Study On The Gene Quantification And Eukaryotic Expression Of Bovine Peptide Transporter Ⅰ

1There were three objectives in the present study. The first objective was to determine bPepT I mRNA expression levels in the mucosa or epithelium of different bovine g.i. tract parts in order to provide persuasive information on the issue of the major absorption point of small peptide in bovine g.i. tract. The second one was try to build a prokaryotic expression system of bPepT I to produce collectable bPepT I protein to produce its antibody, which is the key step to quantify bPepT I protein level by immunal fluorescene method. The third one was to construct a recombinant plasmid for construction of a bPepT I eukaryotic expression system in the next step, which is can be used to study the kinetics of bPepT I transporter in cell level.Part one The study on the mRNA expression of bovine gastrointestinal peptide transporter I.The mRNA expression of PepTI in the limousin bovine gastrointestinal was investingated by relative quantification. We also determined the PepTI gene quantity in Holstein,Bohai, Luxi and Limousin bovine. We listed the tissues according to priority of PepTI mRNA quantity as follows: reticulum, omasum, abomasum, colon, duodenum, jejunum, rumen, caecum, ileum, with the differences being insignificant(P>0.05); The results of PepTI mRNA quantity was different significantly in the small intestinal of bovine of different species(P<0.01). As for duodenum , the gene quantity was much higher in limousin than that in Bohai and Holstein(P<0.05, P<0.05); As for jejunum, the gene quantity was highest in Holstein, with insignificant difference between Bohai and Limousin(P>0.05); And ileum, gene quantity among three species was significantly different(P<0.01), with the highest gene quantity in Holstein, and insignificant difference between Luxi and limousin*Luxi. According to the results of absolute quantification PCR, we listed the tissues according to priority of PepTI mRNA quantity as follows: jejunum, omasum, reticulum, rumen, caecum, duodenum, ileum, abomasum, colon, the difference was extremely significant among all species(P<0.01). As for the stomach, the gene quantity was highest in Omasum, with insignificant difference to reticulum(P>0.05) and extremely significant difference to rumen and abomasum(P<0.01); PepTI mRNA quantity in reticulum was significantly different to rumen and abomasum(P<0.05), with insignificant difference between rumen and abomasum(P>0.05). And the intestinal tract, there is the highest PepTI mRNA quantity in the jejunum, with extremely significant difference to dudenum and ileum(P<0.01); there is insignificant difference among dudenum, caecum and ileum(P>0.05); gene quantity in dudenum, jejunum, and caecum was higher than that in colon with extremely significant difference(P<0.01). Gene quantity in dudenum was higher than that in omasum with significant difference(P<0.05); there is insignificant difference among rumen, dudenum, ileum, and abomasum(P>0.05), which was true of the difference among dudenum, ileum, caecum and abomasum(P>0.05). Combining other experimental results and this study we can conclude that, bPepTI mRNA quantity was highest in jejunum, omaum coming next, with the lowest in the large intestine.Part two Prokaryotic expression of bovine gastrointestinal peptide transporter IBy optimizing the PCR conditions for cloning the CDS of bPepTI to construct pET-32a-bPepTI and pGEX-6P-1-bPepTI prokaryotic expression recombinant plasmid. We expressed the fusion protein with different IPTG concentrations, different induced temperatures, different induced time, and the cells. As a result, we cloned the complete coding region of bPepTI successfully. The DNA sequence reached a consensus with the sequence registered on GENBANK. The test detected no protein expressed from recombinant plasmid pET-32a- bPepTI in any condition, but there was a small amount of soluble protein expressed when pGEX-6P-1-bPepTI was induced in Rosetta, with IPTG concentration of 0.8 mmol/L.We did not get the ideal expression of PepTI. There were eight glycosylation site, one cAMP-dependent protein kinase (PKA) site, and five protein kinase C (PKC) sites in the amino acid sequence encoded by the CDS of bPepTI.Part three Construction of bovine gastrointestinal peptide transporter I Eukaryotic Expression SystemWe amplified the bPepTI gene with the optimum conditions of the PCR amplification from the former experiment, cloning the fragments into pcDNA3.1/V5-his-B to construct the recombinant plasmid pcDNA3.1/V5-his-B-bPepTI. As a result, we amplified gene bPepTI successfully, and sequencing results showed consensus with that registered on GenBank. The study constructed eukaryotic expression recombinant plasmid pcDNA3.1/V5-his-B-bPepTI. successfully, which set the stage for further research of dynamic characteristics of bPepTI.