Cloning And Expression Of Melittin Gene From Apis Mellifera

Bee venom is a complex mixture and a complex aromatic smell,bee venom components are very complicated and have not yet been fully understanded.The major component of bee venom is polypeptide,enzyme,alkamines and other substances.Bee venom plays anti-arthritis role by stimulating pituitary function and promoting the secretion of adrenocorticotropic hormone.Bee venom has shown the character of non-Steroid anti-inflammatory.It has distinct curative effect in anti-beriberoid disease,antiinflammatory,depressurization,And has already achieved in the clinical recognition.But it also need futher study on the pharmacological effects of different composition.Melittin,a main polypeptide component in the bee poison,it takes about40﹪～50﹪parts in bee venom,and has important medical value.From the frame we can know that the T-terminal 20 amino acid residues are hydrophobic and the 6 amino acid residues in the C-terminal are hydrophilic.Under normal circumstances,the C-terminal four amino acid residues has positive charge and the twe in T-terminal takes positive charge.The whole molecule takes six positive charge.The three elements of lysine and twe arginine residues makes bee venom melittin to be a strong alkaLane peptide.In neutral aqueous solution,Melittin is a random monomer curly Structure,but along with changing of pH and ionic strength,Melittin self-crosslinking,a spiral structure of the tetramer.Recent research has found the spiral helical structure of the region is different in different Bee venom solution。The first 21 amino acids of Spiral structure is polarity,they are in the face of spiral,rather than polar amino acids on the other side of the helical.Therefore, this decided melittin can dissolve in water,be combined with the natural membrane, and dissolving cells.Melittin is an important anti-inflammatory substances,it is one of the strongest anti-inflammatory activity so far as we knew.Melittin mainly used to treat arthritis,and other inflammatory,using bee venom instead of bee venom can stimulate the pituitary function.The antibacterial activity of melittin has attracted the attention of people.It also show a high degree of toxicological effects to the tumor cells,and it also can prevention and treatment of radiation,he survival rates of the radiation of animals can significantly improve.In addition,Melittin has a strong surface activity,the speed of permeating lecithin membranes and membrane lipid mixing beyonding any other surfactant, this nature to become an important tool for biofilm researching.Therefore,it is an important raw materials in medicine,agriculture and biological engineering, animal diseases and plant protection treatment.Venom gland of bee is a highly specialized abdominal organs,a tubular structure,composed by the multi-layer cell rich and rough endoplasmic reticulum,the venom of the bottom connected to a swelling of the poison capsule. Melittin is synthesisded as prepromelittin in Venom gland.In the Venom gland of bee,a signal peptide precursor protein is first be synthesized, the venom protein secreted into the extracellular become Melittin precursor protein.bee venom peptide precursor protein is hydrolysised by dipeptidaseⅣbecome melittin and storage in capsule.The bee venom peptide precursor protein has a better dissolubility,thereby reducing the destructive power of the film,which is in the long-term evolution of the venom formed the mechanism of protection of its own.So far,melittin has been mainly obtained by separating and extracting firom bee venom or by chemical synthesis.The spreading of melittin was limited because of the following twe reasons first,the purification difficulty caused by the phospholipase A2,second, the higher cost of protein chemical synthesis The development of biotechnology rovides a new way for melittin production.There is no report about useing genetic engineering methods to express melittin so far.The reason may be that it is difficult to detec small molecules of melittin and it can also kill the genetic engineering expression system.For the reason about,in this study we construct a glutathione transferase(GST)fusion expression vector pGEX-MELmel,the fusion protein was expressed in Escherichia coli.This study established a glutathione transferase(GST)fusion expression vector pGEX-mel,the fusion protein was expressed in Escherichia coli.This is not only can solved Melittin on the werks of death but also efficient expression and accurate detected of recombinant protein.In protein engineering,E. coli was the most extensive system applications,it was widely studied and has a variety of advantages: security,and has a clearly background,growth cycle short,simple operation,easy access to high expression,small size flask culture expression Characteristics of the method can be well applied to large volume commercial production or training.In E.coli expression of heterologous proteins tend to form insoluble bodies inclusion,a series of degeneration process should be needed.ecause of the complex folding method to complex,high cost,so we are soluble fusion protein expression.This study we take Italy bee bee venom for raw aterials,extracted, bee venom total RNA amplification Melittin gene by RT-PCR,it contains a complete open reading frame and 3 'non-coding sequence of 91 bp Nucleotide.we wil be clone it into the vector of pMD-18T, Identificatde it by double enzyme digested and PCR.We Construct Melittin GST fusion expression vector pGEX-MEL ,and transformed recombinant plasmid into E. coli BL21 for fusion expression.After GST affinity chromatography ,we analyse it by SDS-PAGE analysis, the 29 KD has a specific band, with the expected molecular size .It show that bee venom peptide and GST fusion protein in E. coli species have been expressed. By grading-up the conditions of the expression ,we make the most protein of expressed can be solubled.