非洲猪瘟病毒p30蛋白单克隆抗体制备及线性抗原表位定位

【目的】制备非洲猪瘟病毒（African swine fever virus,ASFV）p30蛋白的单克隆抗体（monoclonal antibodies,MAbs）并初步分析其所识别的线性抗原表位,为ASFV及其抗体检测方法的建立及p30蛋白结构和功能的研究奠定基础。【方法】将原核表达并纯化的p30重组蛋白作为免疫原,免疫6—8周龄BALB/c雌鼠,每两周免疫1次,共免疫3次,首次免疫是抗原与等体积的弗氏完全佐剂乳化后免疫,第二次和第三次免疫与等体积的弗氏不完全佐剂乳化,3次免疫后1 w断尾采血,间接酶联免疫吸附试验（ELISA）检测血清抗体效价,选择血清效价最高的小鼠进行加强免疫,3 d后取小鼠脾淋巴细胞与SP2/0骨髓瘤细胞按照4﹕1的比例使用PEG进行常规细胞融合。利用重组p30蛋白作为包被抗原,间接ELISA筛选阳性杂交瘤细胞,有限稀释法进行克隆纯化,直至筛出能够稳定分泌抗体的MAbs。将ASFV接种于猪肺泡巨噬细胞,以筛选的MAbs为一抗、兔抗鼠HRP-IgG为二抗,进行间接免疫荧光试验（IFA）。将感染和未感染ASFV的细胞沉淀处理后进行 SDS-PAGE并转印至硝酸纤维素膜,分别以IFA鉴定为阳性的MAbs上清为一抗、兔抗鼠HRP-IgG为二抗,进行Western blotting分析,筛选获得p30 MAbs。根据已知序列设计引物扩增p30ab与p30bc两段截短基因,其中p30ab代表由第86—153位氨基酸残基的截短体,p30bc代表由第120—187位氨基酸残基的截短体,原核表达部分重叠的截短p30蛋白,最终获得重组蛋白GST-p30ab与重组蛋白GST-p30bc。分别以GST-p30ab和GST-p30bc融合蛋白为包被抗原,以5株MAbs为一抗,以兔抗鼠HRP-IgG为二抗, 通过间接ELISA方法初步定位p30蛋白的抗原表位。【结果】以纯化的重组蛋白为包被抗原,经间接ELISA试验筛选出25株可分泌抗重组 p30蛋白的杂交瘤细胞株。IFA结果显示,5株MAbs（8F4、1D3、1H2、6C3和8E11）与ASFV感染的猪肺泡巨噬细胞IFA 试验呈阳性;Western blotting结果显示,5株MAbs均能够与ASFV感染的细胞呈阳性反应,与未感染病毒的细胞呈阴性反应。试验构建的p30截短体重组蛋白GST-p30ab以可溶和包涵体两种形式表达,而GST-p30bc仅以包涵体形式表达,以两组截短体融合蛋白为包被抗原,通过间接ELISA检测出MAbs 8F4、1H2和6C3与两个重组蛋白均能有效结合,证明MAbs 8F4、1H2和6C3抗原识别区域为两组截短蛋白重叠区域,即第120—153位氨基酸;MAbs 8E11与1D3则只能与GST-p30ab蛋白结合, 证明MAbs 8E11与1D3抗原识别区域为两个重组蛋白的非重叠区域,即第86—119位氨基酸。【结论】本研究可溶性地表达了p30蛋白的第86—153位氨基酸截短体重组蛋白,制备了5株p30 MAbs,定位到2个p30蛋白抗原表位。结合ELISA和IFA,可建立十分可靠的ASFV及其抗体的检测手段。     

