水牛子宫内膜腺上皮细胞和基质细胞的分离·培养和生物学特性研究(英文)

[Objective] The experiment aimed to set up a method for isolating and culturing endometrial stromal cells (BESC) and endometrial glandular epithelial cells(BEGEC) of buffalo as well as laid foundation for studying biological mechanism of embryo implantation and uterine diseases. [Method] The enzymatic digestion method, scraping method, serial filtration and differential velocity adherent technique were used to isolate BESC and BEGEC, then immunocytochemical method and TRYPAN-Blue assay were used to determine the purity and survival rate of isolated cells. [Result] The BESC and BEGEC were successfully isolated and cultured while immunocytochemical method and cell count method demonstrated that the purity was over 90%. The result of TRYPAN-Blue assay shown that survival rate of BESC and BEGEC was 91% and 78% respectively. [Conclusion] The enzymatic digestion method, scraping method, serial filtration and differential velocity adherent technique could isolate BESC and BEGEC with high purity.     

Strain Cultivation Stage and Infectious Concentration on Soybean Cotyledonary Node via Agrobacterium–mediated Transformation System

In soybean cotyledonary transformation,Agrobacterium infection is influenced by many factors.The cultivation stage of Agrobacterium strain and infectious concentration were studied in this research.Results showed that the highest rate of resistant shoots was obtained when the strain cultivation concentration was OD 600 =0.6,and the infectious concentration was OD 600 =0.5.Seven soybean cultivars in North China were also screened for high susceptibility to Agrobacterium and three genotypes were found to be s...     

Expression of Chicken Toll-Like Receptors and Signal Adaptors in Spleen and Cecum of Young Chickens Infected with Eimeria tenella

Toll-like receptors （TLRs） are a group of highly conserved molecules which initiate the innate immune response to pathogens by recognizing structural motifs of microbes. Understanding the changes in chicken Toll-like receptors （ChTLRs） and signal adaptors expression that occur with Eimeria tenella infection will help to elucidate the molecular basis of immune control of coccidiosis caused by Eimeria. The present study detected the dynamic changes in the expression of ChTLRs and associated signal adaptors in the spleen and cecum ofE. tenella-infected chickens during the early stage of infection. The results showed that the expression peak for ChTLRs, MyD88 and TRIF occurred at 12 h post-infection （hpi）, ChTLR3, ChTLRI 5 and MyD88 mRNA expression in the spleen ofE. tenella infected chickens were significantly higher （P〈0.05） than that of negative control chickens, and there were similar tendencies of these molecules expression in the cecum and spleen of E. tenella-infected chickens. The expression of MyD88 was upregnlated at four time points in the cecum of E. tenella-infected chickens. The results of this study indicate that ChTLR3, ChTLR15 and MyD88 play a role in young chickens infected with E. tenella.     

Isolation and Screening of Black Spot-Resistant Endophytic Fungi Strains from Rosa chinensis

[Objective] This study aimed to isolate and screen black-spot-resistant en- dophytic fungi strains. [Method] Various species of healthy Rosa chinensis were col- lected from Xi＇an, Xianyang, Baoji and Weinan City in Shaanxi Province. Endophytic fungi was isolated from stems and leaves, and purified by 2-3 times of inoculation to screen endophytic fungi antagonistic to Marssonina rosae with modified punching method. [Result] Samples collected from Xianyang exhibited the highest colonization rate and isolation rate; endophytic fungi strains isolated from stems presented the highest colonization rate and isolation rate compared with leaves. A total of 67 en- dophytic fungi strains were isolated from Rosa chinensis, including 3 black spot-re- sistant strains which were all derived from Baoji. [Conclusion] This study laid the foundation for further screening candidate strains of biocontrol fungi and environ- ment-friendly fungal biological control.     

Optimum Condition Selection of Xylitol Candida tropicalis Conversion Production

[Objective] The aim was to select the optimum conditions of xylitol Candida tropicalis conversion production. [Method] The effect of cell culture time,conversion time,conversion pH value,conversion initial sugar concentration,speed and inoculation rate were determined respectively.[Result] Optimum fermentation conditions were obtained as follows:cell culture 16 h,conversion time 10 h,conversion pH value 5.5,conversion initial sugar concentration 20 g/L,conversion shaking speed 150 r/min,inoculation rate 10% (volume ratio). The yield of xylitol has increased to 90%. [Conclusion] This study had provided basis for the further study on xylitol.     

