| Disintegrins are a family of small proteins mainly derived from snake venoms.Most of the disintegrins contain an Arg-Gly-Asp(RGD) or Lys-Gly-Asp( KGD) sequence and plentiful cysteines,and the molecular weight is 5~9kD [1]. Most cellular integrins can recognize RGD sequence,while protein component of most extracellular matrix (ECX) contain RGD sequence. So disintegrins become the strong competitive antagonists to the binding of ECX and cellular integrins by force of the RGD recognition sequence and inhibit many physiological actions that the integrins participate in.Adinbitor is cloned from Agkistrodon halys brevicaudus stejneger and characterized as novel disintergrin.In this study, according to the homologous protein sequence of disintegrins published on NCBI, we deduced the DNA sequences and designed two oligonucleotide primers.Total RNA was extracted from venom gland and used in RT-PCR to generate a cDNA,which is 219 bp long. Adinbitor is composed of 73 amino acid residues includng 12 cysteine residues and an RGD three-peptide disintegrin characteristic recognition sequence and its relative molecular weight is 9kD. So it has the inhibitory effect on tumor as the strong antagonists to many integrin receptors.Integrin is a wide range of animals and plants exist in the cell surface of a class of cell adhesion molecules, cells and cell-mediated extracellular matrix between cells and between cells and the interaction involved in the transmission of information both inside and outside cells, cell differentiation, embryonic development , coagulation, cell adhesion and repair organizations, such as the invasion and plays an important role. Integrin and the occurrence of cancer is closely related to the development, integrin can deliver specific signals or to control gene expression induced by tumor cell adhesion, proliferation, invasion and metastasis, and apoptosis, as well as the generation of tumor angiogenesis. Integrin all to have their biological activity must identify the specific RGD sequence, by virtue of this sequence can be used to integrate a variety of integrin receptor strong antagonist, inhibited tumor integrin in the process of the kind of role. Currently integrin has been found to inhibit tumor cells to fibronectin, Bose even protein, laminin and collagen adhesion, thus inhibit tumor cell growth, invasion and metastasis, but also to integrin can induce tumor cells down - The specific role of death.Yeast as a rising Exogenous expression system, as both prokaryotic and eukaryotic cell expression system characteristics, is the field of genetic engineering has been extensively applied, the present study is relatively mature Pichia pastoris (Pichia pastors, P. pastoris). Pichia pastoris is a single-celled eukaryotes the past 10 years, as was the eukaryotic expression system for foreign protein production. Adinbitor expression of the structural and functional closer to the natural Adinbitor is conducive to mass production.This paper will have constructed recombinant plasmid pPIC9K- Adinbitor in Pichia pastoris GS115 to the expression of integrin (rAdinbitor), after column chromatography purification and dialysis, access to the molecular weight of KD 9 to integrin (rAdinbitor), and B16 melanoma cell proliferation, apoptosis, adhesion, invasion and metastasis conduct a study. The experimental results show that rAdinbitor can also inhibit tumor cell proliferation, adhesion, invasion and metastasis, and also can be induced in a dose-dependent manner tumor cell apoptosis.In short, the success of this thesis through genetic engineering tools from China's Lushun mangabey snake venom to get a new integrin family members rAdinbitor, and its inhibitory effect on tumor cells were studied. The results showed that integrin to the biological activity factor, as a new type of anti-tumor substances for the treatment of malignant tumors has opened up a new field for the capture of human cancer provide new means. |
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