天山北坡典型草地土壤呼吸特征及其对环境因子的响应

为揭示天山北坡不同类型草地的土壤呼吸规律,采用土壤碳通量测量系统LI-840A对天山北坡荒漠草原和灌丛草甸土壤呼吸（soil respiration,R_s）日变化和季节动态进行监测,并利用多元回归模型分析温度和水分对草地土壤呼吸的协同影响。结果表明,2种草地类型生长季土壤呼吸速率均表现为日间先增高后降低,夜间较平缓的日变化趋势,日最大排放速率出现在12:00—16:00,最小值出现在6:00—7:00左右;灌丛草甸生长季各个月份的土壤呼吸速率均高于荒漠草原。2种草地类型土壤呼吸速率与温度均呈指数相关;土壤含水量与灌丛草甸土壤呼吸速率的相关性高于与荒漠草原土壤呼吸速率的相关性;土壤呼吸速率受5 cm土壤温度（T）和5 cm土壤含水量（W）的影响显著,荒漠草原土壤呼吸速率与二者之间满足最佳拟合模型R_s=130.515e^0.031TW^2.513,灌丛草甸满足最佳拟合模型R_s =-1.290+0.010T+28.007W+1.199TW。研究结果揭示了荒漠草原和灌丛草甸土壤呼吸的变化规律,为天山北坡草地碳循环研究提供基础数据和理论支持。     

库布齐沙漠北缘不同人工灌木林地土壤肥力质量状况

在库布齐沙漠北缘杭锦旗段选择柠条、沙柳、花棒和杨柴等4种人工灌木林为对象,以流动沙地为对照,研究不同人工灌木林下土壤理化性质、微生物数量和酶活性特征及其相互间的关系,并运用综合指数法对各人工灌木林土壤综合质量进行评价。结果表明:1）总体上,各人工灌木林地土壤容重、蒸发速率和pH值低于流沙地,土壤含水量、入渗率、田间持水量、总孔隙度、速效N、速效P、速效K和有机质含量高于流沙地,土壤盐分与流沙地无显著差异,不同人工灌木林对土壤理化特性的改善存在差异。2）各人工灌木林地土壤酶活性均强于流沙地,总体而言,柠条林地和沙柳林地的建植对土壤酶活性影响较大,花棒林地和杨柴林地的建植对土壤酶活性影响较小。磷酸酶与脲酶、蔗糖酶、多酚氧化酶呈极显著正相关,蔗糖酶与脲酶、多酚氧化酶间极显著正相关。3）各人工灌木林土壤微生物数量均显著多于流沙地,柠条和沙柳林地土壤微生物数量多于花棒和杨柴林地;各类人工灌木林地和流沙地土壤中放线菌数量最多,细菌数量次之,真菌数量最少。细菌、真菌和放线菌间无显著相关性;细菌除了与脱氢酶相关性较低外,与其他酶均极显著正相关;真菌仅与多酚氧化酶显著负相关;放线菌与脲酶、蔗糖酶、磷酸酶显著正相关。 4）各人工灌木林地和流沙地土壤肥力高低为柠条林地＞沙柳林地＞花棒林地＞杨柴林地＞流沙地,人工灌木林的营造改善了土壤理化特征和生物学性质,显著地提高了流沙地土壤肥力质量,营造柠条林效果最佳。     

Immunohistochemical Expression of Potential Therapeutic Targets in Canine Thyroid Carcinoma

Background

Thyroid carcinoma is a common endocrine tumor in the dog. Local invasive growth frequently precludes surgical excision and, in up to 38% of dogs, the tumor has already metastasized by the time of diagnosis. Therefore, it is important to investigate new treatment modalities that may be useful for the large number of dogs with inoperable tumors or metastatic disease.

Hypothesis/Objectives

To investigate the immunohistochemical expression of potential therapeutic targets in canine thyroid tumors.

Animals

74 dogs with thyroid neoplasia.

Methods

Immunohistochemistry was performed for thyroglobulin, calcitonin, vascular endothelial growth factor (VEGF), p53, cycloxygenase‐2 (cox‐2), and P‐glycoprotein (P‐gp).

