Construction Of Lrrc10 Protein Expression Vector And Investigation Of Expression Of Lrrc10 In Differentiated Embryonal Carcinoma Cells

Lrrc10, a novel mouse heart-specific gene, was cloned from mouse embryo heart by application of EST assembly. The cDNA of Lrrc10 is 1410bp and intronless. The gene is mapped to mouse chromosome 10D2. The longest ORF of the cDNA encodes a putative protein of 274 amino acids. Seven leucine-rich repeats are present between 53 amino acid to 212 amino acid . Now the study of LrrclO is little. It was reported that Lrrc10 was the first gene because it was heart-specific gene and was one of LRR family. At present all studies have shown that functions of LRR protein were associated with protein-protein interactions. More than half of LRR proteins are crucial for signal transduction, while human embryo development and many diseases are associated with signal transduction. So Lrrc10 becomes probably a specific and nonside-effect drone to design medicament, which is significant theoretically and practically. So it is very important to study the relation between Lrrc10 and heart.1. Construction of pET-Lrrc10 and Protein Expression of LrrclO Method: To construct pET-Lrrc10, pMD18-T-Lrrc10 was digested by BamH I and Nco I to get Lrrc10, which was ligated with pET-30a(+) digested by BamH I and Nco I. Then the recombinant was transformed into E. coli strain DH5a. Positivestrains were identified by PCR to extract recombinants which were transformed into E. coli strain BL21(DE3) to induce LrrclO protein with IPTG at 37°C.Resul t: Identification by colony PCR and restriction enzymes digestion shows that the recombinant was constructed successfully. The result recombinant plamid was named as pET-LrrclO. After induced by IPTG, the E. coli strain BL21(DE3) transformed by pET-LrrclO expressed recombinant protein which was consistent with expected molecular weight.predicted by LrrclO ORE LrrclO recombinant protein existed as inclusion body and reached its expression peak after induced by IPTG for four hours.2. Research of LrrclO in Differentiation of CardiomyocyteMethod: P19 cells was induced by DMSO to develop cardiogenesis model in vitro. Morphological change of cells was observed by microscope. a-MHC and 6-MHC were used as the markers of cardiogenesis. Total RNA was extracted from undifferentiated and differentiated P19 cells. Expression of LrrclO. was detected by RT-PCRResult: After induced by DMSO, Morphological change of some cells was obvious. RT-PCR showed that a-MHC was detected after differentiated for 6 days and fi-MHC was detected after differentiated for 8 days. These results suggested that differentiation of P19 cells in vitro succeeded. At the second day of differentiation, express of LrrclO was detected by RT-PCR.Conclusion: we constructed pET-LrrclO vector and achieve target protein which was consistent with expected molecular weight. LrrclO expressed in cardiomyocytes differentiated from P19 cells. Based on structure characteristic of the protein, we think that LrrclO is related probably with differentiation of cardiomyocyte.