星突江鲽生长激素基因的克隆及体外重组表达分析  

Molecular cloning and in vitro expression of growth hormone gene from Platichthys stellatus

作　　者：臧坤[1,2] 柳学周[1] 徐永江[1] 张凯 史宝[1] 史学营 李存玉[1,2] 

机构地区：[1]中国水产科学研究院黄海水产研究所,农业部海洋渔业可持续发展重点实验室,山东青岛266071 [2]上海海洋大学水产与生命学院,上海201306 [3]青岛贝宝海洋科技有限公司,山东青岛266000

出　　处：《水产学报》2014年第9期1230-1242,共13页Journal of Fisheries of China

基　　金：国家"八六三"高技术研究发展计划(2012AA10A413);鲆鲽类现代产业技术体系专项(CARS-50);山东省自然科学基金项目(ZR2012CQ025)

摘　　要：利用cDNA末端快速扩增技术(RACE)获得星突江鲽生长激素基因(GH)的cDNA序列全长为957 bp,其中开放阅读框(ORF)长615 bp,编码204个氨基酸,氨基酸序列与牙鲆同源性最高达到73.0%,系统进化显示,星突江鲽GH与其他鲽形目和鲈形目鱼类聚为一个分支。采用实时荧光定量PCR技术对GH基因的组织表达特性进行了分析,结果显示,GH基因mRNA主要在雌雄成鱼垂体中表达,同时在脑、性腺、肝脏、胃和肌肉中均检测到表达,雌鱼胃和肌肉中GH基因mRNA的表达量显著高于雄鱼(P<0.05),表明星突江鲽GH可能主要通过旁分泌和自分泌方式参与性别二态性生长调节。本实验成功构建了体外重组表达质粒GH/pET28a,转化大肠杆菌BL21(DE3)经IPTG诱导可得N端含6个组氨酸的重组蛋白。重组蛋白主要以大小为24.9 ku的包涵体形式存在,Western-blotting免疫印迹呈阳性。包涵体经6 mol/L盐酸胍变性、Ni2+离子亲和柱纯化和尿素梯度复性后可得纯化GH蛋白;5.4和16.2μg/mL重组蛋白添加组中,人胚胎肾细胞HEK293T的增殖受到显著抑制。本研究结果可在分子和蛋白水平解析星突江鲽的生长调控机制。The full-length cDNA encoding growth hormone（ GH） was isolated from the pituitary of Platichthys stellatus using RACE（ rapid amplification of cDNA ends） method. Results showed that Platichthys stellatus GH cDNA sequence is 957 bp in length and encodes 204 amino acids. The highest amino acid identity of Platichthys stellatus GH was 73. 0% with Paralichthys olivaceus. Phylogenetic analysis indicated that Platichthys stellatus GH was clustered with other pleuronectiformes and perciformes species.The tissue expression patterns of GH mRNA in different tissues of female and male adults were analyzed by the quantitative real-time PCR. GH mRNA showed the highest expression level in the pituitary of both sexes with lower levels in the brain,gonad,liver,stomach and muscle. The expression levels of GH mRNA in the female stomach and muscle were significantly higher than those of males（ P〈0. 05）,which implied that it might be involved in sexually dimorphic growth pattern through paracrine and autocrine pathways. The GH /pET28 a recombinant plasmid was successfully constructed and highly expressed in E. coli BL21（ DE3） after being induced by IPTG with special fusion polypeptides containing His6 at their N-terminus. The obtained recombinant GH polypeptide was expressed in form of inclusion bodies with molecular weight of 24. 9 ku and had the antigenicity to His6 antibody by western blotting analysis. The inclusion bodies were denaturalized using 6mol /L guanidine HCl,purified using Ni2 ＋-NTA affinity chromatography and annealed by gradient dialysis in urea,and the verified recombinant GH protein was obtained. The effect of recombinant GH protein on proliferation of human embryonic kidney cells HEK293 T was tested,and the results showed that significant inhibition was only found when protein concentration was higher than 5. 4 μg /mL. Results from the present study could provide basic knowledge for molecular-level and protein-level growth regulation mechanism study of Platichthys stellatus.

关 键 词：星突江鲽 生长激素 基因克隆 组织表达 原核表达 

分 类 号：Q785[生物学—分子生物学] S965.3[农业科学—水产养殖]