| The antimicrobial peptides’characteristics include broad-spectrum bactericidal antimicrobial peptides, anti-tumor, anti-viral and anti-protozoal, as well as the action mode of antimicrobial peptides were unique, so antimicrobial peptides become a hot topic of new antibiotic medications. In order to accelerate the production of antimicrobial peptides, this study cloned the Temporin-1CEa gene from the skin of Rana temporaria chensinensis, the goat mammary gland specific expression vector pBC1-neo-Temporin-1CEa was constructed; and its linear fragment was transferred into goat fibroblasts, the transgenic goat fibroblast cell line be obtained after G418action. Furthermore,5’regulatory sequences of chicken ovalbumin gene was cloned from chicken genomic DNA; and the bicistronic retroviral vector pMX-GFP-5OV-TemporinlCEa, which could specific express Temporin1CEa gene in chicken oviduct epithelia, was constructed. In order to establish the vitro model of regulable exogenous genes expression in the chicken oviduct epithelia, the method of Obturated Interior Digesting was established, as well as the chicken oviduct epithelial cell line was established. In order to detect expression of GFP gene and TemporinlCEa gene in chicken oviduct epithelia, pMX-GFP-5OV-TemporinlCEa vector was transferred into chicken oviduct epithelial cells by liposome. The main results of this study are as follows:1. Construction of goat mammary gland specific expression vector According to the reference sequence in GenBank, Temporin-lCEa gene was be cloned; and the pBC1-neo-Temporin-lCEa vector was constructed by pBCl-neo vector and Temporin-1CEa gene. The results of identification with PCR and restriction enzyme digestion showed that Temporin-1CEa gene was subcloned into pBCl-neo vector, sequencing results showed that goat mammary gland specific expression vector pBCl-neo-Temporin-1CEa was constructed successfully;2. Establishment of goat transgenic fibroblast cell line Goat fibroblasts, which were obtained from goats ear tissue, were transferred into linear pBC1-neo-Temporin-1CEa vector, screened these goat fibroblasts by culture media with300μg/mL G418, obtained trans-pBC1-neo-Temporin-1CEa goat fibroblasts; observed the morphology, calculated cells’population doubling tim cryopreserved and recovered, analysed karyotype, detected vimentin by immunohistochemistry, all the results indicated that goat transgenic fibroblast cell line was established successfully;3. Construction of bicistronic retroviral vector According to the reference sequence in GenBank,5’regulatory sequences cloned chicken ovalbumin gene--5OV was be cloned; and the pMX-GFP-5OV-Temporinl CEa vector was constructed by pMX vector,5OV, Temporin-1CEa gene and GFP gene. The results of identification with PCR and restriction enzyme digestion showed that 50V, Temporin-1CEa gene and GFP gene were subcloned into pMX vector, sequencing results showed that retroviral vector pMX-GFP-5OV-TemporinlCEa was constructed successfully;4. Establishment of chicken oviduct epithelial cell line The OID method was established for isolation and culture primary epithelia from chicken oviduct; as well as, subcultured these primary epithelia, observed its’ morphology, calculated cells’population doubling time, cryopreserved and recovered, analysed karyotype, detected cytokeratin18by immunohistochemistry, detected expression of marker gene and the ability to be transfected; all the results were showed that chicken epithelial cell line could be established by OID method, these cells with normal morphology, good growth characteristics and genetic stability were named XYF-142;5. Detection of bicistronic retroviral vector and XYF-142cell model XYF-142cells were transfected with pMX-GFP-5OV-Temporin-1CEa vector, observed GFP and detected transcription of Temporin-1CEa gene in these cells; the results showed that not only observed GFP in these cells, and detected transcript of Temporin-1CEa gene from these cells’total RNA. It indicated that bicistronic retroviral vector pMX-GFP-5OV-Temporin-1CEa which could specific express Temporin-1CEa gene inchicken oviduct epithelia was constructed successfully, as well as XYF-142cells can be applied as a vitro model of regulable exogenous genes expression in the chicken oviduct epithelia.To sum up, in this study, trans-Temporin-1CEa gene goat fibroblast cell line was established successfully in this study; bicistronic retroviral vector pMX-GFP-5OV-Temporin-1CEa which could specific express Temporin-1CEa gene inchicken oviduct epithelia was constructed successfully; chicken epithelial cell line XYF-142was established successfully, and it can be applied as a vitro model of regulable exogenous genes expression in the chicken oviduct epithelia. This study laid the foundations for further research on producing antimicrobial peptides from goat milk and eggs. |
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