Clonging And HRF Characterization Of L250, A Recombinant Protein From Buccal Gland Of Lampetra Japonica

The purpose of this study is to clone the cDNA sequence encoding translationally controlled tumor protein which we called L-250 according to the information from cDNA library and the primary analysis of expressed sequence tags, and to do the research about expression and purification of this function gene contributing to buccal gland secretion from Lampetra japonica, preparation of polyclonal antibody, immunoblotting analysis to localize the rcombinant protein in buccal gland and identification its activity as histamine-releasing factor.Firstly, total RNA was extracted from buccal gland of Lampetra japonica and used RT-PCR to generate a cDNA , we screened the target gene and cloned it into the expressing vector pET23b, then the function gene was transformed into E.coli strain BL21 for expression under the induction of IPTG .The recombinant proteins L-250 were identified by SDS-PAGE and purified by using the His·Bind affinity chromatography. The results revealed that it had molecular weight of 22.4 KD and concentration of 1.5mg/mL.The polyclonal antibody was obtained by antigen immunizing animals, the antigen was prepared from the purified L-250, and the ploycolnal antibody was examined in the animals'serum by using enzyme linked immunosorbent assay. The speciality of the antibody was identified by the methods of Western blotting. We observed that the polyclonal antibody can recognize protein which was expressed and was in buccal gland secretion by immunoblotting.We can draw the conclusion that the successful expression of L-250 and preparation of polyclonal antibody will provide material for further study.HRF belongs to a class of proteins called translationally controlled tumor protein homologes. We identify the ability of L-250 to induce release of histamine by using rat basophilic leukemia(RBL-2H3) cells. Results from the present study show that L-250 mediates histamine release from RBL-2H3 cells. We all know that histamine might increase endothelial cell-surface expression of thrombomodulin which is a tissue anticoagulant and may modulate some of anticoagulation by activating protein C. Given L-250 induces releasing of histamine and histamine may modulate anticoagulant cascade, we speculate that L-250 may have some potential effects on anticoagulation therapy.