Expression Of Human LIGHT Gene In Escherichia Coli And Pichia Pastoris

Members of tumor necrosis factor (TNF) family can induce multiple biological effects including cell growth, differentiation and death. They play an important role in inflammatory regulation, immune response to infection and tissue homeostasis. LIGHT is one of the special members of TNF family. It not only can regulate tumor cell apoptosis, but also can costimulate T cell activation and regulate T cell immune response, so it is promising to be applied in tumor immunotherapy and research of some autoimmune diseases. The content of LIGHT in human body is very low and it is difficult to isolate and purify. In order to meet the need of research and clinical application, it is a very useful method to produce recombinant human LIGHT by genetic engineering technology. In the present study, the expression of human LIGHT protein were studied in prokaryotic and eukaryotic expression system.Firstly, the extracellular region of human LIGHT gene was cloned into plasmid pET32a (+) to construct prokaryotic expression vector, and the fusion protein of human LIGHT was expressed in E.coli successfully. The expression conditions were optimized prelimarily.Secondly, the fusion protein of human LIGHT was prepared in large scale and was purified by affinity chromatography. Then the purified recombinant protein was used to immunize the mice with Freund's adjuvant and high titer (1:128 000) of mouse anti-human LIGHT polyclonal antibodies with good specificity was acquired finally.Lastly, the yeast expression plasmid pPIC9K-LIGHT-Fc harbouring the extracellular region of human LIGHT gene and Fc gene was constructed sucessfully. After transforming into the host strain GS115 of Pichia pastoris, a genetic engineering yeast which could steadily experess the fusion protein of LIGHT-Fc was identified by PCR and Western blot.The expression of human LIGHT in prokaryotic and eukaryotic expression system was explored successfully in the study, and polyclonal antibodies of mouse anti-human LIGHT was produced. It is the first description of generating human LIGHT protein in Pichia pastoris through recombinant DNA techniques. These results have provided a good foundation for the further functional research of LIGHT and exploring new methods of tumor immunotherapy.