[Cloning, expression, and antibody production of mouse ISP2].

Zhe-Ping Huang, Jian Wang, Hao Yu, Wei-Xiong Shen, Ping Huang, Jia-Ke Tso, Zeng-Ming Yang, Qing-Xiang Shen
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Abstract

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.

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小鼠ISP2的克隆、表达和抗体产生。
利用PCR方法从妊娠第4.5天小鼠子宫着床部位的总cDNA中扩增出一段编码小鼠着床丝氨酸蛋白酶2 ISP2的cDNA,并对其进行测序。A442918。DNA测序结果显示,ISP2 cDNA在3'区除了编码279个氨基酸残基的开放阅读框外,还有一条未报道的204 bp序列,与文献一致。为了获得重组ISP2,构建了表达质粒pGEX-4T-2/ISP2,并将其转化为大肠杆菌BL21(DE3)菌株。表达的融合蛋白GST-ISP2经SDS-PAGE纯化,用凝血酶酶切,再进行SDS-PAGE酶切,回收rISP2。以rISP2为免疫原免疫家兔,获得抗isp2多克隆血清。免疫组化分析显示,ISP2在妊娠早期小鼠子宫内膜上皮中特异性高表达。
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