富含半胱氨酸蛋白61多克隆抗体的制备及其在子痫前期胎盘检测中的作用  

Preparation of Rabbit Polyclonal Antibody against Human Cysteine Rich Protein 61 (Cyr61) and Role of it in the Detection of the Placentas from Preeclampsia

作　　者：苏莉[1,2] 刁振宇[3] 胡娅莉[3] 张艳青[1] 王志群[1] 

机构地区：[1]南京医科大学鼓楼临床医学院,江苏南京210002 [2]扬中市人民医院,江苏扬中212200 [3]南京大学医学院附属鼓楼医院,江苏南京210002

出　　处：《现代生物医学进展》2009年第10期1816-1819,共4页Progress in Modern Biomedicine

基　　金：国家自然科学基金(30872775);江苏省医学重点学科--母胎医学中心(XK200709)基金赞助

摘　　要：目的:制备兔抗人富含半胱氨酸蛋白(Cyr61)多克隆抗体,并用该抗体检测Cyr61蛋白在重度子痫前期(PE)和正常妊娠胎盘中的差异表达。方法:用RT-PCR法从Hela细胞中克隆Cyr61全长mRNA,并在大肠杆菌中表达获得重组蛋白,免疫新西兰白兔得到兔抗人Cyr61多克隆抗体。然后用此抗体对14例重度子痫前期胎盘进行检测,并与孕周匹配的14例正常胎盘比较。结果:Cyr61DNA测序结果与参考序列一致;Western blot竞争法证实制备的兔抗Cyr61抗体与商品化的羊抗Cyr61抗体有共同的靶点;用制备的兔抗人Cyr61蛋白抗体Westernblot检测发现,在重度PE胎盘中Cyr61蛋白含量较正常胎盘含量降低(14.2±1.0vs.29.2±1.1P<0.05)。结论:本研究成功地在大肠杆菌中表达出Cyr61融合蛋白,并制备了特异性高的兔抗人Cyr61抗体。重度PE胎盘中Cyr61蛋白表达水平降低。Objective： To prepare rabbit polyclonal antibody （Ab） against human Cyr61, and investigate the different expression of Cyr61 in the human placentas between severe preeclamptic and normal pregnancies. Methods： Total RNA was isolated from Hela cells and reverse-transcribed to cDNA. The cDNA coding for Cyr61 was amplified by PCR, and cloned in pET-32a expression vector. Recombinant Cyr61 was synthesized in E. coli. New Zealand white rabbits were immunized with the recombinant Cyr61. The levels of Cyr61 protein in the placentas from 14 severe preeclampsia women and from 14 normal pregnancy women were measured by Western blotting analysis. Results： The Cyr61 DNA sequencing was identical to that in GenBank database. Competitive Western blot demonstrated the rabbit Ab recognized the common epitopes with the commercially available goat Ab. Western blot results indicated the Cyr61 protein amount decreased in placentas from severe preeclampsia compared with the normal pregnancies （14.2± 1.0 vs. 29.2± 1.1, P〈0.05）. Conclusion： Recombinant Cyr61 protein expressed in E. coli may induce specific Ab, which has the similar property with the commercial Ab and can be used to detect Cyr61 protein in human. The Cyr61 protein levels in placentas from severe preeclampsia are lower than those of the normal pregnancies.

关 键 词：Cyr61克隆 重组蛋白 多克隆抗体 子痫前期 

分 类 号：Q75[生物学—分子生物学] R714.245[医药卫生—妇产科学]