Expression Of Shiva-1a Gene In Baculovirus And Establishment Of Transgenic Nuclear Donor Cells Of Goat

The object of this study is to obtain transgenic goat fetal fibroblasts, which can be used as nuclear donors in nuclear transfer to produce brucellosis resistance goats. First, expressd the Shiva-1a peptide in baculovirus expression system, and investigated its antibacterial effects. Then constructed the lymphocyte-specific expression vector, which was used to transfect goat fetal fibroblasts, and the positive transgenic cells were selected. The results were as follows:1. Based on the amino acid sequence registered in GenBank, the fragment of Shiva-1a, which was designed by choosing the insect preferred codons and was synthesized and was cloned into the pBacFast Dual vector of BAC-TO-BACTM recombinant baculovirus expression system. The recombinant plasmid was transformed into DH10Bac competent cell. Then prepared recombinant bacmid with liposome was transfected into spodoptera fragiperda 9 (Sf9) cells. Western blot showed that shiva-1a peptide with a molecular weight of 5 kDa was expressed. In vitro antimicrobial test showed that Shiva-1a had antimicrobial activity against E. coli. BL21 strain.2. Combined the PCR amplify sequence of SV40 ployA with the 5' flanking regulation and signal sequence of goat IL-2 gene and together with the sequence of Shiva-1a into the backbone vector pEGFP-C1, to construct the specific expression vector pEGFP-PIS. The double restriction enzyme digestion verification shows that the vector was constructed successfully.3. The lymph-specific expression vector pEGFP-PIS was introduced into the goat fetal fibroblast cells by liposome transfection. The positive cells clone was selected in 700μg/mL G418. The positive transgenic cells were are obtained after selection. Karyotype analysis proved that positive cells can be candidate cells for further study.All these results provided a foundation for further research on the biological activity of shiva-1a antibacterial peptide and laid a pathway for disease-resistance transgenic goat breeding.