寿光鸡腺苷酸琥珀酸裂解酶ADSL基因的表达与克隆化细胞株的筛选  

Expression Study and Establishment of pEGFP-ADSL Cloning Cell Lines of Shouguang Chicken Adenylosuccinate Lyase Gene

作　　者：刘长青[1,2,3] 张洪海[4] 文杰[2] 马月辉[2] 关伟军[2] 唐学玺[1] 

机构地区：[1]中国海洋大学生命科学与技术学部,青岛266003 [2]中国农业科学院北京畜牧兽医研究所,北京100193 [3]蚌埠医学院生物科学系,蚌埠233000 [4]曲阜师范大学生命科学学院,曲阜273165

出　　处：《畜牧兽医学报》2009年第7期982-991,共10页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金(30100132);国家"863"计划项目资助(2006AA10Z198;2007AA10Z170);山东省中青年科学家科研奖励基金(01BS12);国家"973"计划项目(2004CB117506)

摘　　要：利用RT-PCR与RACE方法检测寿光鸡心、肝、脾、肺、肾、脑、腿肌与胸肌8种组织腺苷酸琥珀酸裂解酶（ADSL）基因mRNA的差异表达水平，构建ADSL基因融合表达载体pGEX-ADSL，转化大肠杆菌BL21（DE3），筛选阳性克隆，IPTG诱导表达，通过构建真核表达载体pEGFP-ADSL，利用脂质体介导法转染寿光鸡成纤维细胞，并进行阳性细胞的克隆化筛选与检测。结果表明：ADSL基因开放阅读框序列长为1455 bp，编码485个氨基酸;5′端非转录调控区具有典型管家基因的特征，在邻近起始密码子-28 bp处发生C→T突变，该突变使该位点变为核呼吸因子2（NRF-2）结合位点;经SDS-PAGE电泳显示，pGEX-ADSL重组融合蛋白在分子量约为80.5 ku处有特异的蛋白条带出现，与预期分子量大小一致，等电点为6.79，纯化后用Western-blot检测为寿光鸡ADSL蛋白。pEGFP-ADSL转染寿光鸡成纤维靶细胞后24、48和72 h，转染率在26.4%~41.2%之间，绿色荧光均匀分布于细胞质与细胞核中，随表达量的增加，绿色荧光在细胞核中聚集成团块状或颗粒状，经药物筛选和克隆化培养，获得表达pEGFP-ADSL融合蛋白的阳性克隆细胞株，经RT-PCR扩增与Western-blot检测确认ADSL融合蛋白基因已经整合到寿光鸡成纤维细胞的基因组中，获得正常表达。研究结果为揭示我国优良地方鸡种的风味基因结构，研究其生物学功能奠定基础。The specific expression of ADSL gene in 8 different tissues of Shouguang chicken was investigated by RT-PCR and RACE in this study. The fusion expression vector pGEX-ADSL was constructed and transformed into Escherichia coli BL21 （DE3）, positive cloning screened, induced and expressed by IPTG. Eukaryotic expression vector pEGFP-ADSL was transfected into Shouguang chicken fibroblast cells using lipofectin method. The results of sequences analysis showed that the open reading frame of 1 455 nucleotides encode a protein of 485 amino acids; The promoter of chicken ADSL cDNA showed typical features of house keeping genes,there was a C-28T mutation which caused the site mutate to NRF-2 binding site. The SDS-PAGE electrophoresis results showed that there were specific bands, about 80.5 ku and an isoelectric point of 6.79, and Western-blot analysis showed that the fused protein was ADSL. After transfection 24, 48 and 72 h, transfection rate of pEGFP-ADSL were between 26.4%--41.2%, and green fluorescent could be observed in cytoplasm and nucleus well-distributed except cryptomere vesicle. Both of RT-PCR and Western-blot confirmed that the pEGFP-ADSL had been integrated into the Shouguang chicken fibroblast cell genome, and accessed to the normal fusion protein expression. This research revealed the ADSL gene structure of excellent native chickens of China, established the foundation for further research with its biological function.

关 键 词：寿光鸡 腺苷酸琥珀酸裂解酶 基因结构 基因表达 基因定位 

分 类 号：S831.2[农业科学—畜牧学] Q344.13[农业科学—畜牧兽医]