Cloning And Expressing Of Arresten CDNA In E.CoLi And Pichia Pastoris, And Investigating It's Anti-Angiogenic Activity

The proliferation and metastasis of cancers depend on angiogenesis due to the need for the supplement of nutrition, or there will be no further growth due to starvation when the solid cancer grew up to 2 mm3. This property provided the feasibility for the treatment of cancer by inhibition of angiogenesis, and many angiogenic inhibitors, such as endostatin, angiogenestatin, tumstatin, etc, have been demonstrated to effectively inhibit angiogenesis and consequently the growth of solid cancer. Arresten is the 26kD c-terminal fragment of the globular domain of collagen IV. It mainly localized in the perivascular position around blood vessels, and was also rich in liver. Arresten also inhibited the proliferation of normal capillary endothelial cells. But its action on the proliferation of solid cancer was uncertain. In order to further characterize Arresten and to get its recombinant expression product, human Arresten was cloned and expressed. It have been found that the arresten cDNA at theα1 chain cDNA of the collagen IV showed complicated secondary structure, which hinder the amplification of the arresten gene by PCR. So we amplify the arresten gene with nested polymerase chain reaction (nested-PCR). At first, total RNA from healthy human liver was extracted, and then mRNA was reversely transcripted to cDNA with Oligo(dT)18 primers, and that of the α1 chain of the collagen IV was amplified by PCR with the corresponding specific primer designed with the aid of software Primer Premier 5.0 and RNASTRUCTURE3.5 to characterize its secondary structure. A new gene clone technology was used to construct the expression vector. The PCR product of Arresten was treatedby T4 DNA polymerase with its 3'-5'digestion action to expose the sticky end of interest, which can ligate with T4 DNA ligase to the fragment of plasmid pTYB1 digested by NdeⅠand EcoRⅠ. The pTYB1-arresten was used to transform E. coli ER2566 and the expression of arresten was induced by IPTG, and expression was analyzed by SDS-PAGE. Secondly, we use the classical method T-A clone to construct the vector pGEM-arresten. And it was digested after cloning with Xho Ⅰ and NotⅠto recover the arresten fragment, and then construct the shuttle vector pPIC9-arresten. pPIC9-arresten was used to transform E. coli for its amplification, then the vector was extracted with required amount. The vector was linearized by digestion with Sac I, then the linearized vector was introduced into pichia pastoris for integration into its genome, whose competence cells were promoted by PEG1000. The transformed cell was selected by conditioned medium BMGY, and the insertion of Arresten was confirmed by PCR. This recombinant pichia pastoris was induced by 1% methanol to express Arresten. The expression was analyzed by SDS-PAGE, and Arresten in the supernatant was partially purified to assay for its anti-angiogenic activity. Results: 1. The one-step clone method was successfully used to clone and express the arresten in E. coli. The maximal expression of recombinant arresten was obtained with final concentration of 0.5mmol/L IPTG at 30℃ after 5-hour induction. 2. The recombinant pichia pastoris can express the protein arresten, whose molecular weight by SDS-PAGE is consistent with that expected. 3. The plateau of arresten expression was achieved after 72-hour culture of the recombinant yeast at 30℃,with methanol maintained at 1% by supplement at 12 hours interval. 4. The supernatant after concentration with 50ug in total showed inhibitionactivity on tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S.Conclusion:1. The arresten gene was successfully cloned and expressed in E. coli with the PCR product treated by T4 DNA polymerase to generate the desired sticky end. 2. pPIC9-arresten was successfully constructed, and the recombinant pichia pastoris GS115 expressed the protein arresten efficiently. 3. The expressed a...