化锂转染后,重组载体通过同源重组整合到酵母基因组中,为深入研究pICln奠定了基础。
pICln(p-protein,I-currents,Cl-chloride,n-nucleotide sensitive) is the cDNA of a new protein that was cloned From pig kidney epithelial cell(MDCK cell)by Paulmichl et al.in 1992.Because it caused a significant changes in chloride ion current when it was expressed in Xenopus oocytes,so it is possible that pICln is an anion channel.But there are somebody think that it is a regulatory protein ion channel.
pICln are mainly distributed in the cytoplasmic components in rat heart, Xenopus oocytes and MDCK cells,and in the cell membrane in human reticulocyte. The position of pICln in cells has been controverted.Strange et al.proposed a model in 1996:When the cells volume changed with the external environment changing,pICln transfer between the membrane and cytoplasm.That is,when cells are put in a hypotonic environment,pICln transfer to the cytoplasmic membrane which constitute ion channels,then mediate ion flow;when the external environment of the cells tend to isotonic and the cells volume restore normally, pICln leaves the cell membrane and re-position in cytoplasm.
There is evidence that at hypertonic conditions pICln will also play a regulatory role.The proposed model of this theory seems to solve the position of pICln in the cells,but we still lack of strong evidence to support this theoretical model.
Analyzing of the pICln Sequence,p107-152 sequence is considered the basic function region where pICln can play the action of anti-swelling.The bacteria with this sequence can survive under hypotonic environment.This means that this sequence has the activity of anti-swelling and it is the active region of pICln.
pICln is associated with Sm that prevent Sm associating with SMN.So the Assembly,processing and modification of the spliceosome is affected,pICln can also affect the cytoskeletal proteins.It regulates the cells volume by associating with actin and myosin.
pICln is found in many kinds of tissues and cells,and pICln of different species are highly homologous.This points that pICln has very important function in vivo.At present,we only know that pICln can regulate the cells volume,but in the process of regulation of cells volume,the pICln how to play a role remains unclear.
We know little about the function of pICln,we need to understand the basic chemical structure so that we can learn about the biological function of this protein.The levels of pICln in cells are very low,so it is very difficult for us to know and research much about it.The purpose of this study is that pICln will be built into the eukaryotic expression system and to obtain the high-level expression of fusion protein.The fusion protein was released into the extracellular,and this is very beneficial to recover and purify protein.The advantage of eukaryotic expression system is that this system can modify and process gene and protein,and also can avoid the new synthetic protein degrading. For example the synthetic protein glycosylation.
primers was designed by Primer 5.0 software and was made from the Shanghai Public Health Synthesis Ltd.Upstream primer:5' TGA ATT CTT GGT TCC TGT GGA GCA ATG C 3',The introduction of restriction sites EcoRI:Downstream primer:5 ' ACT CGA GAT CCT CAG TGG TCA ACG TCT G 3',The introduction of restriction sites XbaI。Extract the total RNA from the rat kidney,reverse transcriptase fragment synthesis.1%agarose gel electrophoresis examination,it has the obvious specificity banding about 750bp,match to the theoretical value。After purified by agarose gel extraction kit,and connected with clone pMD18-T simple vector, transformed into theE.coli competent cells of JM109,selected positive clones by blue-white screening,named after the sequencing pMD18-T(+)。Extracted positive colonies from the recombinant plasmid,double digested with EcoRI andXbaI。The fragments were separate by 1%agarose gel electrophoresis,in 2700bp and 750bp place we get the specific band,they consistent with the known fragment size.Expression vector pGAPZαA was digested With EcoRI and XbaI,separately recycle the vector and fragment which are all double-digestion products by gel Recycling Kit,UV detection of sub-ray spectrometer on the target gene fragment and double-digestion products.Mixed by the molar ratio of 1:10,added T4 ligase and reacted overnight in 16℃,the connected products were transformed into E.coli cells,cultivated overnight in 37℃on selection medium containing the Zeocin,picked the positive colonies and Amplified,restriction identified by enzyme digestion,after 1%agarose gel electrophoresis,there are two specific bands at 3100bp and 750bp place under the UV lamp.Name after sequencing pGAPZαA-pICln.Recombinant plasmid linearization with BlnI,then transfected into Pichia pastoris cells SMD1168 with lithium chloride method.Paved on the YPD medium containing Zeocin,cultivate two days in 30℃.The screening of recombinant clones are inoculated into YPD liquid medium,Cultivated 72 hours in 30℃.Removed the part of the culture every 36 hour,after high-speed centrifugation,extract supernatant,stored in 4℃for SDS-PAGE and Western Blot testing.SDS-PAGE detected the expression of pICln shows that the molecular weight of 29KDa to 44 KDa,match with known molecular weight with us.Western Blot results showed that the obtained protein can be combined with the specific polyclonal antibodies.Proved that this protein is the protein we need.
