| Theileria annulata, whcih is a genus Theileria parasite, is transmitted by ticks and parasitized in bovine macrophages, lymphocytes and red blood cells. It can transform the host cell by inducing uncontrolled proliferation and resistance to apoptosis. Galectin-1 is the 14.5 kD protein, which contains a carbohydrate recognition domain with a special affinity for β-galactoside or similar structure. Galectin-1 is widely distributed in different types of cells, tissues and organs, and plays an important role in cell adhesion, growth regulation and immune responses. Previous studies revealed that Galectin-1 is essential to host cell invasion and reproduction. In order to understand the biological function of Galectin-1 in the lymphcytes infected with Theileria anaulata schizonts, the proteins interacted with Galectin-1 were screened by yeast two-hybird system in this study, and the function of Galectin-1 in invasion and cell immortalization were evaluated.The expression level of Galectin-1 was evaluated in lymphocytes infected with Theileria anaulata schizonts and normal lymphocytes by real-time PCR with β-actin for internal gene. Subcellular localization analysis was performed by using laser confocal microscopy. The results showed that the level of Galectin-1 in normal lymphocytes is 8.33 fold higher than in lymphocytes infected with Theileria anaulata schizonts, and Galectin-1 was found in the cell membranes and cytoplasm of two kinds of lymphocytes. However, the Galectin-1 was distributed more close to intracellular membranes other than in cytoplasm.The total RNA was extracted from monoclonal cell line of bovine lymphocytes infected with Theileria anaulata schizonts. The Galectin-1 gene was amplified by RT-PCR. PCR products were digested and ligated into the expression vector pET-30a(+), and then transformed into Escherichia coli BL21(DE3) strain. The recombinant protein was expressed and purified after identification. The polyclonal antibody was prepared by immunizing animals with purified recombinant protein. Western-blot analyses revealed that the anti-Galectin-1 polyclonal antibody have sensitive reaction with both recombinant protein and nature protein.The amplified Galectin-1 gene products were digested with restriction double enzymes, ligated into the vector pGBT9, and then transformed into Y2H Gold strain to testing the bait for autoactivation, toxicity and expression. The results showed that the bait protein was successfully expressed in yeast, and no autoactivation and toxicity to host cells.The mRNA was purified from total RNA, and cDNA library of lymphocytes infected with Theileria anaulata schizonts was constructed by SMART. The ds cDNA library and the linearized pGADT7-Rec vectors were transformed together into yeast 187 competent strains, the Y2H library was constructed by auxotrophic medium screening. The results showed that the titer of the library is 2.0×108 cfu/ml, and the length of ds cDNA inserts is between 300bp to 2500bp.Galectin-1 coding sequence was sub-cloned into pGBT9 vector as the bait, the Galectin-1 interacting proteins were screened in this Yeast Two-Hybrid system, then analysis the functional categorization. The results revealed that 30 potential interacting proteins were obtained, and they could be divided into three types:cellular component, molecular function and biological process.In this study, the expression level of Galectin-1 was evaluated in both normal lymphocytes and lymphocytes infected with Theileria anaulata schizonts, subcellular localization of Galectin-1 was analyzed. The recombinant Galectin-1 protein was expressed and anti-Galectin-1 polyclonal antibody were produced. The Galectin-1 interacting proteins was screened by Yeast Two-Hybrid methods from the library of lymphocytes infected with Theileria anaulata schizonts. Our results provide more valuable information for the Galectin-1 function research in host-parasite interactions. |
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