高产谷胱甘肽酵母菌株的筛选及其抗乙硫氨酸诱变研究(英文)

[Objective] The aim of this study was to screen Saccharomyces for glutathione over-production. [Method] Ethionine-resistant mutants were obtained through UV mutagenesis and rational screening. [Result] A high GSH-producing strain HSJB1 was isolated from soil, and the biomass for this strain by flask shaking fermentation was 3.87 g/L while the GSH yield was 91.87 mg/L. According to the morphological, physiological and biochemical characteristics of cells, this strain was primarily identified as Saccharomyces cerevisiae. An ethionine-resistant mutant YBS77 was obtained through UV mutagenesis of the original strain HSJB1, and the biomass for this strain by flask shaking fermentation was 7.60 g dry cell weight/L while the GSH yield was 211.96 mg/L. [Conclusion] The biomass of the mutant obtained by breeding is increased by 96.38% than that of the original strain, and the GSH yield of the mutant obtained by breeding is increased by 130.72% than that from the original strain, which indicates that the breeding method is feasible.     

Analysis of Sugar Components of Fermented Rice Wine by Ion Chromatography

[Objective] The aim was to analyze sugar components in fermented rice wine by ion chromatography. [Method] The optimal condition for chromatography system of sugar analysis was selected by measuring sugars in fermented rice wine with ion chromatography and pulsed amperometric detection. [Result] The optimal measurement conditions were as follows： Leacheate （Leachate）, consisting of NaOH and CH3COONa, was eluted by gradient concentrations, with column temperature at 35 ℃ and flow rate at 0.4 ml/min. In the condition, sugars in rice wine were ana- lyzed and the results showed that the method is featured by low detection limit, good repetition and high recovery rate. [Conclusion] The research establishes and determines the approaches and optimum conditions for sugar analysis in rice wine by ion chromatography and pulsed amperometric detection, providing references for advancement of research on quality improvement of fermented rice wine.     

链霉素抗性筛选吸水链霉菌高产农用抗生素菌株研究（英文）

[Objective] The research aimed to screen Streptomyces hygroscopicus strains with high production of agricultural antibiotics.[Method] A strain of S.hygroscopicus was screened from the soil of Hainan Island.After natural screening and consecutive ultraviolet induced mutation twice,S6-7 strain was obtained as the original strain then treated by UV irradiation and streptomycin resistance screening,and finally rescreened through shake-flask fermentation.[Result] 7 better strains were selected by primary screening from 62 single colonies which were picked out randomly.After 3 generations of consecutive cultivation on slant media and rescreening,5 strains presented obvious forward mutation.The forward mutation rate reached 8.06%,and the largest production increasing rate came up to 25.11%.[Conclusion] By combining streptomycin resistance screening and conventional ultraviolet induced mutation,both the antibiotic-producing capacity and forward mutation screening efficiency of the original strain were greatly enhanced.     

鸡卡氏杆菌的分子鉴定及其16S rRNA基因序列分析(英文)

Two strains of Gallibacterium, isolated from one laying hen flock in Zhengzhou City of Henan Province, were identified by the morphological observation, genus-specific PCR, and analysis of 16S rRNA gene, which was used to generate the phylogenetic tree, with the 21 members of the 12 genera belonging to Pasteurellaceae to analyze the homology. Two strains were named Yu-ZZ-HL-I-SLG and Yu-ZZ-HL-II-GZ. The comparative result of the 16S rDNA sequence shows that the 2 isolated strains are identical in sequence; the highest identity (99.9%) was observed between the isolated strain and one of the strains of Gallibacterium anatis (AF228002), the homologies between the isolated strain and 3 strains of gallibacterium accessed in NCBI (AF228016, Gallibacterium genomosp.1, AF228017, Gallibacterium genomosp.2, AF228018, Gallibacterium genomosp.1) were above 97.1%, higher than that of the isolated strain and the other strains of the other 11 genera which were between 90.7%-93.2%. It can be seen from the phylogenetic tree that the 2 isolated strains and the other 4 strain of gallibacterium fell into the same branch, furthermore the 2 isolated strains and the strain of Gallibacterium anatis locate in an internal branch, indicating that the 2 isolated strains belong to Gallibacterium anatis.     