引文
[l]Strange K,Emma F,Jackson PS.Cellular and Molecular physiology of volume-sensitization channels.A m.J.Physiol.1996,270(Cell Phsiol.39):C711-C73.
[2]Ackerman MJ,Krapivinsky GB,Gordon E,et al . Characterization of a native swelling-induced chloride current,IC1.swell,and its regulatory protein,pICln,in Xenopus oocytes,physiology.1994,44:17-24.
[3]Pulmichl M,LI Y,Wickman K,et al.New mammalian chloride channel identified by expression cloning.N ature.1992,356:238-241.
[4]A nguita J,Chalfant ML,Givan MM,et al.Molecular cloning of the human volume-sensitive chloride conductance regulatory protein,pICln,from ocular ciliary epithelium.Biochemical and Biophysical Research Communications.1995,208:89-95.
[5]Schwartz RS,Rybicki AC,Nagel RL.Molecular cloning and Expression of a chloride-associated protein pICln in young red blood cells:association with actin.Biochem.J.1997,327:609-610.
[6]Buyse G,DeGreef C,Raeymaekers L,et al.The ubiquitously Expressed pICln,protein form shomomeric complexes in vitro.Biochemical And Biophysical Research Communications.1996,218:822-827.
[7]A.Schmarda,U.O.Nagl,M.Gschwentner,et al.Cell.Physiol.Biochem.1997,7:298-302.
[8]Thomas Voets,Gunnar Buyse,Guy Droogmans,et al.The GXGXG motif in the pICln protein is not important for the nucleotide sensitivity of the pICln-induced C13 current in Xenopus oocytes.FEBS Letters.1998,426:171-173.
[9]Krapivinsky GB,Ackerman M J ,Gordon EA,et al.Molecular characterization of a swelling-induced chloride conductance regulatory protein,pIcln.Cell,2994,76 :439-448.
[10]Strange K,Emma F,Jackson P S.Cellular and molecular physiology of volume-sensitive anion channels.Am J Physiol,1996,270 (Cell Physiol.39):C711.
[11]Tao G-Z,Yohtalou Tashima.A peptide derived from pICln induced a strong hypotonic resistance in Escherichia coli cells.Peptides.2000;21;485-490.
[12]Kramer,A.The structure and function of proteins involved in mammalian pre-mRNA splicing.Annu.Rev.Biochem.1996,65:367-409.
[13]William,T.PU,Grigory B.Krapivinsky,and Lubakrapivinsky et al.pICln Inhibits snRNP Biogenesis by Binding Core Spliceosomal Proteins.Molecular And Cellular Biology.1999,19(6):4113-4120.
[14]DeRobertis,E.M.Nucleocytoplasmic segregation of proteins and RNAs.Cell.1983,32:1021-1025.
[15]Zeller,R.,T.Nyffenegger,E.M.DeRobertis.Nucleocytoplasmic distribution of snRNPs and stockpiled snRNA-binding proteins during oogenesis and early development in Xenopus laevis.ell.1983,32:425-434.
[16]Mattaj.I Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding.Cell.1986,46:905-911.
[17]Fisher,D.E.,G.E.Conner,W.H.Reeves,et al.Small nuclear ribonucleoprotein particle assembly in vivo:demonstration of a 6S RNA-free core precursor and posttranslational modification.Cell.1985,42:751-758.
[18]Liu,Q.,U.Fischer,F.Wang,G.Dreyfuss.The spinal muscular atrophy disease gene product,SMN,and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins.Cell 1997,90:1013-1021.
