The Study Of Gene Cloning, Expression In Pichia Pastoris, Purification And Characterization Of LYVE-1

Lymphatic vessel endothelial hyaluronan (HA) receptor (LYVE-1) is a receptor specificly expressed on lymphatic vessel endothelials. It is a hot spot to study tumor lymphangiogenesis using LYVE-1 as a specificly lymphatic vessel endothelial marker in recent years. Furthermore, now many studies demonstrate that the expression of LYVE-1 in tumor tissues is closely related to lymphonode metastasis. So that making diagnosis of tumor metastasis by LYVE-1 antibody may have great potentiality in clinic application. LYVE-1 is related to HA transmembrane transport and HA takes part in the transport of tumor cells, so LYVE-1 is likely to play a regulatory role in tumor metastasis. To produce LYVE-1 by gene engineering resolves the problem of insufficient source of natural molecule and surmounts the disadvantages of no post-translational modification in prokaryotic expression system such as colibacillus. This method will provide an expression pattern similar to human natural protein and be efficient for the study of mechanism of tumor metastasis, clinical diagnosis and treatment.Pichia pastoris is a kind of Methanol Pichia, which can grow in the medium of methanol as the unique carbon and energy source. The method of expressing exogenous protein of eukaryotic cell with Pichia pastoris not only has the characteristics of fast growth in prokaryotic organism, simple procedure and low cost, but also possesses the function of post-translational processing and modification of mammalia cells and expresses the biological active protein. So far, although many kinds of exogenous protein have been successfully expressed with Pichia system, there is no report of expressing LYVE-1. In order to further study of the possibility of applying LYVE-1 in the diagnosis and treatment of turmor and lymphatic metastasis, we do gene cloning of human LYVE-1, and undertake expression and identification in Pichia pastoris.Firstly, the gene cloning of human LYVE-1 was accomplished. Total RNA of the fresh metastatic lymphonodes got in the radical correction of colon carcinoma was extracted by Trizol, and the total length cDNA of LYVE-1 was obtained by RT-PCR. It was confirmed by agarose gel electrophoresis and sequencing that the cDNA was same as the sequence of the total length cDNA of human LYVE-1, and could be used in construction of the expression recon.Secondly, the recombinant plasmid of pPICZαC-LYVE-1 and transfer the yeast was constructed. The total length cDNA of LYVE-1 after digestion by EcoRⅠand NotⅠand pPICZαC vector plasmid were connected by T4 ligase. The recon was linearized by SacⅠdigestion and transferred into the competent GS115 by BIORADE Gene Pulser. The transferred GS115 was spead on YPDS plate and the positive cloning was selected and identified.Finally, human LYVE-1 protein was expressed in Pichia pastoris and purified. The amplified engineering bacteria were induced to express the interest protein by 1% methanol. The C-terminal His-Taged recombination protein was purified by metal-chelate affinity chromatography (MCAC). It demonstrated that LYVE-1 gene was soluble expressed in yeast cytoplasm and the expressed protein possessed the antigen activity of natural LYVE-1 by the detection of SDS-PAGE and Western Bloting.This study cloned human LYVE-1 gene successfully. Then, the LYVE-1 protein was expressed in bulk in vitro using Pichia pastoris eukaryon expression system, which was similar to human natural molecular in structure and possessed the antigen activity. It provides the experimental foundation for further study of immunological diagnosis and clinical treatment of tumor by LYVE-1.