奶牛子宫内膜组织抗菌肽分离纯化及其抗菌活性研究

目的：研发奶牛抗菌肽生物制剂,防治奶牛子宫内膜疾病。方法：采用酸性尿素聚丙烯酰胺凝胶电泳和琼脂糖弥散法检测子宫内膜组织酸溶性提取物抗菌活性;应用琼脂糖电泳及水平电泳洗脱分离出相应的抗菌条带,HPLC进一步分离纯化,琼脂糖弥散法检测纯化物的抗菌活性,Tricine-SDS-PAGE电泳检测其分子量。结果：粗提物中有两种主要的抗菌成分,其对金黄色葡萄球菌26003、大肠杆菌BL-21、牛致病性大肠杆菌4605、绿脓杆菌10104具有抗菌活性。上层条带的主要色谱峰出现在43、45 min,其中45 min收集的样品有较强的抗菌活性,其相对分子质量约为14 k Da。下层条带主要色谱峰出现在5、35、43、45、46、47、50 min,其中35、43、45 min收集的样品有抗菌活性,其相对分子质量分别约为14-16、6和12 k Da。各个组分对大肠杆菌BL-21和金黄色葡萄球菌26003均具有抑菌活性。结论：从奶牛子宫内膜组织分离出了5个抗菌成分,从相对分子质量、抗菌活性等方面初步认定为抗菌多肽。     

嗜驼璃眼蜱抗菌肽的电泳分析及抗菌活性研究

为探讨嗜驼璃眼蜱血淋巴中抗菌肽的电泳分析方法及其抗菌作用，从半饱血的嗜驼璃眼蜱雌蜱体内抽取血淋巴，用50mL／L的乙酸抽提，经AU—PAGE分析嗜驼璃眼蜱血淋巴中阳离子多肽成分，用电泳凝胶琼脂糖弥散法对各多肽进行抗菌活性的筛选。结果表明，嗜驼璃眼蜱血淋巴中阳离子多肽成分对大肠埃希菌和金黄色葡萄球菌均有很强的抗菌作用。AU—PAGE发现一电泳迁徙率明显高于溶菌酶的阳离子抗菌多肽。提示在嗜驼璃眼蜱的血淋巴中，存在与溶菌酶不同的抗菌多肽，可能是嗜驼璃眼蜱抵御感染的重要分子。AU—PAGE是一种较简单、快速、经济的分离纯化抗菌肽的方法。     

不同乳酸菌分离株抗菌物质及其抗菌活性的体外检测及鉴定

为检测及鉴定乳酸菌分离株的抗菌物质,本研究采用牛津杯法测定乳酸菌的抗菌活性,通过上清液煮沸、过氧化氢排除试验、蛋白类排除试验、有机酸排除试验检测并鉴定了本实验室前期从健康奶牛阴道内分离获得的5株酸菌的抗菌物质。结果显示,这5株乳酸菌对指示菌均具有良好的抗菌活性。坚强乳杆菌、粪肠球菌、屎肠球菌和巴黎链球菌的主要抗菌物质为有机酸,黏膜乳杆菌的抗菌物质为过氧化氢、蛋白和有机酸。本研究表明黏膜乳杆菌可以初步作为防治奶牛子宫内膜炎的潜在益生菌使用。     

奶牛子宫内膜上皮细胞和间质细胞的组织块培养及鉴定

奶牛子宫内膜细胞是研究奶牛子宫内膜生理和病理机制的重要体外模型,国外关于奶牛子宫内膜细胞的分离和培养的研究很多[1-3],而国内尚未见相关报道.本研究选用荷斯坦奶牛的健康子宫内膜,应用组织块培养法培养并分离间质细胞和上皮细胞.为从细胞和分子水平进一步研究奶牛子宫内膜提供必要的条件.     

