The Study On The Construction Of SAK Eukaruon Vector And Expression In Transfected Cells

Staphylokinase(SAK) synthesized by Staphylococcusaureas is a 136-amino-acid single-chain ultramembrane protein. In this paper, staphylokinase gene was amplified from staphyloccusaureus by polymerase chain reaction (PCR), and inserted into pUCm-T Vector with T/A clone. Linear pEGFP-NI and SAK gene were obtained by cleaving the green fluorescence protein eukaryote vector pEGFP-NI and pUCm-T Vector with SAK by Not I and Bam HI. SAK fragment and pEGFP-NI Vector isolated were ligated with T4 ligase. The recombination plasmid was transformed into JM 109 cells. Positive colonies were screened and the plasmid was extracted from JM109 cells. Hela cells were transformed with the recombination plasmid, and were crashed into pieces with ultrasonic wave. After centrifugation, the suspendant and deposit were electrophoresed by SDS-PAGE, and then the protein was separated and identified by Western blot.SAK protein was found expressed in Hela cells.SAK activity was examined by agarose with plasma, and distinctive dissolution circle was found around the inoculated locus , which demonstrated the bioactivity of SAK protein.By observing the development of blastoderm cells in cultures with cytokines without feeding cells, we found that the differentiation of blastoderm cells were retained by cytokines, and can form more blastoderm cell clusters, which helps to establish primordial germ cell lines.By liposome method, PGCs was transformed by SAK gene, and cultured in vitro. With the development of PGCs, SAK was expressed in PGCs, which has a significant implication to the production of SAK protein in eukaryote cells.