Fractionation of bovine spermatozoa for sex selection: A rapid immunomagnetic technique to remove spermatozoa that contain the H-Y antigen

A study was conducted to rapidly fractionate bovine spermatozoa on the basis of cell-surface H-Y antigen (i.e., Y chromosome-bearing spermatozoa). A novel, rapid immunomagnetic method was developed for removal of spermatozoa that bound to anti-H-Y IgG. Fluorescent labeling and flow cytometry were used to measure the efficiency with which spermatozoa binding to anti-H-Y were removed by the immunomagnetic technique. Washed bovine spermatozoa (n=7 bulls) were treated with a mouse monoclonal IgG antibody to H-Y antigen (MoAb 12/49). Fluorescent labeled goat antibody against mouse IgG was added to label those spermatozoa with cell-surface H-Y antigens. Supermagnetized polymer beads coated with an anti-antibody to the MoAb 12/49 were then added to the spermatozoa. After 20 min of incubation, spermatozoa were exposed for 2 min to a magnet, causing the magnetized particles to adhere to the sides of the tube. Nonmagnetized spermatozoa in the supernatent were aspirated and analyzed for fluorescent label by flow cytometry. Approximately 50% of spermatozoa not subjected to immunomagnetic separation were fluorescent labeled, and about one-half of the spermatozoa were observed microscopically to be bound to the magnetized polymer beads prior to magnetic separation (P<0.05). Following magnetic separation, only 1.2% (P<0.05) of the spermatozoa in the magnetic supernatent were fluorescent labeled. Assuming that only Y chromosome-bearing spermatozoa have cell-surface H-Y antigens, the present immunomagnetic fractionation removed almost all of the Y chromosome-bearing spermatozoa, leaving a population that was greater than 98% X chromosome-bearing spermatozoa.     

鸡马立克氏病病毒B抗原片段在大肠杆菌中表达

鸡马立克氏病病毒ＢａｍＨＩ基因文库Ｉ３质粒中含有编码Ｂ抗原膜外Ｄｏｍａｉｎ的ＤＮＡ序列。经ＳｃａＩ和ＳｐｈＩ双酶酶解Ｉ３质粒,分离获得７６４ｂｐＤＮＡ片段,并克隆进Ｍ１３ｍｐ１９中。ＤＮＡ序列测定分析表明克隆片段为ＭＤＶ－Ｂ抗原基因的４９４－１２５８ｂｐ部分序列。进一步分离ＮｃｏＩ－ＨｉｎｄＩＩＩ部分基因片（５３０ｂｐ）,克隆于ＰＬＰｒｏｍｏｔｅｒ控制下的含有修饰型ｃＩｔｓ８５７基因的表达载体中,     

不同感染状态下EB病毒核抗原亚型表达的研究

本实验用免疫印迹法纯化的抗ＥＢＮＡ亚型抗体,结合显微荧光分光光度检测技术,检测在不同感染状态下,三种亚型ＥＢＮＡ抗原在细胞中的表达程度。结果表明,处于潜伏感染状态下的Ｒａｊｉ细胞ＥＢＮＡ－１表达量较大,经巴豆油和正丁酸诱导进入钝挫感染状态后ＥＢＮＡ－１表达减少,而ＥＢＮＡ－２的表达增强。Ｂ＿（９５－８）细胞也有相似的趋势。表明ＥＢ病毒的激活可能与不同ＥＢＮＡ亚型表达量的改变有关。     

人工会成柞蚕杀菌肽D基因的克隆及在大肠杆菌中的表达

以质粒pWR450为载体,克隆了人工合成的柞蚕杀菌肽D基因(122bp),构建的重组子pWR450-Cec转化大肠杆菌JM103、用限制性内切酶酶切鉴定。产物经SDS-聚丙烯酰胺凝胶电泳,结果显示可表达杀菌肽-β-gal融合蛋白。     

Tissue Fixation for Immunohistochemical Detection of Proliferating Cell Nuclear Antigen with PC10 Monoclonal Antibody

PC10 is a monoclonal antibody to proliferating cell nuclear antigen, a nuclear protein associated with the cell cycle. We have evaluated the effects of tissue fixation on PC10 immunoreactivity in sections of paraffin embedded rat tissues. Immunoreactivity was well preserved in tissues after fixation with alcohol-based solutions for 3-24 hr. Fewer PC10-positive cells were detectable in samples fixed with formaldehydecontaining solutions compared with samples fixed with alcohol for the same time. Loss of PC10 immunoreactivity in formaldehyde fixed tissues was progressive, and quantifiable as early as after 3 hr fixation. Consequently, alcohol-based fixatives are strongly recommended for any immunocytochemical prospective study using PC10 antibody. In contrast, loss of PC10-immunoreactivity is always predictable, but difficult to quantitate, using formaldehyde fixed specimens. This aspect should be considered when using PC10 antibody in retrospective studies with routinely-processed archival material.     

Avian epidermis contains ATPase- and Ia-positive Langerhans-like cells

Summary The occurrence of cells resembling mammalian Langerhans cells in the avian epidermis was studied by ATPase histochemistry, Ia immunoreactivity and electron microscopy. The existence of MHC class II antigen-(Ia) expressing, ATPase-positive dendritic cells, which are ultrastructurally similar to mammalian Langerhans cells except for the absence of Birbeck granules, was demonstrated. These cells may be a basic component of the immune system of birds.     

上皮生长因子受体和癌胚抗原分子内血型Y配基表达的免疫生化分析

以抗CEA、EGF-γ和血型前体Y配基抗原的单抗进行分析,结果表明:结肠癌组织中表达CEA和血型前体Y配基较强,而EGF-γ表达水平极低;进一步鉴定分析,识别血型前体Y配基抗原决定簇的单抗C_(14)与肿瘤具有较好的反应性。并发现这种决定簇存在于各种生物大分子中,包括以糖蛋白和糖脂形式存在,并表达于EGF-γ与CEA分子中。这一结果提示血型前体抗原过量表达于肿瘤细胞中,不但是血型组织相容抗原,可能也参与到一些特殊生物分子中,应深入研究和鉴定其功能。     

Binding of anti-H-Y monoclonal antibodies to separated X and Y chromosome bearing porcine and bovine sperm

Studies designed to answer the question whether or not H-Y antigen is preferentially expressed on Y chromosome bearing sperm have resulted in conflicting results. This is probably due to the absence of reliable methods for estimating the percentage of X and Y chromosome bearing sperm in fractions, enriched or depleted for H-Y antigen positive sperm. In recent years a reliable method for separating X and Y chromosome bearing sperm has been published. With this method, separation is achieved by using a flow cytometer/cell sorter, which detects differences in DNA content. This technique provided the first opportunity for testing anti-H-Y antibody binding to fractions enriched for X and Y chromosme bearing sperm, directly. A total of 7 anti-H-Y monoclonal antibodies were tested using sorted porcine sperm and in one experiment also sorted bovine sperm. All monoclonal antibodies bound only a fraction of the sperm (20 to 50%). However, no difference in binding to the X and Y sperm enriched fractions was found. Therefore, the present experiments do not yield evidence that H-Y antigen is preferentially expressed in Y chromosome bearing sperm. © 1993 Wiley-Liss, Inc.