Thatcher M D, Shille V M, Fliss M F, Bazer F W, Sisum W, Randal S
College of Veterinary Medicine, University of Florida, Gainesville 32610.
Biol Reprod. 1991 Jan;44(1):108-20. doi: 10.1095/biolreprod44.1.108.
This study characterized proteins secreted de novo by feline conceptuses collected on Days 10, 12, and 15 (n = 22, preimplantation blastocysts); Days 15, 16, 17, 19, 21, and 25 (n = 6, postimplantation zonary girdle [ZG] i.e. trophoblast and endometrium); and Days 30, 36, 39, and 50 (n = 5, postimplantation ZG and free chorioallantois [CA]) and cultured in Minimal Essential Medium. De novo secretion was shown by incorporation of 3H-leucine into proteins detected in culture media by 2D-PAGE and fluorography. Western blotting, and NH2-terminal amino acid microsequencing. Major radiolabeled proteins identified as they appeared temporally on fluorographs were as follows: feline conceptus protein 1 (fCP1), Mr = 20,000, pI 5.0-5.3; fCP2, Mr = 80,000, pI 6.5-7.2; fCP3a, Mr = 67,000, pI 6.3-6.5; fCP3b, Mr = 67,000, pI 5.9-6.3; fCP4, Mr = 56,000, pI 5.0-6.0; and fCP5, Mr = 29,000, pI 5.0-5.8. The fCP1 was produced by blastocysts on Days 10-15, ZG on Days 16-25, and CA on Day 30; on Days 39-50, CA synthesized 5 proteins, possibly fCP1 isomers. The fCP2, fCP3a and b, and fCP4 were produced by blastocysts on Day 15, ZG on Day 25, and CA on Days 30-50. The fCP5 was made by ZG on Days 16-36 and by CA on Days 30-39. Western blotting identified fCP1 as retinol-binding protein (RBP), fCP2 as alpha fetoprotein, fCP3a as albumin, and fCP3b as transferrin. Amino acid sequence homologies between fCP1 and rabbit and human plasma RBP and porcine conceptus RBP2 were 93, 96, and 100%, respectively, at the first 37 NH2-terminal amino acids. The identities of fCP4 and fCP5 have not been established. Antiviral activity detected in all media was less than 3 units/ml when tested with feline fibroblast cells infected with vesicular stomatitis virus.
本研究对在第10、12和15天收集的猫科动物孕体（n = 22，植入前囊胚）；第15、16、17、19、21和25天收集的孕体（n = 6，植入后带状环[ZG]，即滋养层和子宫内膜）；以及第30、36、39和50天收集的孕体（n = 5，植入后ZG和游离绒毛膜尿囊[CA]）分泌的新生蛋白质进行了表征，并在最低必需培养基中培养。通过将³H-亮氨酸掺入二维聚丙烯酰胺凝胶电泳（2D-PAGE）和荧光显影、蛋白质免疫印迹以及氨基末端氨基酸微测序检测到的培养基中的蛋白质，证实了新生分泌。在荧光照片上按时间顺序出现的主要放射性标记蛋白质如下：猫科动物孕体蛋白1（fCP1），相对分子质量（Mr）= 20,000，等电点（pI）5.0 - 5.3；fCP2，Mr = 80,000，pI 6.5 - 7.2；fCP3a，Mr = 67,000，pI 6.3 - 6.5；fCP3b，Mr = 67,000，pI 5.9 - 6.3；fCP4，Mr = 56,000，pI 5.0 - 6.0；以及fCP5，Mr = 29,000，pI 5.0 - 5.8。fCP1由第10 - 15天的囊胚产生，第16 - 25天的ZG产生，第30天的CA产生；在第39 - 50天，CA合成了5种蛋白质，可能是fCP1的异构体。fCP2、fCP3a和b以及fCP4由第15天的囊胚产生，第25天的ZG产生，第30 - 50天的CA产生。fCP5由第16 - 36天的ZG产生，第30 - 39天的CA产生。蛋白质免疫印迹鉴定fCP1为视黄醇结合蛋白（RBP），fCP2为甲胎蛋白，fCP3a为白蛋白，fCP3b为转铁蛋白。在最初的37个氨基末端氨基酸处，fCP1与兔和人血浆RBP以及猪孕体RBP2的氨基酸序列同源性分别为93%、96%和100%。fCP4和fCP5的身份尚未确定。当用感染水疱性口炎病毒的猫科动物成纤维细胞进行测试时，在所有培养基中检测到的抗病毒活性均低于3单位/毫升。
