人输卵管蛋白全长cDNA克隆及真核细胞中的表达

Cloning of Human Oviductin cDNA and Its Expression in HeLa Cells

目的:克隆人输卵管蛋白(hOviductin)全长cDNA序列,真核细胞中表达,并了解hOviductin是否与大鼠卵母细胞结合。方法:构建人输卵管黏膜上皮细胞cDNA文库,以^(32)P标记的兔输卵管蛋白全长cDNA序列为探针,自文库中筛选hOviductin全长cDNA序列。将获得的hOviductin编码区cDNA序列插入pEGFP-N1真核表达载体,转染HeLa细胞,表达分泌重组的绿色荧光蛋白(EGFP)-hOviductin,并估量其浓度。激光扫描共聚焦显微镜下观察EGFP-hOviductin与大鼠卵巢的卵丘细胞-卵母细胞复合物(COC)或去除透明带后的裸卵的结合情况。结果:所构建的cDNA文库重组率为98.5％,滴度为1.1×10~6pfu/mL。克隆所得hOviductin全长cDNA约为2500 bp,Gen-Bank的登录号为AY189737。EGFP-hOviductin在条件培液中的浓度为0.236 nmol/L,能结合在去透明带的大鼠裸卵质膜外表面,而未见其与COC结合。结论:hOviductin能在HeLa细胞中表达分泌,其分泌产物可结合于大鼠裸卵质膜外表面。

Objective To clone full-length cDNA of human oviductin (hOviductin), express the recombinant hOviductin in eukaryotie cells and determine whether hOviductin, can bind to rat oocyte. Methods: A cDNA library was constructed using oviductal mucosa epithelial cells (OMEC) derived from hysterec- tomized patients, and the transcripts of hOviductin gene in the total RNA derived from OMEC were analysed by Northern blotting method Using ^(32)P-labelled rabbit oviductin cDNA as probe. The probe was used to screen hOviductin cDNA from the cDNA library. The cDNA fragment containing open reading frame derived from hOviductin cDNA was inserted in to eukaryotic vector pEGFP-N1 to construct pEGFP-N1/hOviductin, which then was transfected into HeLa cells to express and secrete EGFP-hOviductin fusion protein. Rat ovarian cumulus cells-oocyte complexes (COC) and zona pellucidae-free nude oocyte were incubated in the transfected HeLa cells conditioned medium (TCM) to observe the binding sites of EGFP-hOviductin onto COC or nude oocyte under laser scanning confocal microscope. Results: The constructed human OMEC cDNA library contained 98.5% recombinants; the titer of the phages in the cDNA library was 1.1×10~6 pfu/mL. The result of Northern blotting analysis revealed that the mRNA of hOviductin gene is about 2.5 kb. The full-length hoviductin cDNA was secreened out of the cDNA library and cloned, and has been submitted to GenBank (Accession No. : AY189737). The transfected HeLa cells expressed and secreted EGFP-hoviductin whose concentration in TCM was 0. 236 nmol/L. We observed hoviductin situated at the outer surface of rat nude oocyte membrane, but not at the cumulus cells and zona pellucidae. Conclusion: hOviductin could be expressed and secreted in HeLa cells, and bind to the outer surface of rat nude oocyte membrane. This study was a precursor work for the further study on the effect and mechanism of hOviductin in early embryo development.