[19]Matthias Grimmler,Liane Bauer, Marjaana Nousiainen,et al.Phosphorylation regulates the activity of the SMN complex during assembly of spliceosomal U snRNPs. European Molecular Biology Organization. 2005,6(1):70-76.
[20]Laich AJ,Furst UO,Nagl M,et al.The transposition of the swellingdependent chloride channel ICln from the cytosol to the membrane is regulated by the cell volume.Soc.Gen.Physiol.Meeting Abstracts,2Aa.
[21]Schonbeck U,Mach F,Sukhova GK, et al.Regulation of matrix metalloproteinase exp reeion in human vascular smooth muscle cells by lymphocytes:a role for CD40 signaling in plaque rupture[J].Circ Res,1997,81(3):448-454.
[22]Musch MW,Luer CA,Davis-Amaral EM,et al.Hypotonic Stress induces translocation of the osmolyte channel protein pIcln,in embryonic skate (raja eglanteria) heart.The Journal of Experimental Zoology.1997,460-463.
[23]Emma F,Breon S,Morrison,et al.Effect of cell swelling on memberane and cytoplasmic distribution of pICln.Am.J.Physiol.1998,274(6):C1545-C1551
[24]Ballanyi K,Grafe P.Cell volume regulation in the nervous system.Renal Physiol Biochem,1998,325-142.
[25]Tao G-Z,Komatsuda A,Miura AB,et al.pICln predominantly localizes at luminal surface membranes of distal tubules and Henle's ascending limbs.Biochem Biophys Res Commun 1998,247:668-673.
[26]Francesco Emma,Roberto Sanchez-Olea,Kevin Strange.Characterization of pICln binding proteins:identification of p17 and assessment of the role of acidic domains in mediating protein-protein interactions.Biochimica et Biophysica Acta.1998,1404:321-328.
[27]M.A.Sells,J.Chernoff,Trends Cell Biol.1997,7:162-167.
[28]chwartz RS,Rybicki AC,Nagel RL.Molecular cloning and Expression of a chloride channel-associated protein pICln in human young red blood cells:association with actin.Biochem J.1997,327:609-616.
[29]Li Y-Y,Tao G-Z,Nagasawa H,et al.pICln and cytosolic F-actin constitute a heteromeric complex with a constant molecular mass in rat skeletal muscles.J Biochem 1999,126:643-649.
[30]Nilius B,Gerke V,Prenen J,et al.Annexin Ⅱ modulates volume activated chloride currents in vascular endothelial cells.J Biol Chem 1996,271:30631-30636.
[31]Okada Y.Volume expansion-sensing outward-rectifier Cl~- channel:fresh start to the molecular identity and volume sensor.Am J Physiol 1997,273:755-789.
[32]Tilly BC,Edixhoven MJ,Tertoolen LGJ,et al.Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton. Mol Biol Cell 1996,7:1419-1427.
[33]Zhang J,Larsen TH,Lieberman M.F-actin modulates swelling activated chloride current in cultured chick cardiac myocytes.Am J Physiol 1997,273:1215-1224.
[34]Ishibashi K Sasaki S,Uchida S,et al.Tissue expression of mRNA of chloride channel from MDCK cells and its regulation by protein kinase.Biochemical and Biophysical Research Communications.1993,192(2):561-567.
[35]Abe T,Takeuchi K,Takahashi N,et al.Intrarenal mRNA expression of the rat MDCK-type chloride channel.Nephron.1996,73:23-26.
[36]马颖哲.pICln蛋白的分布及功能研究[D].吉林:吉林大学,1999.
[37]孙英,李辉,田方曦,等.毕赤酵母表达重组人白细胞介素11的连续培养研究[J].中国生物工程杂志,2003,23(2):91-93,100.
[38]欧阳立明,张惠展,张嗣同.巴斯德毕赤酵母的基因表达系统研究进展[J].生物化学与生物物理学进展,2000,27(2):151.
[39]彭毅,杨希才,康良仪.影响甲醇酵母外源蛋白表达的因素[J].生物技术通报,2000,4:33.
[40]路蓉,安云庆.巴斯德毕赤酵母表达系统及其分泌型蛋白表达的研究进展[J].临床和实验医学杂志,2004,3(1):43-47.