Results

Fifty‐four (73%) tumors were classified as follicular cell thyroid carcinomas (FTCs) and 20 (27%) as medullary thyroid carcinomas (MTCs). Eighty percent of FTCs and all MTCs had a high percentage (76–100%) of neoplastic cells immunopositive for VEGF. Thirteen percent of FTCs and 50% of MTCs expressed cox‐2. Seven percent of FTCs and 70% of MTCs expressed P‐gp. No tumor was immunopositive for p53 expression. Expression of VEGF (P = .034), cox‐2 (P = .013), and P‐gp (P < .001) was significantly higher in MTCs compared to FTCs.

Conclusions and Clinical Importance

VEGF is a potential therapeutic target in both FTC and MTC in dogs. Cox‐2 and P‐gp may be useful molecular targets in canine MTC.     

牛瑟氏泰勒虫p23-IL-18融合基因的原核表达

为了研发牛瑟氏泰勒虫病基因工程亚单位疫苗,本实验将p23-IL-18融合基因克隆到pET-28a原核表达载体,构建pET-28a-p23-IL-18重组原核表达质粒,经BamHⅠ和EcoRⅠ双酶切鉴定后在37 ℃用IPTG诱导表达,对表达产物进行SDS-PAGE和Western blotting分析。结果表明,成功构建牛瑟氏泰勒虫pET-28a-p23-IL-18原核表达质粒,目的基因在大肠杆菌中成功获得表达,融合蛋白的分子量约为48ku,并被牛瑟氏泰勒虫阳性血清所识别,具有良好的反应原性。本研究结果为牛瑟氏泰勒虫病基因工程亚单位疫苗的制备奠定了基础。     

Gonadotropins I and II do not stimulate thein vitro secretion of 17α,20β-dihydroxy-4-pregnen-3-one 20-sulphate by rainbow trout gonads during final sexual maturation

Thein vitro secretion of 17,20-dihydroxy-4-pregnen-3-one 20-sulphate (17,20-P-sulphate) and the free steroid 17,20-dihydroxy-4-pregnen-3-one (17,20-P), by rainbow trout (Oncorhynchus mykiss) gonads, in response to gonadotropin (GTH) I and GTH II, were studied during the final stages of sexual maturation. Substantial amounts of 17,20-P-sulphate were produced, by both mature ovaries and testes, indicating considerable 20-hydroxysteroid sulphotransferase (20-HST) activity within these tissues. In the post-ovulatory ovary the level of 17,20-P-sulphate (36.6 ng ml^–1) greatly exceeded that of 17,20-P (8.59 ng ml^–1). The amount of 17,20-P-sulphate produced in incubations of both mature ovary and testes was unaffected by either GTH I or GTH II treatment at physiological concentrations up to 100 ng ml^–1. Similarly, incubations of maturing ovary and testes, treated with GTH I or GTH II, in the presence of added 17,20-P at 100 ng ml^–1 of medium, produced levels of 17,20-P-sulphate that were similar to those of the controls. In incubations of mature ovarian follicles at the stages of germinal vesicle breakdown and preovulation, both GTHs significantly stimulated secretion of 17,20-P, although GTH II was always more potent than GTH I. GTH II significantly elevated the levels of 17,20-P in testicular incubations from mature males more than 4-fold relative to GTH I and controls, which did not differ from one another.In conclusion, 20-HST, the enzyme responsible for the sulphate conjugation of 17,20-P, was found to be active in the ovaries and testes of rainbow troutin vitro. However, the levels of this enzyme do not appear to be regulated by either GTH I or GTH II.     

Hydrogen sulfide ameliorates high glucose-induced endothelial cell senescence by suppressing oxidative stress

