Cloning, Expression And Activity Detection In Pichia Pastoris Of Trypsin-like Serine Protease CDNA Of Tenebrio Molitor Larvae

Tenebrio molitor L. in insect taxonomy belonging to Coleoptera, Tenebrionidae, Tenebronini. At present, the related research has shown that Tenebrio molitor L. contains variety types of important biological active substances, including protein, amino acid, lipid, trace elements, and vitamins and so on. 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. For the moment, research about Tenebrio molitor L major on its biological characteristics and artificial breeding technology, as a kind of high protein nutrition, to study the function of protein is rare. In the foreign country, it has caused widespread concern. Some related researches of its digestive enzyme, and a few of its antimicrobial and thrombolytic effects. 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. This study is based on the fibrinolytic enzyme gene cloning early in the research group, to further discuss the TMTLSP gene cloning and expression in Pichia pastoris. Pichia Pastoris is a methylotrophic yeast which has an ideal secretion system and post-translational processing mechanism so that it can express heterologous proteins effectively, especially eukaryotic proteins.In this thesis, TMTLSP fragment was firstly obtained by PCR amplification using a plasmid which contains TMTLSP cDNA as a template; the amplified fragment was then cloned into P. Pastoris expressing vector pPIC9K. Correct by sequencing, obtaining a recombinant expression plasmid pPIC9K-TMTLSP. The plasmid linearized and then transformed in P. Pastoris cell GS115by electroporation. The positive recombinant was screened and confirmed by PCR.A single colony was used fermented which grew at30℃in a shaking incubator. Cultured and induced methanol to a final concentration of0.5%every24hours. At the same time, take the sample and transferred the supernatants for freezement in-80℃. After144h. the supernatants were analyzed for protein expression by SDS-PAGE. The results showed that pPIC9K-TMTLSP/GS115showed26KD in SDS-PAGE atlas, but empty including pPIC9K can’t product the protein. After dialyze purified fibrin-plate method showed that the products had activity.Tenebrio molitor L digestive enzyme is a new and strong thrombolytic protease, is of great significance to star its study of eukaryotic gene expression. This research aims to construct the TMTLSP recombinant with P. Pastoris express system and for the next shift become the foundation of genetic engineering drugs.