Type I strain of Toxoplasma gondii from chicken induced different immune responses with that from human,cat and swine in chicken

In this study,four strains of Toxoplasma gondii with the same genetic type(Type I) originated from chicken,human,cat and swine were used to compare the immune responses in resistant chicken host to investigate the relationships between the parasite origins and the pathogenicity in certain host.A total of 300,10-day-old chickens were allocated randomly into five groups which named JS(from chicken),CAT(from cat),CN(from swine),RH(from human) and a negative control group(—Ve) with 60 birds in each group.Tachyzoites of four different T.gondii strains(JS,CAT,CN and RH) were inoculated intraperitoneally with the dose of 1×10~7 in the four designed groups,respectively.The negative control(-Ve) group was mockly inoculated with phosphate-buffered saline(PBS) alone.Blood and spleen samples were obtained on the day of inoculation(day 0) and at days 4,11,25,39 and 53 post-infection to screen the immunopathological changes.The results demonstrated some different immune characters of T.gondii infected chickens with that of mice or swine previous reported.These differences included up-regulation of major histocompatibility complex class Ⅱ(MHC Ⅱ) molecules in the early stage of infection,early peak expressions of interleukin(IL)-12(IL-12) and-10(IL-10) and long keep of IL-17.These might partially contribute to the resistance of chicken to T.gondii infection.Comparisons to chickens infected with strains from human,cat and swine,chickens infected with strain from chicken showed significant high levels of CD4~+ and CD8~+ T cells,interferon gamma(IFN-γ),IL-12 and IL-10.It suggested that the strain from chicken had different ability to stimulate cellular immunity in chicken.     

E.tenella吉林株单卵囊的分离及PCR鉴定

[目的]为了获得E．tenella纯株,进行相关的分子生物学研究。[方法]对已经建立的球虫单卵囊分离技术进行改进,用分离的单卵囊胶囊接种雏鸡。同时应用PCR方法对收集的单卵囊分离株进行球虫种类鉴定。[结果]20只试验鸡中有15只粪便中分离到卵囊,感染成功率为75％。PCR扩增结果表明该单卵囊分离株为Etenella。[结论]采用单卵囊胶囊进行接种,操作简便,既节省了接种时间,又降低了接种难度,且大大提高了接种成功率,为球虫的分子生物学研究提供了材料。     

E．tenella吉林株单卵囊的分离及PCR鉴定

1材料与方法1．1材料①试验动物。1日龄海兰公雏,购自长春市北方鸡场。饲养于150℃,消毒1h的隔离器中,自由采食和饮水。饲喂不添加任何抗球虫药的全价饲料,喂前80℃高温消毒30min。②试验虫种。单卵囊分离用虫种,为该实验室从球虫严重临床发病鸡盲肠中分离。③试剂。基因组DNA提取试剂盒购于天根生化科技（北京）有限公司,D12000及PCR相关试剂购自大连宝生物工程有限公司。其他试剂均为国产分析纯。④仪器。     

肠艾美尔球虫单卵囊分离及ITS-1序列测定

[目的]建立一种有效的鉴定球虫种类分子生物学方法。[方法]采用单卵囊分离技术,分离肠艾美尔球虫。根据GenBank中发表的艾美尔属球虫18s和5.8srDNA序列,设计特异性引物,扩增内转录间隔区1（ITS-1）,PCR产物直接测序。[结果]成功地分离出肠艾美尔球虫,其卵囊扩增出434bp清晰条带,且最低能检测出27孢子化卵囊。[结论]该研究结果为球虫虫种及虫株的准确鉴定奠定了基础。     

