Study On Cloning Angiostatin Gene And Its Expressionin E.coli DH5α

Angiostatin(AS) is a kind of polypeptide identified as a product of plasminogen cleavaged by protease. It blocks neovascularization by directly inhibiting endothelial cell proliferation and migration. In this manner, Angiostatin inhibits the growth of primary and metastatic tumors in vivo. It is a promising drug for angiogenesis-dependent diseases especially in pre-clinical and clinical cancer trials. Angiostatin has already attracted intense interest for its potent action, low toxicity, broad-spectrum and the. absence of drug resistance. In our experiment, we use pEZZIS, a secretory and fusion plasmid vector, to construct the recornbinant plasmid containing the angiostatin gene and express it hi E.coli DH5a, then identify the recombinant protein.The first part of our experiment is to extract and purify the total RNA from the human liver tissue in the condition without RNase and to identify its integrity and purity. We amplify the angiostatin gene by RT-PCR and introduce the restriction endonulease sites of EcoR I and Xba I and their corresponding protective bases in the primers. In this part, we succeed in obtaining genes of angiostatin (Kringle 1-4 domains) and PLG Kringle 1-5 domains and sequence the PCR product of Kringle 1-5 domains. Except for a small uncertain fragment after the sequencing primer, the result is consistent with the corresponding sequence obtained from Gene Bank.The second part of our experiment is the construction of recombinant plasmid pEZZ18-AS. First, the purified pEZZIS and PCR product of angiostatin are digested by EcoR. I and Xba I. After purifying the digested products respectively, we ligate these two kinds of DNA by T4 DNA ligase and construct the recombinant plasmid pEZZ18-AS. Then transform it to the competent E.coli DH5a. We screen recombinatant clon through Amp+ LB plates. Then taking recombinant E.coli DH5a solution as template we do the PCR and perform single and double enzyme digestion for further identification. We sequence the inserted gene fragment of the indentified recombinant clone. The result is: Angiostatin gene ORF (Open Reading Frame) linkswith the ORF in expression vector correctly. But the first base of the codon AAA coding for Lys414 in PLG Kringle 4 domain mutates from A to G which leads Lys change to Glu.The third part of the experiment is preliminary expression of the recombinant plasrnid in Ecoli DH5a and the identification of the recombinant protein. We culture the Ecoli DH5a containing recombinant plasmid pEZZ18-AS and the Ecoli DH5a containing plasmid pEZZIS in the Amp+LB solution at the same time. When the ODsoo of the bacterial solution is 0.5, we add IPTG to the control tubes and make their terminal concentration to Immol/L. Then continue to culture them for another 2 hours. Collect the inducing and non-inducing E.coli DH5a by centrifuge and SDS-PAGE. There is no difference in the expression amount of the recombinant protein between the inducing and non-inducing E.coli DH5a. Besides, in comparison with the E.coli DH5a containing plasmid pEZZIS, there is a new band which is a little smaller than the 66 kDa standard protein in the E.coli DH5ct containing plasmid pEZZl 8-AS. This result is corresponding to the theoretical molecular weight of fusion protein ZZ-AS. In addition, after ultrasonic disintegrate the recombinant E.coli DH5a and E.coli DH5a containing pEZZIS we centrifuge them. Take these two kinds of supernatant to perform ELISA. There is statistics significance in the difference between the OD450 of the two groups. (P<0.01) That can certify the expression of the recombinant protein inthe E.coli DH5ct.In short, we succeed in the construction of the secretory expression plasmid pEZZ18-AS which contains angiostatin gene and express it in E.coli DH5a. We also confirm the expression of angiostatin in the recombinant E.coli DH5a by SDS-PAGE and ELISA. Our experiment undoubtedly lays a solid foundation for further relevant research.