摘要
目的:构建猪LIF原核表达载体,表达并纯化重组猪LIF蛋白,制备兔抗猪LIF多克隆抗体并进行免疫定位。方法:根据GenBank中猪LIFcDNA基因序列扩增出目的基因片段,将扩增的片段克隆进PET-31b表达载体的NdeⅠ和XhoⅠ酶切位点之间,经转化使其在大肠杆菌BL21中表达,产生重组猪LIF蛋白,纯化后作为抗原免疫新西兰大白兔,制备多克隆抗体;经ELISA和Western blotting分析鉴定抗体的效价和特异性;最后利用所制备的抗体进行LIF蛋白在猪体内的免疫定位。结果:酶切和DNA测序显示,所构建的原核表达质粒PET-31b-mpLIF含有编码成熟型猪LIF蛋白的cDNA序列;表达结果显示,诱导样品在相对分子质量约20000的位置上有明显的增强表达条带;纯化结果显示,重组蛋白在相对分子质量约20000的位置上有一条清晰、单一的条带;通过免疫新西兰大白兔,获得了特异性较高、效价达1∶10000的多克隆抗体;应用该抗体进行的免疫定位显示,LIF蛋白在心肌细胞、脾索和脾被膜的单层上皮细胞、肺成纤维细胞中有弱表达。结论:重组猪LIF蛋白在大肠杆菌中进行了高效、正确表达;得到了效价较高、特异性较强的多克隆抗体;LIF蛋白在心肌细胞、脾索和脾被膜的单层上皮细胞、肺成纤维细胞中有弱表达。
Objective To construct the expression plasmid of porcine leukemia inhibitory factor （LIF） in the prokaryotic system, and purify the expressed recombinant porcine LIF protein as antigen to immune rabbit for preparation of rabbit anti-pig LIF polyclonal antibody, and immunolocalize of LIF in pigs. Methods The cDNA fragment encoding mature porcine LIF protein was amplified by PCR, then cloned into the prokaryotic expression vector PET-31b at restriction sites Nde I and Xho I. After transformed into E. coli BL21 for expression, the recombinant porcine LIF protein was purifed for preparation of anti-pig LIF polyclonal antibody as immunogen. Western blotting and ELISA were used to test the specificity and titer of the purifed IgG. Immunostaining was used to assay the expression of LIF in pigs. Results The constructed expression plasmid was identified by DNA Sequencing and restriction enzyme digestion. The expression of the recombinant porcine LIF protein was performed exactly in E. coli BL21. By immuning the rabbit, the polyclonal antibody was successfully prepared. The results of ELISA and Western blotting showed that the antibody had high titer （1 ： 10000） and specificity. By immunostaining, LIF protein was faintly expressed in porcine myocardial cell, splenic cord and monolayer covering epithelium cell and pulmonary fibroblast. Conclusion The prepared polyclonal antibody has high titer and specificity; LIF has faint positive immunoreactivity in the normal porcine, myocardial cell, splenic cord and monolayer covering epithelium cell and pulmonary fibroblast.
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2008年第1期82-86,共5页
Journal of Jilin University：Medicine Edition
基金
吉林省科技厅基金资助课题(20010522)
吉林省长春市科技计划项目资助课题(2006075)
关键词
白血病抑制因子
多克隆抗体
免疫定位
leukemia inhibitory factor
polyclonal antibody
immunolocalization
