Study On Expression Character And Expression Regulation Element Of Parotid Secretory Protein In Swine

Mammalian salivary glands belong to external secretion glands, but they have part function of endocrine glands. Most secretions can reach mouth by the salivary genal tube, and work in stomach when pH>4. While a fraction of secretions can reach directly into the bloodstream. There are lots of salivary secreted proteins, in which a protein may have multifunction and several proteins always share the same function. So mammalian salivary gland can be used as bioreactors.Parotid secretory protein (PSP) is expressed especially in the parotid gland, as it is antibiotics in function, many people have interested in it. To understand the expression of PSP and the relationship with α -Amylase (AMY), we research the experession of these two genes in different postnatal stages in mouse and pig by RT-PCR and Q-PCR (fluorescence quantitative Polymerase Chain React). Result shows, PSP expressed highly in almost postnatal stages in mouse and pig, and expression level changing with the time. Furthermore, the trend of PSP and AMY keeps the same in all the time, suggest they shares coregulation. And results from Northern and Western blot show the same as Q-PCR.We analysis the full amino acid sequence of pig PSP, then four potential peptides were found as antigenic determinants, their sequence and location at pig PSP were list as fellows: 22-37aa, 41-59aa, 180 -195aa, C-terminal. Among them, Peptide- I (22-37aa,) shown the best, then it was chosen as antigen to to conjugate with KLH, to develop antiserum in rabbit. A polyclonal antibody specific for pig PSP was generated. We research PSP expression in protein level; result shows PSP expression in higher level than that of GAPDH. And a new variant of PSP was found in mouse, which can reacted with PSP-antiserum.In the second part, we located the transcription start site by 5'RACE ,result shows, we got 39bp longer cDNA sequence than the reported one, and we correct the first code of PSP ORF to its -22bp upstream;And we located the transcription start site on -19bp of the new ORF. We predicted the core promoter of PSP, result shows, PSP promoter is untypical one, TATA box was not include, And eight Inrs were found near the transcription start site, it proved the result of 5'RACE, and fit the character of Inr.We located PSP promoter by instant transfection. Firstly ,we successfully rebuild plsmid pEGFP-N1, and set up a serious of cell transfection vectors including different length of 5'regulation region by PCR, clone and subclone.We take Acc-M cell as transfection subject, and take primary parotid epithelium cells as control. And I found suitable transfection condition, the best ratio of lipofectamine/DNA was 1/3 pmol: 2μ l using serum-free medium, and the optimal time to observe was 24 hours after transfection.