蒙古鲌ob基因克隆及表达-水产养殖专业毕业论文.docxVIP

华中农业大学2005年硕上研究生学位论文摘要 华中农业大学2005年硕上研究生学位论文 摘要 本文中的试验鱼——蒙古舶购自南湖渔场，健康无病，雄鱼体长23．1cm，体重 151．39；雌鱼体长为25．2cm，体重155．69。根据已知的人和鼠ob基因序列设计引 物，引物中分别引入EcoR I和HindIII酶切位点，通过R1二PcR从蒙古舶脂肪组织 RNA中扩增到ob基因片段。将此片段与pBluescrip SK(+)载体同时用EcoR I和Hind IⅡ取酶切，电泳回收各自片段后连接并转化大肠杆菌DH5 a感受态细胞，涂布于含 IPTG、X．gal和Amp+的琼脂糖平板，挑取乳白色的菌落，进行PCR扩增和双酶切 鉴定，阳性克隆命名为pSK-ob，将此质粒送大连宝生物公司测序，首次获得蒙古舶 ob基因编码区438bp的序列。ob基因序列在不同物种之间具有很高的保守性。将 此序列与人、鼠和猪的ob基因序列进行比较，发现蒙古柏与人、鼠和猪ob基因核 苷酸编码序列的同源性分别为82％、84％和99％；蒙古鹪ob基因编码的蛋白lcptin 氨基酸序列与人、鼠和猪leptin蛋白氨基酸同源性分别为80．8％、78．8％和95．2％。 定量取蒙古舶不同组织O．29，提取其总RNA后，用半定量RT-PCR技术分析组织 ob基因表达特异性，结果表明：ob基因在脂肪和肝脏组织中表达量最大，在心脏、 脾脏、肌肉、脑、卵巢中表达量次之，在肾脏中表达很少，而在精巢和肠道组织中 不表达。将含ob基因的克隆载体pSKoob与表达载体pET-28a同时用EcoR I和Hind III双酶切，回收所需片段连接并转化后，涂布于含1PTG、x．gal和Kan+的琼脂糖平 板，挑出乳白色的菌落，进行PCR扩增和酶切鉴定。鉴定过的阳性克隆子命名为 pET-28 a．ob，转化大肠杆菌BL21(DE3)，通过IPTG诱导表达，SDS—PAGE电泳分 析。在分子量20KD处有一融合蛋白，而融合蛋白前体分子量为4KD，则剩余蛋白 分子量为16kD，此蛋白分子量与期望值一致，且在一定时间内，表达量随着时间 的延长，蛋白含量增加。 关键词：蒙古舶； 肥胖基因； 瘦素； 组织；表达 一 一 ．堂壁翌!茎直塾塑!!苎里至堕丝塞竺 Abstract In this study,Culter rhongolicus(Basilewsky)were taken from South Lake fishing ground；the length and weight of male and female fish were about 23．1cm，151．39 and 25．2cm，155．69 each．Tissues such as adipose，liver,hean，spleen，muscle，brain，ovary, kidney,spermary had been obtained from the double fish and deserved in the refrigerator at-70℃after cleaned by distilled water．Each tissue’s total RNA were extracted by one step method and the extracted total RNAs were identified by Eppendorf Bio-photometen All the total RNA that ratios of OD2so／ODz60 were from 1,8 to 2．0 were diluted into 0．5／29似L．The primers were designed according to the consensus region of human and mouse ob genes and were employed to perform RT-PCR．The primer was included EcoR I and HindIII enzyme cut site at sequence．The product of RT-PCR and pBluescript sK(+)vector were both digested by EcoR I and Hind III，then linked and transformed into DH5 competent cell．The recombined plasmid was extracted and then identified by PeR and double enzyme digestion．The p