梅山猪LH-β基因真核表达系统的构建及体内外转染表达效果的研究  

The Eukaryotic Expression of Porcine LH-β Gene in vitro and the Effect of Transgenetic Expression in vivo on the Reproduction of Mice

作　　者：吕学斌[1] 李江凌[1] 高荣[2] 武梅[2] 杨毅[2] 王丽焕[2] 刘世贵[2] 

机构地区：[1]四川省畜牧科学研究院,成都610066 [2]四川大学生命科学学院,成都610064

出　　处：《畜牧兽医学报》2005年第9期877-881,共5页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：四川省应用基础项目

摘　　要：将克隆的梅山猪LH-β基因亚克隆到真核表达型载体VR1020上,通过质粒PCR的方法在转化子中筛选阳性克隆,用BamHⅠ、BglⅡ双酶切鉴定重组子。得到的重组真核表达质粒转染Cos7细胞株,RT-PCR检测猪LH-β表达情况。然后用VPLH-β质粒、VR1020分别肌肉注射小鼠,研究了小鼠的生殖特性情况。结果显示:通过质粒PCR和双酶切分析,得到猪LH-β基因以正确方向插入的重组真核表达质粒VPLH-β。重组质粒VPLH-β经脂质体介导转染Cos7细胞,培养不同时间后提取mRNA进行RT-PCR,发现转染细胞RNA中含有与LH-β基因对应的mRNA;结果表明已成功构建了PLH-β的真核表达质粒,能正确转录表达LH-β。动物试验结果显示PLH-β免疫小鼠后的各项指标均高于对照处理。克隆构建的猪VPLH-β质粒能发挥明显的生殖生理调节作用,可较好地促进雌性动物的排卵、怀孕和正常产仔。VR1020 was first used as the eukaryotic expression plasmid to construct LH-β eukaryotic expression system. The recombinant plasmid was detected by the plasmid PCR and BamH I ,Bgl Ⅱ digestion to identify the correct insert orientation, the new plasmid was named VPLH-β. VPLH-β was transformed into E. coli DH5α competent cells. The ability of VPLH-β to express full-length porcine LH-β in vitro was confirmed by RT-PCR in Cos7 cells that were transiently transfected with VPLH-β in the entrapment of Lipofectamine. 48 BALB/c female mice at the age of 2 weeks were divided into 3 groups and were vaccinated intramuscularly in the left and right quardriceps respectively with 100μg VPLH-β, with 100μg VR1020; Saline solution was used as control treatment. The blood of mice were collected from tail vain on 7, 14, 21, 28 days after inoculation to determine the dynamic changes of the content of LH-β and FSH in the sera. The reproduction of mice was also detected. The experimental results indicated that the VPLH-β, we first recombined, could correctly express in vitro by using the Lipofectamine entrapment system. In the animal research, the amount of LH and FSH were significantly raised in the mice inoculated with VPLH-β plasmid, which was correlated with reproduction performance of the mice that produced more young mice than the control mice. These results indicate that the inoculation with VPLH-β could remarkably raise the content of LH and FSH in the sera of mice and promote the reproduction performance.

关 键 词：猪 LH—β基因 真核表达 转染 生殖 小鼠 

分 类 号：S828.2[农业科学—畜牧学]