Expression Of Hyaluronidase HPH20in Arabidopsis And Safflower Oil-body System

With the development of genetic engineering techniques, the use of plants to producepharmaceutical proteins has become an area of great interest. Using plants as expression systemsfor the production of recombinant proteins has distinct advantages, such as safety, ease, low costand high yields. Recombinant protein could long-term stably without degraded in seeds.Hyaluronidases (Hyals) were widely distributed in animal tissues and microorganisms.They were a class of glycosidases that predominately degrade hyaluronic acid (HA), withlimited ability to degrade chondroitin and chondroitin sulfates. HAase widely used in theadjuvant treatment, such as ophthalmologic operation, tumour and treatment of myocardialinfarction.Commercially available HAase most derived from animals (cattle, sheep) testicular tissue.They low purity, high immunogenicty, have great risk to treatment. Express hPH20in animalcells cost high and esay to react with endogenous protein. Here, we expressed humanhyaluronidase PH20in the Arabidopsis and safflower oil body, result are:1. The hPH20gene was found in GenBank and was transformed with a plant preferencecodon. The hPH20gene was inserted into the pOTBar plasmid. The new recombinant plasmid,named pOTBar-hPH20, was transformed into Agrobacterium EHA105.2. The pOTBar-hPH20plasmid was transformed into A. thaliana by the Agrobacteriumtumefaciens method. Primary transformant (T1, T2) seeds were screened on1/2MS basal platessupplemented with11mg/mL Basta, obtained12independent Basta-resistant lines were used fordetection at the molecular and protein levels.(1) As a result SDS-PAGE and Western blotindicated that expression of target protein oleosin-hPH20in A. thaliana seeds.(2)Enzymeactivity test results that the transgenic A. thaliana seeds measured activity range were4~6U/mL.This indicated that the recombinant oleosin-hPH20expressed in Arabidopsis was biologicallyactive.3. The transformed safflowers were obtained by Agrobacterium-mediated transformation.We obtained transgenic plants in screening medium containing Basta.The survival of seedlingsgrafted with4strains of positive transgenic safflower. Detection the transgenic safflower at themolecular level: PCR amplification results show that the target protein oleosin-hPH20has beensuccessfully integrated into the genome of safflower. This is the first trial of human PH20production in an oleosin-fusion expression system,and the recombinant protein exhibited typical activity. This result might be useful for the massproduction of human PH20. We have confirmed that the oleosin-fusion expression system canbe utilized effectively for the production of hPH20.