[41]易绍宣,吴军,贺伟峰,等.人CTLA4胞外区突变体在毕赤酵母中的高效表达[J].第三军医大学学报,2003,25(21):1902-1905.
[42]Wickman,K.,W.T.Pu,and D.E.Clapham.Unpublished data.
[2]Ackerman MJ,Krapivinsky GB,Gordon E,et al . Characterization of a native swelling-induced chloride current,IC1.swell,and its regulatory protein,pICln,in Xenopus oocytes,physiology.1994,44:17-24.
[3]Pulmichl M,LI Y,Wickman K,et al.New mammalian chloride channel identified by expression cloning.N ature.1992,356:238-241.
[4]A nguita J,Chalfant ML,Givan MM,et al.Molecular cloning of the human volume-sensitive chloride conductance regulatory protein,pICln,from ocular ciliary epithelium.Biochemical and Biophysical Research Communications.1995,208:89-95.
[5]Schwartz RS,Rybicki AC,Nagel RL.Molecular cloning and Expression of a chloride-associated protein pICln in young red blood cells:association with actin.Biochem.J.1997,327:609-610.
[6]Buyse G,DeGreef C,Raeymaekers L,et al.The ubiquitously Expressed pICln,protein form shomomeric complexes in vitro.Biochemical And Biophysical Research Communications.1996,218:822-827.
[7]A.Schmarda,U.O.Nagl,M.Gschwentner,et al.Cell.Physiol.Biochem.1997,7:298-302.
[8]Thomas Voets,Gunnar Buyse,Guy Droogmans,et al.The GXGXG motif in the pICln protein is not important for the nucleotide sensitivity of the pICln-induced C13 current in Xenopus oocytes.FEBS Letters.1998,426:171-173.
[9]Krapivinsky GB,Ackerman M J ,Gordon EA,et al.Molecular characterization of a swelling-induced chloride conductance regulatory protein,pIcln.Cell,2994,76 :439-448.
[10]Strange K,Emma F,Jackson P S.Cellular and molecular physiology of volume-sensitive anion channels.Am J Physiol,1996,270 (Cell Physiol.39):C711.
[11]Tao G-Z,Yohtalou Tashima.A peptide derived from pICln induced a strong hypotonic resistance in Escherichia coli cells.Peptides.2000;21;485-490.
[12]Kramer,A.The structure and function of proteins involved in mammalian pre-mRNA splicing.Annu.Rev.Biochem.1996,65:367-409.
[13]William,T.PU,Grigory B.Krapivinsky,and Lubakrapivinsky et al.pICln Inhibits snRNP Biogenesis by Binding Core Spliceosomal Proteins.Molecular And Cellular Biology.1999,19(6):4113-4120.
[14]DeRobertis,E.M.Nucleocytoplasmic segregation of proteins and RNAs.Cell.1983,32:1021-1025.
[15]Zeller,R.,T.Nyffenegger,E.M.DeRobertis.Nucleocytoplasmic distribution of snRNPs and stockpiled snRNA-binding proteins during oogenesis and early development in Xenopus laevis.ell.1983,32:425-434.
[16]Mattaj.I Cap trimethylation of U snRNA is cytoplasmic and dependent on U snRNP protein binding.Cell.1986,46:905-911.
[17]Fisher,D.E.,G.E.Conner,W.H.Reeves,et al.Small nuclear ribonucleoprotein particle assembly in vivo:demonstration of a 6S RNA-free core precursor and posttranslational modification.Cell.1985,42:751-758.
[18]Liu,Q.,U.Fischer,F.Wang,G.Dreyfuss.The spinal muscular atrophy disease gene product,SMN,and its associated protein SIP1 are in a complex with spliceosomal snRNP proteins.Cell 1997,90:1013-1021.
[19]Matthias Grimmler,Liane Bauer, Marjaana Nousiainen,et al.Phosphorylation regulates the activity of the SMN complex during assembly of spliceosomal U snRNPs. European Molecular Biology Organization. 2005,6(1):70-76.
[20]Laich AJ,Furst UO,Nagl M,et al.The transposition of the swellingdependent chloride channel ICln from the cytosol to the membrane is regulated by the cell volume.Soc.Gen.Physiol.Meeting Abstracts,2Aa.
