The Human Endostatin And Mouse Endostatin Gene Syntheses And The Prokaryotic Expression And Function Verification

Objective:Endostatin originally obtained from the isolation and purification of the mice hemangioendothelioma cells medium, the endostatin is a C-terminal fragment of collagen 18, it’s inhibit angiogenesis through inhibiting proliferation of the vascular endothelial cell but doesn’t influence the function of the mature vessel, therefore it’s an ideal new angiogenesis inhibitors, clinically used to inhibit tumor angiogenesis in order to achieve tumor treatment, the recombinant endostatin mainly acquired through the E.coli prokaryotic expression system and mostly exists in the inclusion body.The human and mouse endostatin gene is highly homology and has the similar biological function, the article will determine the human and mouse endostatin gene sequences and investigate the soluble expression problem of the endostatin through construct prokaryotic expression vectors with different protein tags, finally confirmed the inhibition effect of hunman recombinant endostatin to angiogenesis.Methods:according to reports in the literature and NCBI database to determine the Human Endostatin (HE) and Mouse Endostatin (ME) cDNA sequences, they were obtained through DNA synthesis respectively, add in different enzyme sites respectively and design primers, recycled after PCR amplification, the Human Endostatin (HE) double enzyme processing with prokaryotic expression plasmid pGEX-4T1 which carry GST label, the Mouse Endostatin (ME) double enzyme processing with prokaryotic expression plasmid pET-32a which carry TrxA label, then T4 DNA ligase processing respectively, constructed prokaryotic expression plasmid pGEX-4T1-HE and pET-32a-ME, then expressed through induction in escherichia coli BL21 (DE3) with IPTG, determining the inducing condition of the Human Endostatin and Mouse Endostatin, disposing the induced bacteria with ultrasonic method and centrifugate to get the supernanant and sediment then proceeding SDS-PAGE to determine the protein distribution, through washing, denaturation, renaturation, and concentration of the inclution body to get the recombinant protein, finally confirmed the inhibition effect of hunman endostatin to angiogenesis by chick chorioallantoic membrane test.Results:sequencing analysis shows that the gene synthesis of the Human Endostatin and Mouse Endostatin successed determined by PCR amplification, both of the gene is about 560bp,conforms to the theory,through the molecular cloning methods connect the Human Endostatin and Mouse Endostatin gene to prokaryotic expression plasmid pGEX-4T1 and pET-32a respectively, affirmed by PCR, double enzyme digestion and sequencing analysis, the prokaryotic expression plasmid pGEX-4T1-HE and pET-32a-ME was successful constructed, the inducing expression of the Human Endostatin and Mouse Endostatin was successful in escherichia coli BL21(DE3) at 37 ℃ with 1 mmol/L IPTG.through the SDS-PAGE analysis, compared with the blank group, the experimental group appears the purpose stripe, the Human Endostatin recombinant protein is about 46 kDa and the Mouse Endostatin recombinant protein is about 39 kDa, coincide with the theory, the recombinant protein mainly exists in inclusion body, there is no recombinant endostatin protein exists in supernanant of the ultrasonic bacteria liquid. Through washing, denaturation, renaturation of the inclution body to get the the Human Endostatin and Mouse Endostatin recombinant protein. In chick chorioallantoic membrane test with the human endostatin recombinant protein, compared with the controls, the test group has a lesser blood vessel number and thinner blood vessel diameter.Conclusion:the results showed that the length of 554 bp of the Human Endostatin gene and the length of 565 bp of the Mouse Endostatin gene have been synthesized successfully, The human, murine endostatin gene expression vectors with different labels was successful constructed and induced expression in Escherichia coli. The hunman and murine recombinant protein have been acquired successfully, the chick chorioallantoic membrane test prove the anti-angiogenesis activity of the purified refolded human endostatin recombinant protein. Our results also show that TrxA tags and GST tags have no difference in the increase of soluble expression of recombinant endostatin, both of the recombinant protein with different tags are mainly existed in inclusion body.