健康奶牛生殖道乳酸菌的分离鉴定及其抑菌活性研究

【目的】 从健康奶牛生殖道分离筛选益生乳酸菌,为未来防治奶牛子宫内膜炎提供益生菌菌株并为开发防治此病的微生态制剂奠定基础。【方法】 采集10份健康奶牛的生殖道分泌物样品,用MRS培养基进行分离培养,通过形态学观察和16S rDNA序列对分离出的菌株进行鉴定,将所得序列结果与NCBI核酸数据库参考菌株进行相似性比对,确定各分离菌株的种属。利用牛津杯法分离筛选出具有较好抑菌活性的菌株,研究这些筛选出的菌株的生长曲线、产酸能力及抗菌药敏感性,并对其主要抗菌物质进行检测。【结果】 从10个样品中共分离出13株乳酸菌,经染色镜检和16S rDNA测序分析鉴定出13株乳酸菌,分别为植物乳杆菌11株、殊异肠球菌和鼠肠球菌各1株。单株抑菌试验得到5株对大肠杆菌抑菌能力较好的乳酸菌,5株乳酸菌生长特性及产酸能力均良好,对氨苄西林、四环素、红霉素、利福平、甲氧苄啶、氯霉素和青霉素7种抗菌药物敏感性较强。其中21和35号菌株的抗菌物质为过氧化氢和细菌素,而25、33和34号菌株的抗菌物质除过氧化氢和细菌素外还包括有机酸。【结论】 从奶牛生殖道中分离得到的3株乳酸菌有望作为微生态制剂用于奶牛子宫内膜炎的临床防治,其具体防治效果有待进一步探究。     

日本鳗鲡脾脏抗菌肽抗菌活性检测方法的比较

本试验旨在建立一种在日本鳗鲡脾脏抗菌肽分离纯化过程中简便、灵敏的检测其抗菌活性的方法。试验以日本鳗鲡脾脏分子质量小于10 ku的蛋白质为抗菌肽样品,选择琼脂板孔穴扩散法和微孔液体培养法2种传统方法及微量液体培养法为检测方法,比较这3种方法在抗菌肽对3种鳗鲡常见致病菌株(迟钝爱德华菌、气单胞菌、嗜水气单胞菌)抗菌活性检测中的灵敏度和优缺点。结果表明:与琼脂板孔穴扩散法及微孔液体培养法相比,微量液体培养法简便、易于观察结果,具有最高的灵敏度且样品用量最少,适合在鳗鲡脾脏抗菌肽分离纯化过程中跟踪检测抗菌活性,尤其适合分离纯化后期获得微量单一样品抗菌活性的检测。由此得出,微量液体培养法适用于检测日本鳗鲡脾脏抗菌肽的抗菌活性。     

奶牛子宫内膜细胞体外培养及应用研究概况

子宫内膜细胞体外培养技术在研究动物子宫内膜生理功能、繁殖障碍疾病机制及治疗药物筛选等过程中具有重要的作用和意义。论文就国内外有关奶牛子宫内膜细胞体外培养技术中常用的分离、纯化和鉴定等方法以及该细胞体外模型相关应用进行综述,为探索简便、高效的奶牛子宫内膜细胞体外培养方法及国内有关奶牛子宫内膜生理病理的分子细胞学研究提供参考。     

健康奶牛阴道乳酸菌分离鉴定及作为候选益生菌株的体外评价

为分离健康奶牛阴道乳酸菌并鉴定其益生特性,本研究采集健康奶牛阴道临床样品并分离筛选到4株乳酸菌,对其抑菌活性、产酸性能、产H_2O_2能力及对抗生素敏感性进行检测,通过16S rDNA序列分析鉴定乳酸菌分离株。结果显示,4株乳酸菌具有较好的抑菌活性和产酸性能,在体外均能够产生H_2O_2,并对万古霉素具有抗性;经鉴定4株乳酸菌分别为唾液乳杆菌1株、黏膜乳杆菌2株、坚强肠球菌1株。本研究分离鉴定的4株乳酸菌均可以作为候选益生菌株用于奶牛子宫内膜炎防治。     

多黏类芽孢杆菌抗菌肽的分离纯化及特性研究

试验旨在分离纯化多黏类芽孢杆菌（Paenibacillus polymyxa）BLCC1-0402代谢产物中的抗菌肽,为抗菌肽制备及其制品检测提供参考。采用离心、不同分子质量卷式膜超滤浓缩、Superdex peptide 10/300GL凝胶过滤层析对多黏类芽孢杆菌发酵上清液进行逐级分离纯化,对不同时段的收集液做抑菌试验,以大肠杆菌O78标准菌株为指示菌,采用打孔法进行抑菌活性检测,比较评价分步层析效果,以Tricine-SDS-PAGE进行分子质量检测。结果显示,通过5和3 ku卷式膜超滤获得的3~5 ku组分蛋白质样品抗菌活性较强;对于3~5 ku组分经凝胶过滤层析分离纯化,纯化后的抗菌肽A3抑菌活性最强,经Tricine-SDS-PAGE小分子多肽电泳检测,已达到电泳纯,分子质量为4 ku;抑菌活性检测结果显示,该抗菌肽A3对大肠杆菌O78标准菌株具有较强的抑菌作用。同时,抗菌肽A3表现出较好的耐热性,90~100 ℃处理15 min,抑菌活性可保持在96%左右;具有较好的酸碱稳定性,在pH 2.0~9.0下,抑菌活性保持在90%以上;经胃蛋白酶作用后抗菌肽A3抑菌活性降低20%,胰蛋白酶作用后抗菌肽A3抑菌活性降低18%,蛋白酶K对抗菌肽A3的抑菌活性几乎无影响。本研究结果表明,分离得到的抗菌肽A3是一种对大肠杆菌O78具有抑菌活性的新型抗菌肽,具有一定的开发潜力,可为下一步抗菌肽的结构分析、理化特性分析等深入研究提供一定参考依据。     

