Cloning And Expression Of Mast Cell Degranulating Peptide Gene From Apis Mellifera

Honeybee venom is a wide range of natural ancient and modern drugs, a wide range of medical value, such as anti-inflammatory, anti-radiation, antibacterial, anti-cancer, sedation, desensitization, and lower blood pressure, such as immunosuppression, effective in the treatment of rheumatoid arthritis, inflammation, cardiovascular and other diseases. Some honeybee venom on the membrane composition, ion channels, receptor, calmodulin, enzymes of unique features, has become a common international standard biochemical reagents; In addition, as with insecticidal honeybee venom, anti-bacterial and Inhibition of virus activity, but also an effective biological pesticide.Bee venom as an important anti-inflammatory substance, the clinical has been widely used for centuries, the modern medicine that bee venom contains a variety of substances, the most important peptide material of which is Melittin and Apamin. In recent years people isolated from the bee venom another important peptide: mast cell degranulation peptide. Mast cell degranulating peptide has a variety of important immunology and pharmacology effect. In particular, its similar to the allergens, can be used as a study on inflammatory cells, interleukin cells and basophils tablets cells of the secretion of mechanism, so treatment can be developed with potential drugs. It is recognized that the potential of its clinical application. As conventional separation steps not only cumbersome, and the yield is not high, and lower purity of bee venom, bee venom caused prices high. So through the method of molecular biology mast cell degranulation peptide expression has broad prospects. Development and Utilization of bee venom resources, it can not only increase the income of beekeeping, and promote the development of bee-keeping industry, but also to create greater social and economic benefits.Mast cell deganulating Peptide also known as peptide 401, which is a 22 amino acid containing alkaline polypeptide, accounting for the entire bee venom of 1%-2%, molecular weight of 2.48 kDa. MCDP molecular contains two arginine, five lysine, five hydrophobic amino acids and a phthalocyanine-amine end of the spindle. MCDP was purified by the integrity of its sequences showed that theα-helices with a complete structure of a three-dimensional spherical structure, molecular surface distribution of eight positive charge and isoelectric point of 12. As MCDP contains four cysteine residues, and this will in the internal molecular disulfide bond formation of the two, one in the 3rd and the 15th amino acids between, and another at No. 5 with the 19th amino acids , so that has two elements closely linked to the asymmetric double loop structure. Experiments show that MCDP biological activity is determined by its unique amino acid skeleton of the decision, the three-dimensional structure of biological activity is that it must, when the two disulfide has been opened or carboxymethylation, MCDP biological on the activity affected, replacement of the two arginine residues can also significantly affect certain biological activity; MCDP molecular changes in the five lysine residues of theε-subgrade Mission ammonia can also to the loss of activity. Thus, a relatively MCDP elements in a number of different functional areas, the distribution of elements in different parts and different elements in the implementation of its regional variety relative biological effectiveness can play an independent role.Mast cell deganulating Peptide is a peptide antagonistic activity of the peptide. Preliminary research suggests that a two mutually antagonistic MCDP the biological effects, on the one hand, it has obvious anti-inflammatory effect, on the other hand, low concentration of MCDP can prompte Mast cells threshing, caused inflammatory response. In addition, there are two important pharmacological properties, a role in the central nervous system (CNS), to block voltage-dependent potassium channel, a nerve toxin, and the other is the impact on the cardiovascular that can significantly lower blood pressure in rats.Based on the above, we launched Mast cell deganulating Peptide gene cloning and expression studies to accelerate our research and the use of bee venom provide a theoretical basis, and will also create a bioreactor using bee venom production of biological products of genetic engineering industry for theoretical basis.Main results are as follows:1,by RT-PCR method cloned precursor MCDP gene, the sequence of 342 bp, including a complete open reading frame and 3'non-coding region of 189 bp nucleotide sequence.2,using GST fusion gene expression in E. coli BL21 system to the mast cell deganulating peptide gene was highly expressed. Expression products by SDS-PAGE analysis, in about 29 kD, a specificity of the expression of the zone, with the expected molecular weight of the same size.3,the conditions of the optimization results show that: pGEX- MCDP induced expression of OD600, IPTG concentration, temperature and time have a certain selectivity. Through the optimization of conditions in the cell concentration OD600=0.8 when induced by adding IPTG induction of the final concentration of 0.4mmol/L, 25℃induced expression under four hours, it can be highly efficient protein expression, the expression protein of the soluble protein.4,by using GST affinity chromatography system successfully purification the GST- MCDP fusion protein, for the future experiments provide the basis.