【作者】 郗艳丽；

【导师】 马颖哲；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 硕士

【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。更多还原

【Abstract】 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.更多还原