Cloning Serine Anticoagulation AL-174、AL-71Gene And Prokaryotic Expression Of AL-71Gene From Aedes Albopictus

Today,cardiovascular and cerebrovascular cerebral thrombosis disease is growing threat to human health and life,to explore and research antithrombotic drug has become a hotspot in medical circles all over the world.In recent years,although the drugs can inhibit platelet activity and dissolution of thrombus development, these drugs have some disadvantages,for example,side effects are a lot,the treatment is not remarkable,cycle is long,etc.So we still need to explore and research the anticoagulation drug which is more suitable for clinical application,As we all know,Mosquitoes transmit many diseases such as dengue fever、Japanese encephalitis、yellow fever、filariasis and malaria,it’s widely distributed in the South of China and Southeast Asia.When mosquitoes are sucking blood,the anticoagulant substances is secreted by saliva,these anticoagulant substances not only modulate host immune response,coagulation and inflammatory reaction help the smooth completion,but all also it is a group of potential pharmacological active substances.In mammals,antithrombin is mainly secreted by the liver cells,it is an important member of the serine protease inhibitor family,to maintain the dynamic balance between coagulation and anticoagulation has important significance.With the rapid development of molecular biology and genetic engineering,if we can realize safe and effective protease inhibitor extraction in mosquitoes to prevention,diagnosis and treatment of thrombotic diseases,we will provide a theoretical basis for the development of human anticoagulant and thrombolytic drugs.For exploring the treatment feasibility of anticoagulant substance of the mosquitoes to thrombotic diseases, this study successfully cloned the Aedes albopictus serine anticoagulant AL-174gene and AL-71gene, and the prokaryotic expression of AL-71gene,this Study built the foundation for mosquito anticoagulant substance.The results as followed:This experiment successfully cloned the Aedes albopictus serine anticoagulant AL-174gene,sequence is1383bp.After homology analysis,the sequence homology of Aedes albopictus serine anticoagulant AL-174gene is84.26%,amino acid sequence homology is82.57%.In conclusion,nucleotide sequence and amino acid sequence homology were high, it indicates that AL-174gene sequence is conserved in the mosquito species.From the position between the phylogenetic tree:Aedes albopictus serine anticoagulant AL-174nucleotide sequences in the phylogenetic tree trunk,the gene distantly relates with other mosquito species. At the same time,first structure and Secondary structure transmembrane region and tertiary structure model of AL-174were predicted by biological analysis software. Result showed AL-174protein structure contains two disulfide large, alpha-helix,beta-sheet and random coil,Six cysteine molecules to connection mode of1～6、2～4、3～5three of two disulfide bonds,The active site contains His、Arg and Ser, Multiple sequence alignment of the catalytic domain showed high similarity,And the catalytic site amino acid residue with serine protease inhibitor gene sequence is consistent.This experiment successfully cloned the Aedes albopictus serine anticoagulant AL-71gene, sequence is1362bp. After homology analysis,the sequence homology of Aedes albopictus serine anticoagulant AL-71gene is88.85%,amino acid sequence homology is90.24%.In conclusion,nucleotide sequence and amino acid sequence homology were high,it indicates that AL-71gene sequence is conserved in the mosquito species.From the position between the phylogenetic tree:Aedes albopictus serine anticoagulant AL-71nucleotide sequences in the phylogenetic tree trunk,the gene distantly relates with other mosquito species.At the same time, first structure and Secondary structure transmembrane region and tertiary structure model of AL-71were predicted by biological analysis software.Result showed AL-71protein structure contains two disulfide large, alpha-helix, beta-sheet and random coil,Multiple sequence alignment of the catalytic domain showed high similarity,And the catalytic site amino acid residue with serine protease inhibitor gene sequence is consistent.We successfully constructed the Prokaryotic expression vector PET-AL-71,and expression vector can express target proteins by IPTG induction.The SDS-PAGE results showed that the recombnant protein molecular weight is about50KD, the final concentration of0.8mmol/L IPTG,High expression of protein induced by5h at37℃.