Expression Of Human Endostatin In Cultured Cells And Larvae Of Silkworms(Bombyx Mori)

Human endostatin is a novel antiangiogenic molecule,which can inhibit the proliferation and development of new blood vessels,and cause nearly complete regression of established tumours in experiments．The recombinant human endostatin has a potent inhibitory activity in vitro and in vivo angiogenesis assays,It has no evidence of toxicity,also has no resistance to therapy in animal study．Therefore,recombinant human endostatin has a good prospect as a new bio－medicine in human anticancer therapy． Baculovirus expression vector system(BEVS)is a high－level eukaryotic expression system founded in 1980's．Up to now thousands of genes have been high expressed by or using this system．Silkworm(Bombyx mori)BEVS has some additional advantages as comparedto normal BEVS． In this paper,the cDNA encoding human endostatin was cloned into pBacPAK8,a baculovirus shuttle vector．The recombinant shuttle vector pBacPAK－Endo was co－infected with Bm－BacPAK6 DNA into BmN cells．The recombinant virus was screened,plaque－purified and identified by PCR and DNA dot hybridization． BmN culture cells were infected with the BmBac-Endo at a MOI of 10．The cells were collected after 72 hours,then lysed with PBS for preparation of cell extract．SDS－PAGE analysis indicated that the LacZ gene has been replaced by endostatin gene in the recombinant viruses．Biological activity assay results showed that the expression product of rBm－BacEndo has a high level of biological activity,the rate of inhibiting endothelial cells proliferation can reach 90．1％．In ELISA assay, the expression product of cell can show activity with 53 P／N value of4．3． The recomblnantvlrus was also expressed Inthe larvae ofsllkworm by Inoculation ofrecomblnant virus Into the sib Instar silkworm．Hemolymph of the Infected larvae was collectedevery day．The ELISA assay showed a maximum activity In day 5二 theblologlcal actlvltyassay showthatthe expresslonproductln larvae Is about 50－fold hlgherthanthat expressed In culturedcells．as expected,SDS－PAGE andwestern blottinganalysls showed a pattern ofmolecular weight about 20kD．...