Aim: To construct the human endostatin gene, which is the angiogenesis inhibitor, into non-fusion expression vector in Escherichia coli. Methods: The experiment was completed in the Laboratory of Medicine and Molecular Biology, School of Life Science, Sun Yat-sen University from February to December 2004. The expression plasmid was stored in the Laboratory of Medicine and Molecular Biology of Sun Yat-sen University, liver cells were obtained from the induced fetal liver in Zhujiang Hospital of Southern Medical Unviersity. Human endostatin gene was acquired by means of reverse transcription-polymerase chain reaction, it was cloned into PGEM-T vector and ligated at 16 °C for 16 hours, the ligation production was transformed into Escherichia coli DH5α, and the obtained clones were amplified and cultured, and the obtained plasmid was identified by EcoR I and BamH I, restriction enzyme. The recombinant plasmid with inserts were denominated as PGEM-Tendo, its positive clone was amplified and cultured, plenty of plasmids were extracted; T7 and SP6 were taken as sequencing primers for forward and backward sequencing identification automatically. PGEM-Tendo plasmid and expression vector pBV220 were orientationally connected after EcoR I and BamH I restriction enzyme, and then transformed into Escherichia coli DH5α, positive clone was identified with polymerase chain reaction, and denominated as pBV220-endo. Tbe DH5α engineering bacteria containing pBV220-endo was cultured at 30 °C by a small amount, and was collected after heat inducement at 42 °C for 4 hours; Polyacrylamide gel electrophoresis was performed on the collected bacteria and that before inducement. The bacteria body was collected after centrifugation and sonication, Polyacrylamide gel electrophoresis analysis of the pellet and supernatant was processed. Results: Endostatin gene including restriction enzyme sites obtained by reverse transcription polymerase chain reaction was 573bp. It was digested by EcoR I and BamH I restriction enzyme, the endostatin segment at 552 bp and PGEM-T vector at 3 000 bp could be observed on 10 g/L agarose gel electrophoresis, it was indicated that Endostatin was successfully constructed on PGEM-T vector. After automatically forward and backward sequencing, Genbank analysis proved that the obtained 552 bp gene fragment belonged to the functional section of human endostatin gene, the aminophenol code base at the 471 mutated from G to A, but the coded amino acid was still arginine. Polyacrylamide gel electrophoresis analysis showed that specific protein strap was observed, the size was concordant with that of endostatin protein. The non-fused recombinant proteinendostatin mainly expressed as non-dissolvable inclusion body, and the amount of expression was 14.5%. Conclusion: The cloned Endostatin gene of 552 bp has almost the same gene sequence with the endostain cDNA of collagen XVIII in GenBank, which indicates that human endostatin gene obtains non-fused expression in Escherichia coli, it lays an experimental methodological foundation for the anti-angiogenesis therapy with endostatin gene.
|Original language||English (US)|
|Number of pages||3|
|Journal||Chinese Journal of Clinical Rehabilitation|
|State||Published - May 21 2005|
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