Cloning And Expression Of Recombinant Chicken PEPT1 And Its Transport Regulation

Nine experiments were conducted to study the mechanism of regional and ontogenetic expression of chicken; and clone,expression and purification of epitope of C-terminus of chicken PEPT1; constructed cell model of cPEPT1 in 293-T cell and study the effect of hormone and regulative factors on cell model transportation.Exp.1 This study was to establish the Real-Time PCR methods to Quantitative investigate Peptide transporter PEPT1 mRNA expression rate in chicken small Intestine: method Real-Time PCR was established to evaluate gene expression of peptide transporter in small intestine of chick. The methods of Real-Time PCR to quantitative investigate peptide transporter (PEPT1) including RNA extraction and reverse transcription in chicken small intestine. The primers of 18SrRNA and PEPT1 were designed according to the gene sequence reported to detect target gene. A series of experiments were executed to optimize concentration of template and primers, also established a rational anneal temperature.Exp.2 Regional and Ontogenetic Expression of PEPT1 mRNA in Small Intestinal of Broilers: In order to analysis the development of Peptide transporter 1 (PEPT1) mRNA in different intestinal segments of broiler. 100 1-day-old male Arbor Acres (AA) broilers were randomly divided into 5 replicates. Peptide transporter 1 (PEPT1) mRNA in different intestinal segments(duodenum, jejunum, ileum) and in different day(9,18,27,36,45) were determined by Real-Time PCR. The results showed that: (1) Regional expression of PEPT1 mRNA in duodenum, jejunum, and ileum was declined gradually; (2) Ontogeny patterns of PEPT1mRNA in duodenum and jejunum were different. The abundance of PEPT1 mRNA in duodenum was relatively higher on day 9,18,27 while in jejunum on day 36,45 . The abundance of PEPT1mRNA in duodenum on day 27 was significant higher than on day 45 (P<0.05); in jejunum day 36 and 45 was significant higher than on day 9,18 (P<0.05).Exp.3 Regional and Ontogenetic Expression of PEPT1 mRNA in Small Intestinal of Beijing-you Chicken: In order to analysis the development of Peptide transporter 1 (PEPT1) mRNA in different intestinal segments of Beijing-you chicken. 100 1-day-old male Beijing-you chicken were randomly divided into 5 replicates. Peptide transporter 1 (PEPT1) mRNA in different intestinal segments (duodenum, jejunum, ileum) and different day (18,36,54,72,90) were determined by Real-Time PCR. The results showed that: (1) Regional expression of PEPT1 mRNA in duodenum, jejunum, and ileum was declined gradually; (2) Ontogeny patterns of PEPT1 mRNA in duodenum were different with jejunum and ileum. The abundance of PEPT1 mRNA in duodenum was relatively higher on day 18,36 were higher than day 54,72,90, and the day on 18 was top of them all, while the lowest day on 72. In jejunum , the higher day was on day 90, it was higher than day 18,36 significantly, while the PEPT1 mRNA expression on day 54,72 was high than day 18,36, but the result was not significant. The abundance of PEPT1 mRNA in ileum on day 72 was the highest day and on day 54,72,90 was significant higher than on day 18,36. Exp.4 Construction of the Vector of Beijing-you chicken intestinal peptide transporter PEPT1: Construct the pEASY-T3-PEPT1 recombine plasmid. According to the PEPT1 gene sequence in GenBank, a pair of specific sequences as primers was designed to amplify the PEPT1 gene isolated from intestinal of chicken jejunum. By reverse transcription polymerase chain reaction (RT-PCR), PEPT1 gene was amplified successfully. The PCR products were cloned into pEASY-T3 vector. Sequence analysis indicated that the length of the gene PEPT1 was 2145bp, by TMPRED analysis the gene including 12 Transmembranes and with a large hydrophilic loop. The PEPT1 epitope was mainly focus on the C-terminus.Exp.5 Construction of the Expression Vector in Rosetta (DE3) for PEPT1epitope gene of Poultry intestinal peptide transporter PEPT1: This study was to clone chicken intestinal PEPT1 epitope gene. According to the PEPT1 gene sequence in GenBank, two pair of specific sequences as primers were designed to amplify the PEPT1 epitope fragment gene used cDNA template isolated from intestinal of chicken duodenum cell by reverse transcription polymerase chain reaction (RT-PCR), PEPT1 gene was amplified successfully and