The Coding DNA Sequence Cloning Of Hirudin Gene Into A Multicopy Vecor And The Expression Of The Cloned DNA In Pichia Pastoris

Natural hirudiin is a kind of small molecule active peptide secreted from the salivary of Himdo medicinals, a most strong anticoagulin so far, and it can effectively cure thrombus disease. While the resource of natural hirudin is limited, low production, high production cost, thus restricting the application in clinic. The development of recombinant hirudin by using gene technology can hurdle the shortcome. With the intensive application of genetic engineering in the medical, recombinant hirudin get a lot of expression in bacteria and yeast. Pichia pastoris expression system is the best one.It has been reported that more than 400 kinds of exogenous protein were expressed successfully in Pichia pastoris.This article aims to build multi-copy expression vectors in order to get high expression strains and antithrombin activity of hirudin,for the study of hirudin and clinical applications.We use genome-wide chemical synthesis synthesize European medicinal leech (Hirudo medicinalis) of hirudin,which was cloned into the Pichia pastoris expression vector pAO815 vector. Pichia pastoris expression vector pAO815 was developed by Invitrogen as intracellular multi-copy expression vector,it could be integrated into the yeast cells by increasing the exogenous gene copy number to increase the production of exogenous protein expression, but because of the lack of secretion of the carrier signal, its expression product exists in yeast cells, thus making it easy to be degraded, reducing the expression of output at the same time does not facilitate the separation and purification products. In order to obtain a higher expression, the product easier separation and purification of the expression system, pAO815 and exogenous gene was transformed and constructed. In study, we used two vectors pPIC9K and pAO815, which are widely used yeast expression plasmids, the former is a secretory plasmid, upstream of the cloning site containing the yeast a factor signal peptide coding sequence to the cellular protein could be secreted things, greatly simplifying the protein purification methods;the latter is intracellular expression plasmid, provided the conditions for building multi-copy plasmid, making expression vector copy in building when it is more greatly enhanced the integration of multiple copies generated when the probability to enhance the expression of material production.First we synthesize hirudin then cloned it into the Pichia expression vector pPIC9K. And then design primers to amplifyα-facor-Hirudin from pPIC9K-Hirudin inserted into the vector pAO815, thus pAO815 have become secreted from the expression of intracellular expression.Then, multi-copy plasmid was linearized and transformed Pichia pastoris GS115, PCR screening expression of multiple copies of strain, after methanol induction and optimal expression condition, induced by methanol at 30℃120h of the medium the expression of recombinant hirudin reach peak. Urea containing SDS-polyacrylamide gel electrophoresis showed that medium containing a single molecular weight of 15kDa protein band, although with the calculated molecular weight of recombinant hirudin 7kDa do not match, but after analysis and activity test which we believe hirudin with the active form. Activity test showed that the leech has a pretty good expression of the anticoagulant activity, anticoagulant activity reached 1600ATU/ml.Finally,we got the hirudin secreted high expression strains, and obtained with anti-thrombin activity of expression product, as hirudin and clinical application of foundation.