Functional Investigation On The Chemotactic Activity And Calcium-Flow Of Swine Theymus Activation Regulating CCL17

Thymus activation regulating chemokine CCL17 is one member of the CC chemokine family which plays a vital role in autoimmune diseases and inflammation reaction diseases and so forth. Research on the swine-origin CCL17 is not only favorable to study its basic functions but also conducive to make beneficial explorations concerning the potential value of CCL17; especially it enjoys a broad prospect regarding HPPRRS(Highly pathogenic porcine reproduvtive and respiratory syndrome). On the premise of CCL17 expression in prokaryotic strains, the inclusion body is induced by supernatant and the swine CC17 prokaryotic nucleoprotein can be purified; in the meantime, the activity can be evaluated using the calcium-flow experiment and chemotaxis assay. The swine-origin nucleoprotein is analogous with the eukaryotic protein with respect to biological activity; therefore, it deserves promising scientific potential. Analysis on the activity of CCL17 can provide theoretical basis for further work, and the results obtained in this work were as follows:1. Induction of prokaryotic swine CCL17 protein and its purificationIt is discovered that protein expression is mainly based on inclusion body in the inducement of protein. Reasons for the formation of inclusion body are as follows: 1. The protein expression is too fast to be folded (this bears relation to the nutrient concentration of the medium and the oxygen concentration of the bacteria living environment); 2. The composition of amino acid of the recombinant protein (objective reasons cannot be changed); 3. Temperature of the expression (the protein will not be expressed if temperature changes). Enough protein can be acquired through induction and purification by reducing to 80% of the normal level of the medium concentration, which can avoid the low purification rate of inclusion body and the complex operation. After diluting the concentration to 80% of the normal level, the needs of both the supernatant induction of swine CCL17 and its concentration in supernatant can be met. Considering little difference existed between the structure and function of the prokaryotic protein and eukaryotic protein in small molecule weight, this experiment provides a scientific basis for the purification of prokaryotic protein. Meanwhile, it can pave the way that functions of a protein were investigated by using the prokaryotic protein instead of eukaryotic protein.2. Chemotaxis assay of prokaryotic swine CCL17 protein to swine monocytic cell line of 3D4/31 strainGiven that CCL17 can induce and activate the deformation of the monocytes and generate pseudopodia which cause the movement of CCL17 from low concentration to high concentration. Five concentrations (1000 ng/ml, 100 ng/mL, 10 ng/mL, 1 ng/mL, and 0.1 ng/mL) in a gradient were applied in the present work to evaluate the activity of swine monocytic cell line of 3D4/31 strain. These results indicated that neither the high dose nor the low dose can facilitate movement and chemotaxis to swine monocytic cell line of 3D4/31 strain which can be stimulated only when the concentration of CCL17 is 1000 ng/mL and 100ng/mL. These experimental data are valid becase the chemotactic index is greater than 23. Experiment of Calcium-flow of prokaryotic swine CCL17 protein to swine monocytic cell line of 3D4/31 strainDue to the fact that CCL17 can couple with the CCR4 receptor of protein G on the surface of swine monocytic cell line of 3D4/31 strain to activate the calcium iron channel and to enable the extracellular calcium iron flow into cytoplasm, the calcium-flow experiment was applied for investigating the activity of CCL17. According to the data obtained in chemotactic assay, CCL17 exhibited the best activity on the swine monocytic cell line of 3D4/31 strain when tested at 1000ng/mL and 100ng/mL, respectively. Calcium-flow experiment was conducted at the positive concentration in chemotaxis assay. These results showed that CCL 17 can remarkably stimulate the inward flowing of calcium ion in swine monocytic cell line of 3D4/31 strain when tested at 1000ng/mL and 100ng/mL of swine CCL17, and the fluorescent intensity in the groups treated with 1000ng/mL and 100ng/mL were much higher than that in control groups.