Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Cloning and Expression of Cytotoxin Ⅲ from Naja Naja Atra and Its Mechanism on Inhibition of Tumor Cells

Author ChenXingYong
Tutor XuKangSen;LiuJing
School University of Science and Technology of China
Course Biochemistry and Molecular Biology
Keywords Chinese Cobra Cytotoxic Fusion protein pPIC9K HepG2 cells K562 cells Apoptosis Allantoic membrane
Type PhD thesis
Year 2009
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The main component of the venom mixture with a variety of bioactive proteins and peptides. Some of the protein as a neurotoxin, cytotoxic and tissue necrosis-inducing agent having a lethal effect, others can cause a variety of different pharmacological response, and toxicity is low. All of these protein toxins can attack certain specific sites in cells and affect physiological processes. Of Chinese Cobra the cytotoxic (Cardiotoxin CTXS) is a class with alkaline cell membrane polypeptides, cause a variety of pharmacological effects by binding to the surface of the cell membrane phospholipids or membrane proteins. In recent years, the purification of various Cobra cytotoxic component has been widely used for the treatment of solid tumors and blood diseases. The purpose of this paper is to study the cytotoxic component 3 (CTX III) Cloning, expression and purification of recombinant cytotoxic activity analysis and cell toxin as a feasibility study of the clinical anticancer drugs. Mainly include the following three parts: the study the CTX Ⅲ of the physical and chemical nature, and determination of the N-terminal 15 amino acid residue sequence cloned CTX III gene. Respectively loaded two prokaryotic E.coli expression vector and the eukaryotic yeast expression vector for expression. CTX III is expressed in the pET30a vector in the intracellular formation of inclusion bodies, the presence of an insoluble form. The CTX III gene into pThioHis B carrier, and thioredoxin (Trx) fusion expression, the fusion protein can be released from the sites of cleavage by enterokinase. On a laboratory scale, per liter of culture broth can be expressed 7mg recombinant cardiotoxin, purified and recovered by the HisTrap column, yields of up to 75%, the enterokinase cleavage gel column recoveries of 70%, about 37% of the total recovery rate. CTX III gene inserted into the eukaryotic yeast secretory expression vector (PIC9K), induction of recombinant protein expression level of 9.5 mg / L, the culture liquid gel column directly after centrifugation recovering the recombinant target protein yield up to 65%. Compared to natural toxins, recombinant protein N-terminal redundant three amino acids (Gly-Tyr-Thr), RP-HPLC analysis of the recombinant protein purity greater than 95%. Hemolytic activity and K562 tumor cell proliferation inhibition of detection of of recombinant cytotoxic and natural cytotoxic activity, the hemolytic score corresponding to the mass concentration of 50% (HU50) 6.53μg/ml and 7.4μg/ml, 12-hour IC50 for 3.56μg/ml 2.63μg/ml show that the activity of the recombinant protein close to the natural protein, suggesting that correct folding of secretory expression of recombinant proteins. The choice of the eukaryotic yeast secretory expression vector for optimal expression. 2, using the method of analysis of different biological killing effect of cytotoxic in vitro cell line K562 in blood diseases through the inhibition of cell proliferation, Annexin V / PI double staining experiments, Caspase activity assay and DNA fragmentation was observed CTX III-induced apoptosis of K562 cells situation. The results show that the the CTX III role in K562 cells allows cells to arrest in the G2 / M phase and activation of Caspase-8 and cytochrome C dual signaling pathways induced downstream Bid molecules cutting, prompted K562 apoptosis. MAPK specific blocker and Western blot analysis showed that CTX III-induced apoptosis of K562 cells, activation of MAPK signaling pathway, through phosphorylation of p38 and JNK protein. Chorioallantoic membrane (CAM) K562 tumor models in vivo experiments, the high-dose CTX III does not affect embryo development, indicating that CTX III non-toxic side effects, CTX III can also effectively inhibit the CAM on the growth of K562 cells formed tumor nodules, a process that associated with Caspase-8 and cytochrome C pathway. By analyzing the formation mechanism of the the K562 blood disease that CTX III may be combined through a competitive ATP, thereby blocking the abnormal tyrosine kinase activity in K562 cells. 3, the paper also analyzed the effect of the of CTX III role in the application of solid tumors, liver cancer, in lower doses, CTX III can effectively inhibit the proliferation of HepG2 cells (24 hours ICs 50 = 2.58μg/ml) but had no obvious effect on Bel-1402. Further studies showed that DAPI staining and Annexin V / PI double staining experiments and DNA fragment analysis prove that the CTX III promote apoptosis, the process of apoptosis is regulated by the death receptor pathway and mitochondrial pathway are two mechanisms. CTX III's early role in HepG2 cell cycle arrest in the S phase, semi-quantitative PCR and western blot found that cyclin protein cyclin D1, cyclin A and cyclin E in the process of transcription and translation, the protein and mRNA levels were lowered, Bcl The proportion of protein and mRNA in -2/Bax always make changes. Tail vein injection of CTX III cause rapid death of mice, the LD 50 to 936.7 ± 38μg/kg. Nude mice in vivo the HepG2 tumor models, the results show that oral administration, CTX III (2 mg / kg / d) can significantly inhibit tumor growth inhibition rate of up to 39.4%. Pathological examination of the heart, liver, spleen, lung, kidney and small intestine and other organs and found no hemorrhage or necrosis, indicating that CTX III animals by oral non-toxic side effects. CTX III in vivo, the outer role can effectively inhibit the proliferation of tumor cells (K562, HepG2), the eukaryotic yeast expression of the resulting recombinant cell toxins exhibit similar biological activity of natural toxins, therefore, the use of recombinant expression techniques not only provide a source for the venom anticancer drugs, and protein structure - function relationships provide a wealth of material for research.

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