Study Of High Efficient Secretory Expression Of Endostatin Via Adenoviral Vector

OBJECTIVEIn this study, we would construct four recombinant adenoviral vector containing human endostatin gene leaded by a series different modification and optimization sequence based on IL-2 signal peptide, and to compare the secretory expression level of endostatin gene. To lay a foundation for product a long-term, stable, low side effects adenovirus-mediated human endostatin gene therapy drugMETHODS1 Primarily,We obtained 4 kinds of Endostatin gene,which is respectively followed by four different sigal peptide sequence:wild-type IL-2 signal peptide, optimization-based IL-2 signal peptide, Kozak sequence combined with wild-type IL-2 signal peptide, Kozak sequence combined with optimization-based IL-2 signal peptide individually;2 Secondly constructed four types of co-transfection of recombinant plasmid pDC315 (io)-IwE, pDC315 (io)-ImE, pDC315 (io)-KIwE and pDC315 (io)-KImE;3 Via "HI-IQ" recombinant system, obtained four different recombinant adenovirus: rAd-IwE, rAd-ImE, rAd-KIwE and rAd-KImE, and then amplified, purified and determinated theire titer respectively;4 The four recombinant adenovirus vector was expressed in the NCI-H460 cells,and then compare the four expression levelsRESULTS1 Obtained four signal peptide-guided Endostatin gene;2 Constructed four types of recombinant plasmid including:pDC315(io)-IwE, pDC315(io)-ImE, pDC315(io)-KIwE and pDC315(io)-KImE;3 Obtained four recombinant adenovirus including rAd-IwE, rAd-ImE, rAd-KIwE and rAd-KImE, and the titer of virus are 3.9×108,3.7×108,3.1×108 and 3.5×108 respectivley;4 the results of comparision shows that Endo can be highly effectively experessed by both Ad-ImE and Ad-KImE,the highest expression is 3 times and 4 times higher than of Ad-IwE respectively.CONCLUSIONSWe obtained a more efficient secretion of endostatin-mediated expression of recombinant adenovirus vector.