奶牛子宫内膜上皮细胞和间质细胞的分离、培养及鉴定

为了分离培养奶牛子宫内膜上皮细胞和间质细胞,给建立奶牛子宫内膜体外模型提供条件,研究采用胰蛋白酶和胶原酶消化法分离细胞,随后传代培养纯化细胞,并通过光镜观察和免疫细胞化学染色对其进行鉴别.结果表明:0.3%胰蛋白酶消化组织块1 h后可得到上皮细胞,纯度＞80%;上皮细胞呈多边型,核大而圆,具有细胞角蛋白免疫反应性.用0.1%胶原酶Ⅱ继续消化2 h,可得到间质细胞,纯度＞90%;间质细胞贴壁后可见梭形和多角形两种形态,免疫细胞化学染色显示这两种细胞均具有波形蛋白免疫反应性,而细胞角蛋白无免疫反应性,提示它们为来源于内膜基质的基质细胞,说明采用酶消化法能够成功地培养奶牛子宫内膜间质细胞和上皮细胞.     

乌鳢体表粘液抗菌肽CSM14的分离纯化及部分生物学活性研究

为建立分离纯化乌鳢体表粘液抗菌肽的方法并研究其抗菌活性,本研究通过嗜水气单胞菌对乌鳢进行刺激,采用甲醇提取、SephadexG-50凝胶层析和反向高效液相色谱从其体表粘液中提取抗菌肽,经质谱分析测定分子量;并对抗菌肽的抑菌谱、最小抑菌浓度(MIC)、热稳定性、溶血活性进行了初步研究。结果表明:分离纯化的乌鳢体表粘液抗菌肽分子量约为3024.15ku,并具有广谱的抗菌活性,热稳定性好,没有溶血活性。     

Signal transduction systems mediated by protein kinase-C and estradiol receptors in cow placenta and caruncle.

Characteristics of signal transduction systems mediated by protein kinase-C (Ca2+/phospholipid-dependent protein kinase) and estradiol receptors in cow placenta and caruncle were conducted by evaluating protein kinase-C activity and estradiol receptor concentrations and by exploring substrate proteins for the enzyme. The enzyme activity was detected in cytosolic and total particulate fractions of both tissues. The activity levels in these fractions were comparable to those in other cow tissues such as liver and mammary gland. The enzyme activity was inhibited by palmitoylcarnitine, gossypol and adriamycin, known phospholipid-interacting inhibitors of the enzyme. Phosphorylation by the enzyme and subsequent autoradiography revealed that only 125K protein in placental cytosol and two low molecular weight proteins in caruncular cytosol were found to be substrates for protein kinase-C. Ca2+ acts as inhibitor of the phosphorylation of several phosphoproteins other than the substrates. Estradiol receptor concentrations in cytosolic and nuclear fractions were similar in both tissues, and the cytosolic concentrations were also comparable to those in pregnant uterus. However, the nuclear concentrations were extremely low when compared to those in the uterus. The signal transduction systems mediated by protein kinase-C and by estradiol receptors seem to be concertedly suppressed in cow placenta and caruncle.     

不孕奶牛宫颈黏液相关ASA免疫印迹分析

为探讨和分析免疫性不孕奶牛和可孕奶牛宫颈黏液ASA能够识别的精子膜抗原及其差异,本试验通过提取精子膜总蛋白,对24头免疫性不孕奶牛和14头可孕奶牛宫颈黏液ASA能够识别的精子抗原进行了比较分析。免疫印迹试验结果显示,不孕奶牛宫颈黏液ASA对分子质量为88-90ku、66ku、55~57ku、25-27ku的精子膜抗原有特殊免疫性反应,识别抗原率为100％（24／24）、100％（24／24）、87．5％（21／24）、70．8％（17／24）;而可孕奶牛宫颈黏液AsA对分子质量为88-90ku、55-57ku、25-27ku的识别率仅为21．4％（3／14）、14．2％（2／14）、14．2％（2／14）,可孕奶牛宫颈黏液不能识别分子质量为66ku#~精子膜抗原。结果表明,不孕奶牛宫颈黏液ASA~g够识别更多的精子膜抗原,这些精子膜抗原可能与免疫性不孕有密切相关,对其深入的研究将有助于免疫性不孕相关精子膜抗原的筛选和鉴定。     

