Recombinant Expression Of Endotoxin Cry4BLB From Bacillus Thuringiensis And Evaluation Of Its Effect On Flies Eradication

In the livestock and poultry breeding industry,the raging of house flies has a bad impact on the production of livestock and poultry.Chemical pesticides usually were used kill house flies in farmer,but the housefly population cannot be controlled.In addition,chemical pesticides contain a large number of toxic components that are difficult to degrade.With long-term usage of large doses of chemical pesticides,it may even endanger the surrounding environment of the farm and the health of humans and animals.Biological insecticides are mainly divided into three categories:Bacillus thuringiensis,insect viruses and plant extracts.Compared with chemical insecticides,they have low toxicity,low residue,easy degradation,and are unsuitable for drug resistance.Selective and other characteristics will not cause adverse effects on the health of humans and animals and the ecological environment,meet the needs of ecological development,and have broad application prospects.B.thuringiensis,as a new type of biological insecticide,has received great attention at home and abroad due to its wide source,low cost,high efficiency,safety and non-polluting characteristics,but it keeps the stage of research and development and application now.The endotoxin produced the effect on killing the target insects which is located at the midgut epithelium,and it damages the intestinal wall and increases the blood p H,which makes the infected pest neurological disorder,unable to eat and eventually leads to death.In this project,the evaluation of the killing effect of the endotoxin Cry4BLB gene of BTi on adults and larvae of housefly was carried out.The experiment proved that the endotoxin Cry4BLB gene of B.thuringiensis Israel subsp.Israel has a strong killing effect on the adult and larvae of housefly.In this experiment,the preserved.B thuringiensis subsp.Israel was recovered and purified,and its genome was extracted;the Cry4BLB gene was amplified by PCR technology,and the recombinant plasmid p ET-28a-Cry4BLB was constructed,and transformed into the competent cells Trans-1,after identified by PCR technology,the PCR product were sequenced and the sequence was analyzed by blast tool to evaluate its homology;Then the recombinant plasmid p ET-28a-Cry4BLB was transformed into E.coli Rosetta competent cells to constructed prokaryotic expression plasmid,and the positive clones were screened by PCR technology.The target protein was expressed by using IPTG induction,and the expression product was identified by SDS-PAGE immunoblotting techniques respectively;The toxicology test was made with the recombinant protein Cry4BLB against housefly to evaluate its killing effect.The result showed that the target gene Cry4BLB was successfully amplified in this experiment,and its size was about 1947bp.The recombinant engineering bacteria Cry4BLB was constructed in the living cells,and after induction by IPTG,the target protein was successfully expressed,and its fusion protein was about 77KD.It was2.831×10⁶cfu/piece,but the killing effect on fly maggots was better,and its LD₅₀was2.018×10⁵cfu/piece.Therefore,the recombinant protein Cry4BLB can kill houseflies and fly maggots at a certain degree,it will lay a foundation for the development of new biological insecticides.