【作者】 李晓霞；

【导师】 陈祥贵；

【作者基本信息】 西华大学 ， 食品科学， 2006， 硕士

【摘要】 Lrrc10基因是采用EST介导的基因克隆和表达谱分析,成功克隆的一个在小鼠心脏特异表达的新基因,该基因cDNA全长为1410bp,定位于小鼠染色体10D2,无内含子,其最大开放阅读框编码的假定蛋白有274氨基酸组成,含有7个亮氨酸重复继续基序。Lrrc10基因是第一个被报道具有心脏特异性的LRR家族成员,目前的研究表明所有LRR蛋白的功能均与蛋白质相互作用有关,至少一半的LRR蛋白参与信号转导通路,而人类的胚胎发育和很多疾病都与信号转导通路密切关联。该基因有可能成为药物设计中特异性的、副作用小的靶标,具有重要的理论和实际意义。因此,本文通过构建Lrrc10原核表达载体以期获得重组Lrrc10蛋白,为进一步的功能研究提供基础,同时在研究胚胎癌细胞P19的体外心肌分化过程中Lrrc10基因表达,为探讨Lrrc10与心肌发育的关系提供依据。 1.重组质粒pET-Lrrc10的构建和Lrrc10蛋白表达 方法:Nco Ⅰ和BamH Ⅰ双酶切载体pMD18-T-Lrrc10,回收目的片段与经同样酶切后的表达载体pET-30a(+)连接,转入E.coli strain DH5a,菌落PCR筛选阳性菌落,提取阳性菌质粒并进行PCR和酶切鉴定;将重组子转入E.coli strain BL21(DE3),于37℃振荡培养,利用1mmol/L IPTG诱导目的蛋白的表达。 结果:PCR鉴定和酶切鉴定的结果表明,成功构建了融合表达载体并命名为pET-Lrrc10。转入E.coli strain BL21(DE3)进行目的蛋白的表达,获得与预期分子量大小相一致的融合表达蛋白Lrrc10;Lrrc10蛋白以包涵体形式存在于宿更多还原

【Abstract】 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.更多还原