AIM：To explore the effect of hydrogen sulfide on the senescence of human umbilical vein endothelial cells (HUVECs) induced by high glucose. METHODS：Senescence model was established by treating HUVECs with 33 mmol/L glucose for 48 h. The parameters were detected to demonstrate the effect of hydrogen sulfide on senescence and the mechanism involved was also investigated. RESULTS：In the cells treated with high glucose, the proliferation was attenuated with a higher number of senescence-associated β-galactosidase (SA-β-Gal) positive cells, and plasminogen activator inhibitor 1 (PAI-1) protein expression, malondialdehyde (MDA) production and NF-κB p65 activity were increased significantly, but the expression of superoxide dismutase 1 (SOD1) was decreased. However, the cell number and SOD1 expression were increased, and the number of SA-β-Gal positive cells, PAI-1 protein expression, MDA production and the activity of NF-κB p65 were decreased after sodium hydrosulfide (100 and 200 μmol/L) treatment.CONCLUSION：Exo-genous hydrogen sulfide prevents HUVECs against high glucose-induced senescence by suppressing oxidative stress and NF-κB p65 activity.     

Increase in plasma total antioxidant capacity of grazing Japanese Black heifers and cows in forestland in Japan

Blood total antioxidant capacity (TAC) has become a key bio‐marker for animal health. Forest‐grazing cattle are known to forage various native plants that have high TAC. This study evaluated differences of plasma TAC between forest‐grazing (FG) and pasture‐grazing cattle (PG). Experiment 1 monitored the plasma TAC levels of 32 Japanese Black cattle. The level in PG did not change throughout the grazing period. However, that in FG, which increased from summer, was significantly higher than that in PG through fall (P < 0.05). In experiment 2, we used nine Japanese Black heifers and investigated their blood antioxidant parameters and the TAC in plants that the cattle consumed in late June and September. The plasma TAC levels in FG were significantly higher than those in PG in both periods (P < 0.05). Plasma levels of lipid peroxidation in FG tended to be lower than that in PG (P = 0.098). Furthermore, the TAC levels in various species of shrubs and trees consumed by FG were higher than those in pasture grasses. Results of this study show that plasma TAC of grazing Japanese Black cattle in forestland increase from summer through fall.     

p53调控Nfkbiz基因表达的研究

核因子-κB（NF-κB）在炎症反应中起着关键的促进作用,并且受其他多种因素的调节。Nfkbiz基因编码的IκB？蛋白是IκB家族成员之一,在炎症反应中起着调节NF-κB的作用。本研究采用荧光实时定量PCR分析Nfkbiz基因的表达情况,发现在p53特定刺激源作用下Nfkbiz基因mRNA丰度显著降低。分析Nfkbiz基因序列,发现其基因中存在可能的p53反应元件,通过构建荧光素酶报告质粒及双荧光素酶报告试验和染色质免疫共沉淀试验,证明Nfkbiz基因中含有p53反应元件并且受p53蛋白调节。以上结果初步证明Nfkbiz是受p53蛋白负向调控的靶基因。     

盛行风作用下柴木达盆地典型多花柽柳灌丛资源岛特征

[目的] 探究盛行风作用下柴达木盆地诺木洪地区典型多花柽柳(Tamarix hohenackeri)灌丛资源岛特征,旨在深入了解在类似的生境下柽柳灌丛的生态学意义,为盐碱地改良和荒漠—绿洲的生态保护提供借鉴。[方法] 通过对柴达木盆地诺木洪地区荒漠—绿洲过渡带典型多花柽柳灌丛周围土壤系统采样,全面了解冠下、冠缘和间地110 cm深不同土层土壤有机碳(SOC)、土壤含水率(SMC)、土壤pH和EC值特征,探讨盛行风对灌丛资源岛的影响。[结果] ①典型多花柽柳灌丛周围,冠下枯落物和地下生物量显著大于冠缘和间地,SOC含量在冠下显著大于冠缘,具有资源岛特征; ②盛行风背风向冠下SMC、冠缘和间地SOC含量显著大于其他方向,且冠缘0—5 cm土层有着较低的土壤pH值; ③背风向地上生物量和冠缘枯落物量显著大于其他方向,减少了水分蒸发,促进了SOC的增加。[结论] 柴达木盆地荒漠—绿洲过渡带多花柽柳灌丛周围土壤不仅冠下具有资源岛特征,且背风向土壤会形成更适合植物生长的SMC,SOC含量和较低土壤pH值,即风影区资源岛。