Prokaryotic Expression Of Mus Musculus Tim-3 Protein And Generation Of Polyclonal Antibody Against This Molecule

Objective1.Cloning Mus musculus TIM-3 gene and constructing a prokaryotic expression vector of this molecule.2.Expressing TIM-3 gene in Escherichia coli of Rosseta(DE3)and preparing polyclonal antibody of Mus musculus TIM-3.Methods1. The extracellular TIM-3 gene was cloned from the spleen cells of BALB/c mice by RT-PCR and connected into the vector pRSET-B in order to construct prokaryotic expression vector pRSET-B-TIM-3.2. Mouse TIM-3 protein was expressed in Escherichia coli of Rosseta(DE3)by IPTG. induction and purified by Ni-NTA column. Polyclonal antibody against this TIM-3 protein was generated by immunizing rabbit with the routine method. The specificity and sensitivity of this antibody was confirmed by ELISA, western blot,immunohistochemistry assay and FACS.Results1.The fragment of 510bp extracellular DNA sequence was obtained from the spleen cells of BALB/c mice by RT-PCR and connected into the vector pRSET-B. After the recombinant was confirmed by double endonuclease digestion, PCR and direct sequencing, the prokaryotic expression vector pRSET-B-TIM-3 was constructed successfully.2.Induced by IPTG in Rosseta(DE3), the recombinant TIM-3 protein could be expressed effectively in the form of inclusion bodies. The interest protein of about 24-25kD was confirmed by western blot.3.After immunization of rabbit with the recombinant TIM-3 protein which was purified with Ni-NTA column, the polyclonal antibody against mouse TIM-3 protein with a high affinity and specificity was obtained. By using ELISA,,the titer of this polyclonal antibody was estimated about 1:320000.Conclusion1.The prokaryotic expression vector pRSET-B-TIM-3 was constructed successfully. In the prokaryotic expression system of Rosseta(DE3), the mouse TIM-3 protein was expressed effectively in the form of inclusion bodies.2.The polyclonal antibody with high affinity and specificity was generated successfully.SignificanceThe polyclonal antibody against mouse TIM-3 obtained in this study can be used for detecting the expression of mouse TIM-3 in vitro and for blocking the signal pathway of TIM-3/ TIM-3L in vivo. It will help to further study the function and mechanism of TIM-3 in acute and chronic virus infection.