[21]Schonbeck U,Mach F,Sukhova GK, et al.Regulation of matrix metalloproteinase exp reeion in human vascular smooth muscle cells by lymphocytes:a role for CD40 signaling in plaque rupture[J].Circ Res,1997,81(3):448-454.
[22]Musch MW,Luer CA,Davis-Amaral EM,et al.Hypotonic Stress induces translocation of the osmolyte channel protein pIcln,in embryonic skate (raja eglanteria) heart.The Journal of Experimental Zoology.1997,460-463.
[23]Emma F,Breon S,Morrison,et al.Effect of cell swelling on memberane and cytoplasmic distribution of pICln.Am.J.Physiol.1998,274(6):C1545-C1551
[24]Ballanyi K,Grafe P.Cell volume regulation in the nervous system.Renal Physiol Biochem,1998,325-142.
[25]Tao G-Z,Komatsuda A,Miura AB,et al.pICln predominantly localizes at luminal surface membranes of distal tubules and Henle's ascending limbs.Biochem Biophys Res Commun 1998,247:668-673.
[26]Francesco Emma,Roberto Sanchez-Olea,Kevin Strange.Characterization of pICln binding proteins:identification of p17 and assessment of the role of acidic domains in mediating protein-protein interactions.Biochimica et Biophysica Acta.1998,1404:321-328.
[27]M.A.Sells,J.Chernoff,Trends Cell Biol.1997,7:162-167.
[28]chwartz RS,Rybicki AC,Nagel RL.Molecular cloning and Expression of a chloride channel-associated protein pICln in human young red blood cells:association with actin.Biochem J.1997,327:609-616.
[29]Li Y-Y,Tao G-Z,Nagasawa H,et al.pICln and cytosolic F-actin constitute a heteromeric complex with a constant molecular mass in rat skeletal muscles.J Biochem 1999,126:643-649.
[30]Nilius B,Gerke V,Prenen J,et al.Annexin Ⅱ modulates volume activated chloride currents in vascular endothelial cells.J Biol Chem 1996,271:30631-30636.
[31]Okada Y.Volume expansion-sensing outward-rectifier Cl~- channel:fresh start to the molecular identity and volume sensor.Am J Physiol 1997,273:755-789.
[32]Tilly BC,Edixhoven MJ,Tertoolen LGJ,et al.Activation of the osmo-sensitive chloride conductance involves P21rho and is accompanied by a transient reorganization of the F-actin cytoskeleton. Mol Biol Cell 1996,7:1419-1427.
[33]Zhang J,Larsen TH,Lieberman M.F-actin modulates swelling activated chloride current in cultured chick cardiac myocytes.Am J Physiol 1997,273:1215-1224.
[34]Ishibashi K Sasaki S,Uchida S,et al.Tissue expression of mRNA of chloride channel from MDCK cells and its regulation by protein kinase.Biochemical and Biophysical Research Communications.1993,192(2):561-567.
[35]Abe T,Takeuchi K,Takahashi N,et al.Intrarenal mRNA expression of the rat MDCK-type chloride channel.Nephron.1996,73:23-26.
[36]马颖哲.pICln蛋白的分布及功能研究[D].吉林:吉林大学,1999.
[37]孙英,李辉,田方曦,等.毕赤酵母表达重组人白细胞介素11的连续培养研究[J].中国生物工程杂志,2003,23(2):91-93,100.
[38]欧阳立明,张惠展,张嗣同.巴斯德毕赤酵母的基因表达系统研究进展[J].生物化学与生物物理学进展,2000,27(2):151.
[39]彭毅,杨希才,康良仪.影响甲醇酵母外源蛋白表达的因素[J].生物技术通报,2000,4:33.
[40]路蓉,安云庆.巴斯德毕赤酵母表达系统及其分泌型蛋白表达的研究进展[J].临床和实验医学杂志,2004,3(1):43-47.
[41]易绍宣,吴军,贺伟峰,等.人CTLA4胞外区突变体在毕赤酵母中的高效表达[J].第三军医大学学报,2003,25(21):1902-1905.
[42]Wickman,K.,W.T.Pu,and D.E.Clapham.Unpublished data.