Studies On Cloning And Prokaryotic Expression Of Trypsin-like Serine Protease CDNA Of Yellow Mealworm (Tenebrio Molitor)Larvae

With the development of society and the improvement of people’s living standard, due to A type of common cardiovascular and cerebrovascular disease which cased by thrombosis,2million people die while3million people need medical care. The Mortality rate up to40.7%and become the biggest reason of death, even Transcend the cancer. Thus, cardiovascular and cerebrovascular disease have become one of biggest threaten of human health. Thrombolytic is prevention and treatment of disease of heart head blood-vessel antithrombotic drugs, mainly through the fibrinolytic enzyme original activators activate fibrinolysis enzyme original into fibrinolysis enzyme, fibrinolysis enzyme catalysis of the thrombus main matrix fiber protein hydrolysis; And some direct role in protein fiber, make its hydrolysis, achieve finally make the purpose of recanalization. Promptly dissolve suppositories treatment, make block revascularization, vital organs quickly get to the blood reperfusion, stem the happening of the viscera fail is very good treatment. So, looking for good effect and side effects and the development of small fibrinolytic therapy drugs, to the more effective treatment for the serious harm human health of disease of heart head blood-vessel has the very vital significance.Tenebrio molitor L. include variety types of protein, amino acid, lipid, Trace elements and vitamins. In the passed test, Health care products from Tenebrio molitor L have the effect on improving immunity, Fatiguing resistance, lower blood lipids, anticancer, promoting the new superseding the old. Tenebrio molitor L has been used as feed, but little report about the efficiency protein. Nowadays, just a little reports about the isolation, effect and gene studies of the Tenebrio molitor L trypsin-like serine proteinase (TMTLSP), and little report about its cloning. In this study, the full-length cDNA of TMTLSP is cloned and Prokaryotic Expressed.Firstly, two methods are used to isolate the total RNA. Concentration, purity, integrality are compared. Thus, high quality total RNA is obtained.Secondly, the template cDNA which was reversely transcribed from the total RNA of Tenebrio molitor larvae, was carried out temperature gradient Polymerase Chain Reaction (PCR) with the degenerate primers of Eupolyphaga sinensis fibrinolytic enzyme. The full-length cDNA, achieved with Rapid Amplification of cDNA End method (RACE) from the amplification product, was named Tenebrio molitor Trypsin-like Serine Protease (TMTLSP). TMTLSP includes869basic pairs (bp)(GenBank accession No. JN6624614) with the open reading frame of777bp and258coding amino acids. It also has the unique Trypsin-like initiation sites and expected binding sites of substrate active sits. The results of comparative analysis illustrated that the gene encoding of amino acid sequence was highly similar as those within a variety of insects.Thirdly, pET28a (+) vector and E. coli BL21are used in the Prokaryotic Expression of TMTLSP. It also is purified and initially renaturation. Based on the results, this study could be considered as a valid reference for the further investigation of Trypsin-like serine protease extraction and examination.