水牛生长分化因子9成熟肽基因克隆及重组蛋白质和多克隆抗体的制备  

Cloning of buffalo growth differentiation factor 9 mature peptide cDNA sequence and preparations of the recombinant protein and polyclonal antibody

作　　者：刘玉鹏[1,2] 彭中友[2] 郭日红[2] 雷明明[3] 于建宁[3] 陈瑞爱 施振旦[3] 

机构地区：[1]广东省兽用生物制品生物技术研究与应用企业重点实验室,肇庆大华农生物药品有限公司,广东肇庆526238 [2]华南农业大学动物科学学院,广东广州510642 [3]江苏省农业科学院畜牧研究所,江苏南京210014

出　　处：《江苏农业学报》2013年第2期346-351,共6页Jiangsu Journal of Agricultural Sciences

基　　金：广东省肇庆市科技计划项目(2010C004);江苏省第八批“六大人才高峰”项目[苏农函(2012)35号]

摘　　要：为了获得水牛生长分化因子9(Growth differentiation factor 9,GDF9)重组蛋白质和抗GDF9抗体,根据GDF9基因编码区序列(GenBank:FJ529501.1)设计1对引物,用水牛基因组DNA为模板扩增水牛GDF9成熟肽基因序列,并与其他反刍动物的GDF9成熟肽基因序列进行同源性比较。将该序列克隆到表达载体pRSET-A的BamHⅠ和HindⅢ酶切位点之间以构建重组表达载体,并将重组表达载体转化大肠杆菌E.coli BL21(DE3),转化菌经IPTG诱导表达重组蛋白质。经Ni-NTA凝胶纯化后的重组蛋白质与矿物油佐剂混合免疫新西兰兔制备抗GDF9抗血清,使用ELISA方法检测血清抗体效价,从抗血清中进一步纯化GDF9抗体。结果显示,成功获得了水牛GDF9成熟肽基因序列,该序列在牛和其他反刍动物之间高度同源。成功构建了重组表达载体并转化大肠杆菌E.coli BL21(DE3),E.coli BL21(DE3)经IPTG诱导后表达了分子量为1.96×104的GDF9重组蛋白质,其最高表达量达到菌体蛋白质总量的30%左右。成功制备了抗GDF9抗血清,血清效价达到1∶51 200,抗血清经纯化后得到了高纯度的GDF9抗体。GDF9重组蛋白质和抗GDF9抗体有望应用于提高羊繁殖性能和促进动物胚胎发育的研究和技术开发。In order to obtain recombinant GDF9 （growth differentiation factor 9 ） protein and anti-GDF9 antibody, a pair of primers designed according to the buffalo GDF9 gene mature peptide sequence （GenBank- FJ529501.1 ） was used to amplify the cDNA sequence of water buffalo GDF9 mature peptide with water buffalo genomic DNA as the template, and the alignment was made between the cDNA sequence and this region of other ruminant. The cDNA sequence was cloned into the BamH I and Hind m sites of plasmid pRSET A to generate the expression vector pR-GDF9, which was further transformed into bacteria Escherichia. coli BL21 （DE3）. The transformed bacteria were in- duced to produce a recombinant GDF9 protein by IPTG. Then, the recombinant GDF9 protein was purified by 50% Ni-NTA agrose chromatography and homogenized into mineral oil adjuvant. The mixer was used to immunize New Zealand White rabbits to raise anti-GDF9 antibody. The titer of the antiserum was determined by ELISA. The results showed that buffalo GDF9 gene mature peptide sequence which shared high homology with other ruminant molecules was cloned successfully. E. coli BL21 （DE3） expressing GDF9 protein with the expected molecular mass of 1.96× 10^4 was obtained, and the highest expression level achieved 30% of total bacterial protein. Anti-GDF9 serum with titre of 1 ： 51 200 was obtained successfully and further purified to get high-purity anti-GDF9 antibody. The recombinant GDF9 protein prepared in this study can be used for immunizing sheep to improve ovulation rate and reproductive performance, and the anti-GDF9 anti- body can be applied for improving animal embryo development in vitro.

关 键 词：生长分化因子9 成熟肽 克隆和表达 多克隆抗体 

分 类 号：S823.83[农业科学—畜牧学]