Uterine secretions of the cow contain proteins that are immunochemically related to the major progesterone-induced proteins of the sheep uterus

Proteins that cross-react with antiserum to the major progesterone-induced proteins found in the pregnant sheep uterus, the uterine milk proteins (UTM-proteins), were detected as radiolabelled secretory products of endometrium from pregnant cows. Cross-reactive proteins included a form at 57,000 molecular weight as well as other lower-molecular-weight variants found in lower amounts. Similar proteins were also detected in uterine fluid from a cow at day 270 of gestation. Using immunohistochemical procedures, proteins that cross-reacted with antiserum to sheep UTM-proteins could be localized to the epithelial cells of endometrial glands in the cow. Results indicate that UTM-protein-like molecules are secreted by the endometrium of the cow during pregnancy.     

大口黑鲈不同组织中抗菌肽粗提及抑菌活性测定

研究了大口黑鲈不同组织中抗菌肽的提取及其粗提物对不同菌种的抑菌作用。以新鲜的大口黑鲈为试验材料,用5％乙酸作为提取液,对大口黑鲈粘液、皮肤、肝脏、脾、卵、鳃组织进行提取,并对粗提物进行抑菌活性检测。结果表明大口黑鲈粘液、皮肤和肝脏组织提取物对嗜水气单胞菌有抑菌作用,皮肤和肝脏组织提取物对大肠杆菌有抑菌作用。肝脏组织抗菌肽提取物经SephadexG-25凝胶过滤层析柱分离后,其收集液对嗜水气单胞菌仍具有抑菌作用。大口黑鲈皮肤和肝脏组织提取物对嗜水气单胞菌、大肠杆菌均有抑菌作用。     

Effects of Hemophilus somnus on the pregnant bovine reproductive tract and conceptus following cervical infusion

Three strains of Hemophilus somnus were infused into the posterior cervix of six pregnant cows. The organism persisted in the cervicovaginal region for eight to 87 days, and at parturition H. somnus was isolated from chorioallantois in four of six cows; placentitis developed, and fetal membranes were retained. All calves were born alive and no H. somnus was recovered from them. One cow died 14 days after parturition. The death was attributed to severe necrotizing metritis; H. somnus was not isolated from the uterus at death, but was isolated from the placenta at parturition and cervicovaginal mucus two days days later.     

赤点石斑鱼血清与皮肤黏液的SDS-PAGE蛋白电泳分析

以正常生活状态下的非免疫赤点石斑鱼(Epinephelus akaara)为材料,采用SDS-PAGE蛋白电泳技术,分析和比较了全血清和皮肤黏液中的蛋白区带数。结果表明:赤点石斑鱼血清与皮肤黏液蛋白图谱不完全相同,其中相同的蛋白区带数为9条,血清中特有蛋白带为16条,皮肤黏液中特有蛋白带为10条。计算机辅助分析蛋白图谱,结果提示:赤点石斑鱼血清及皮肤黏液中免疫球蛋白(Ig)的轻链大小为25.0 ku,重链为73.2 ku。该试验为进一步研究赤点石斑鱼皮肤黏液Ig的理化性质奠定基础。     

Separation of mounting-inducing pheromones of vaginal mucus from estrual heifers

There is evidence that mucus of the female bovine genital tract contains pheromones that induce physiological and behavioral responses in other animals. To study these pheromones, vaginal mucus was collected from heifers either at estrus or during diestrus. The mucus was then applied to the hindquarters of the same animal during diestrus or to the hindquarters of herdmates during diestrus. The behaviors of the treated animal and its herdmates were then observed. To attempt to isolate the mounting-inducing substance, mucus was dialyzed or separated on ion-exchange resins. Diestrous heifers to which their own estrual mucus has been applied were nearly always mounted by herdmates (P less than .01). But, heifers to which another's estrous mucus had been applied were not mounted. This suggests that vaginal mucus contains not only estrus-related pheromones, but also individual distinctive odors. The dialyzable fraction of vaginal mucus and the neutral fraction, prepared by ion-exchange chromatography of the dialyzable solution of vaginal mucus, had a mounting-inducing activity on the herdmates, as did the application of an animal's own vaginal mucus. These findings suggest that mounting-inducing pheromones are relatively low molecular weight, neutral substances.