小鼠着床丝氨酸蛋白酶2cDNA的克隆、表达及抗体制备  

Cloning,Expression, and Antibody Production of Mouse ISP2

作　　者：黄哲平[1] 王健[1] 余浩[2] 黄萍[1] 沈维雄[1] 左嘉客[1] 杨增明[2] 申庆祥[1] 

机构地区：[1]上海市计划生育科学研究所国家计划生育药具重点实验室,上海200032 [2]东北农业大学生命科学学院,哈尔滨150030

出　　处：《生物化学与生物物理学报》2003年第7期649-654,共6页

基　　金：国家重点基础研究发展规划项目 (973计划 ) (No .G19990 5 5 90 3 )~~

摘　　要：以孕 4 .5天小鼠子宫着床位点总cDNA为模板 ,用PCR方法扩增获得着床丝氨酸蛋白酶 2 (ISP2 )的cDNA。序列分析表明 ,该cDNA序列中的开放阅读框编码 2 79个氨基酸 ,并与文献报道完全相同 ,但在 3′非翻译区多 2 0 4bp ,已登录于GenBank(登录号 :AF4 4 2 819)。为了获得重组ISP2 (rISP2 ) ,构建了pGEX 4T 2 ISP2表达质粒 ,进而在大肠杆菌BL2 1(DE3)中表达融合蛋白质GST ISP2 ,经SDS PAGE回收的融合蛋白质用牛凝血酶酶切 ,再经SDS PAGE回收获得rISP2。用rISP2作为免疫原免疫家兔获得ISP2多克隆抗血清。用该抗血清进行免疫组化研究表明 ,ISP2在妊娠早期的小鼠子宫内膜腺上皮有特异性的高表达。A cDNA encoding mouse implantation serine proteinase 2 (ISP2)was amplified from total cDNAs of mouse uterus implantation sites on D4.5 of pregnancy by PCR, and sequenced(GenBank accession No. A442918). DNA sequencing indicated that the ISP2 cDNA had an unreported 204 bp sequence at 3′ untranslated region besides the open reading frame encoding 279 amino acid residues, which was identical with literature. In order to obtain recombinant ISP2(rISP2), an expression plasmid pGEX 4T 2/ISP2 was constructed and transformed into E.coli BL21(DE3) strain. Expressed fusion protein GST ISP2 was purified by SDS PAGE and digested with thrombin, and the digestion mixture was subjected to SDS PAGE again to recover rISP2. Rabbits were immunized using rISP2 as immunogen, and the polyclonal anti ISP2 antisera were obtained. Immunohistochemical analysis using this antisera showed specific and high expression of ISP2 in mouse endometrial gland epithelium in early pregnancy.

关 键 词：小鼠 着床 表达 丝氨酸蛋白酶 融合蛋白质 DNA 抗体制备 克隆 3′非翻译区 开放阅读框 

分 类 号：Q78[生物学—分子生物学] R392[医药卫生—免疫学]