The Expression And Ion Transfer Function Of CFTR Gene In The Intestinal Of Diarrhea Mice

CFTR is one of the ATP binding cassette transporters,as a kind of Cl-ion transport channel,it provides the transport way which Cl-across the cell membrane and adjust the Cl-transfer speed,at the same time CFTR is the center of the salt transport across epithelial membrane and fluid flow and ion concentration,therefore,CFTR plays an important role in regulating in diarrhea.Cl-In this topic,firstly,built the models of Osmotic diarrhoea in mice,put the duodenum jejunum ileum and colon as the research object,to test the expression of CFTR gene,distribution and orientation detection,built the CFTR expression vector in vitro cell culture experiments to research CFTR recombinant protein transported function of ion associated with diarrhea,to clarify the relationship between the CFTR protein and diarrhea.First,using mannitol gavage methods to construct mice diarrhea model,40 minutes later,the mice began to appear symptoms of diarrhea,listlessness,slow,soft feces present state;With the extension of time,the diarrhea symptoms get worse and stool showing fecal water samples.Collected the diarrhea mice of duodenum,jejunum,ileum and colon tissue.Cloned NBD domain of CFTR and NKCC1 gene,PCR and immunohistochemistry results showed that the three genes expressed in the duodenum,jejunum,ileum and colon tissue of the normal group,3h and 9h,s diarrhea groups.Real time PCR results showed that the three genes expressed differently in different intestinal tissues,which might be due to the presence of hypertonic environment makes the expression of the three genes changed.Secondly,using mouse genomic DNA as a template to synthesis CFTR-NBD1,CFTR-NBD2 genes,degenerate primer,Amplificate,clone,sequence CFTR protein nucleotide domain of the mouse and getted the 690 bp,666bp gene fragments respectively,translated into 230 and 222 amino acid sequence respectively.Through the comparison of the amino acid sequence finded that the obtained CFTR-NBD1 and CFTR-NBD2 amino acid sequences have characterized sequence of ABC protein family.Therefore,ensured the the PCR amplified nucleotide product is a ATP binding region gene of the mouse ABC protein preliminarily.To double digest the cloning of the recombinant plasmid of CFTR-NBD1,CFTR-NBD2 gene and PEGFP-N1 eukaryotic expression plasmid respectively.The digested products were connected by T4 ligase to construct the recombinant eukaryotic expression plasmid pEGFP-N1-CFTR-NBD1?pEGFP-N1-CFTRNBD2,Through PCR,double enzyme digestion and sequencing,Succes-sfully constructingthe eukaryotic expression vector CFTR-NBD1 and CFTR-NBD2.Last,Using liposome transfection method to import recombinant eukaryotic expression plasmid pEGFP-N1-CFTR NBD1,pEGFP N1-CFTR-NBD2 into IEC cells of the mice,the empty plasmid pEGFP-N1 transfecting into mouse IEC cells and IEC cells without any treatment were the control group and the blank group respectively,the results showed that transfection group and the control group had fluorescence while the blank group had nothing under the fluorescence microscope.It indicates that the CFTR-NBD gene successful expressed in IEC cells.The collected cells were Fractured into suspension by ultrasonic cracker for intracellular fluid,the cell culture medium was collected as the extracellular fluid,the concentration of the three kinds of ions in intracellular and extracellular fluid deteced by Ion detection kit,Analysis of significant difference with SPSS16.0 software.the results show that the 3 kinds of ion transported from intracellular to extracellular under function of the CFTR-NBD protein,Therefore,ensured that the CFTR-NBD were the nucleotide binding region of the mouse CFTR proteingene and CFTR-NBD protein had the function of transporting ions.In summary,This is the first study to investigate the expression of CFTR-NBD and NKCC1 gene in osmotic diarrhea mice model and construct the eukaryotic expression vector pEGFP-N1-CFTR-NBD in osmotic diarrhea mice model.This study found that introducing CFTR-NBD gene into mouse IEC then expressed successfully by the mouse IEC culture and CFTR-NBD protein has the function of transporting the three kinds of ion through detecting the concentration of three kinds ion.The successful cloning and expression of mouse CFTR-NBD gene give hope to find the target sites of diarrhea treatmented medicine by revealled the function and the transport mechanism of CFTR transporters in mouse,Finally achieved the purpose of drug treatment and prevention of diarrhea in livestock and poultry.