Construction Of Pichia Pastoris Expression Vector And Transfection Of PICln Gene

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.