the PCR products were cloned into pEASY-T3 vector. Keep the transmembrane epitope domain between 9 and 10 of C-terminus. The PEPT1epitope gene was subcloned into expression vector pET-22B(+)with a His-tag at the C-terminus and expressed in Rosetta (DE3). The products was purified by Ni-NTA chromatography column and identified by SDS-PAGE, Western blot and mass spectrogram. The recombinant expression vector pET22B-6-His-PEPT1 was constructed and the 6-His-PEPT1 protein was successfully expressed in an insoluble form. After expression and purification by metal chelate affinity chromatography, purified 6-His-PEPT1 fusion protein was obtained.Exp.6 Construction of the Expression Vector of Pichia Pastoris Yeast for PEPT1 epitope gene of Poultry intestinal peptide transporter PEPT1: According to the PEPT1 gene sequence in GenBank,a pair of specific sequences as primers were designed to amplify the PEPT1 gene isolated from intestinal of chicken jejunum. PEPT1 gene was amplified successfully. The PCR products were cloned into pEASY-T3 vector. Sequence analysis indicated that the length of the gene PEPT1 gene was 1000 bp, analysis the epitope by TMPRED; in order to produce secretary protein, keep the last two transmembrane domain of C- terminus. The PEPT1 epitope gene was subcloned into the pichia pastoris expression vector pPIC9K. The study obtained the recombinant plasmid successfully by PCR and restriction enzyme validation. The obtained recombinant plasmid was designated as pPIC9K-PEPT1(s). The sequence was corrected by MS mensuration.Exp 7: Construction of the Expression Vector of Beijing-you chicken intestinal peptide transporter PEPT1 and its transfecting condition optimization: This study was carried out to constructed pcDNA3-PEPT1 recombinant gene and use double transfection system to observe cell condition by EGFP after target gene transfecting. Established Real-time PCR system detects gene expression system. By optimize the cultural condition of 293-T, we got a rational transform condition for PEPT1 cell model.Exp8:Cloning and constructing recombinant Beijing-you chicken PEPT1 in 293-T Cell: This study was to establish Beijing-you chicken PEPT1 cell model. According to PEPT1 gene sequence in GenBank, a pair of specific sequences as primers were designed to amplify the PEPT1 sequence isolated from intestinal of Beijing-you chicken jejunum cell by RT-PCR, The PEPT1 gene was amplified successfully and the PCR products were cloned into pEASY-T3 vector. Sequence analysis indicated that the length of PEPT1 gene was 2145bp and its sequence was correct. This gene was inserting into expression vector pcDNA3.0. The constructed pcDNA3.0-PEPT1 was expressed in 293-T cells with pcDNA3.0-EGFP plasmid by lipofectin methods. Fluorescence level was detected by flow cytomete(rfcm)in 16h,20h,24h to detected positive ratio and extracted total RNA of the cell, wipe out contaminated DNA by DNAseâ… and get cDNA by reverse transcription, a pair of specific sequences as primers were to verified the PEPT1 expression level, use plasmid pEASY-T3-PEPT1 to construct standard curve, by Real-Time PCR survey we found that there are steady transcription level in 16h,20h,24h and 44h after pcDNA3-PEPT1 transfection. We can conclude that Beijing-you chicken PEPT1can be expressed efficiently by eukaryotic expression vector pcDNA3.0-PEPT1 in 293-T cells.Exp9:Clone and construct Beijing-you chicken PEPT1 (cPEPT1) gene in eukaryotic expression vector and express it in 293-T cells, detecting transport activity of recombinant cPEPT1 protein. Methods: Culture the 293-T cell density to 90-95% then demeshed by trypsinization, transfers to 24 wells plates. Infection protocol was used when cellular fusion is about 90-95%, added 1ul pcDNA3-cPEPT1 plasmid each well and detected the mRNA expression by Real-Time PCR assay, at the same time added 3H marked Gly-Leu dipeptides with different concentration of H+,GH and glucagons, abeyance the reaction in 30 and 60 minutes, the radioactivity measured by liquid scintillation counting. Results: By Real-Time PCR assay we obtained a steady mRNA expression rate in the transfected system; the result show that, compared with H+ and glucagons group, the GH group significantly promote the cell ability to absorb 3H marked Gly-Leu. Conclusion: The experiments were successfully cloning and establishing a transient expression system.