转基因柔嫩艾美耳球虫对生态环境的影响

【目的】从表达M2e-EYFP融合蛋白转基因柔嫩艾美耳球虫的稳定性,及其在环境中的传播能力、竞争能力和存活能力方面,分析转基因球虫对生态环境的潜在风险。【方法】稳定性试验包括:转基因球虫感染非靶动物,观察临床症状和卵囊产出情况;转基因球虫继代繁殖,检测荧光率;转基因球虫与毒害艾美耳球虫混合感染,PCR检测卵囊中的外源基因。传播能力试验主要是分析感染鸡和不感染同居鸡的卵囊产量和荧光率。竞争性试验则是用不同混合比例的2种球虫感染鸡,检测卵囊产量和荧光率;存活能力试验是用不同处理方式的卵囊感染鸡,检测卵囊是否产出以分析卵囊存活能力。【结果】转基因球虫不感染小鼠和家鸽,与野生型柔嫩艾美耳球虫宿主特异性相同;连续繁殖传17代后外源基因稳定表达。与毒害艾美耳球虫混合寄生时在异种球虫体内未检测出外源基因。传播能力试验中,46d攻毒时转基因球虫感染与不感染同居组的卵囊产量差异不显著,但与不感染对照组相比均显著减少,且1∶1感染和不感染同居组卵囊荧光率检测结果维持在51.6%67.9%。竟争性试验中,攻毒时各比例感染组卵囊产量均极显著少于不感染攻毒对照组,荧光率检测结果各比例组变化幅动不大,且1∶1组哨兵鸡卵囊荧光率检测结果为41.2%61%。存活能力试验,转基因球虫与野生型球虫在夏季室温条件下均不能存活90d,且与其他不同方式处理的卵囊外界存活时间接近。【结论】说明转基因球虫具有良好的稳定性,在环境中的传播能力、竞争能力和存活能力未呈现显著优于野生球虫的趋势。     

安徽部分地区鸡柔嫩艾美耳球虫单卵囊的分离与鉴定

为获得纯种柔嫩艾美耳球虫虫株,从安徽省巢湖市、霍邱县、定远县3个地区患球虫病的鸡场采集3株混合球虫,培养至孢子化,分离单卵囊接种1～7日龄的雏鸡,感染后7～12 d,收集卵囊,然后通过生物学特性和PCR方法进行虫种鉴定。结果显示,3个地区的单卵囊感染成功率分别为50%、57%和29%,各虫株经生物学特性和PCR鉴定均为柔嫩艾美耳球虫。研究表明:1.0%琼脂单卵囊分离法简便且成功率较高,利用特异性引物PCR鉴定虫株,为纯种地方株的研究奠定了基础。     

鸡柔嫩艾美耳球虫单卵囊分离技术的构建及致病性研究

构建了一种新的鸡柔嫩艾美耳球虫单卵囊分离技术 ,并对杨陵柔嫩艾美耳球虫地理株进行了分离 ,对继代增殖的纯种卵囊进行了致病性试验。结果表明 ,该单卵囊分离技术简单易行 ,单卵囊感染成功率可达4 0 % ;该虫株对雏鸡有很强的致病性 ,经口接种 1× 10 5个孢子化卵囊 ,致死率高达 80 %。     

柔嫩艾美尔球虫库尔勒株免疫原性初步研究

本实验采用单卵囊分离技术,得到1株柔嫩艾美尔球虫(Eimeria tenella),对该株E.tenella的免疫原性做了初步研究,分别用100个卵囊/只,1,000个卵囊/只,10,000个卵囊/只的剂量免疫,以100,000个卵囊/只的剂量攻毒,结果显示,1000个卵囊/只的剂量免疫效果最好。     

柔嫩艾美耳球虫子孢子抗原的制备

[目的]为进一步研究柔嫩艾美耳球虫（E.tenella）的单克隆抗体奠定基础。[方法]给肉鸡雏接种E．tenella纯种孢子化卵囊：第5～10天收集鸡的粪便并标记、粗提，经卵囊孢子化、纯化孢予化的卵囊、计数卵囊后，最终制备出子孢子抗原。[结果]该方法制备E．tenella子孢子抗原较其他方法简单易行，不需太多专门仪器和培养液，一般实验室均可进行，效果良好。但所用时间较长。[结论]该试验成功分离提纯出了鸡的E．tenella卵囊，并获得了蛋白浓度较高的抗原，为单克隆抗体的制备提供了有力保障。     

肠艾美尔球虫单卵囊分离及ITS-1序列测定

[目的]建立一种有效的鉴定球虫种类分子生物学方法。[方法]采用单卵囊分离技术,分离肠艾美尔球虫,提取卵囊基因组DNA。根据GenBank中发表的艾美尔属球虫18SrDNA和5.8SrDNA序列,设计特异性引物,扩增内转录间隔区1（ITS-1）,PCR产物直接测序。[结果]成功分离出肠艾美尔球虫,PCR扩增出434bp清晰条带,且最低能检测出27个孢子化卵囊。[结论]该研究结果为球虫虫种及虫株的准确鉴定奠定了基础。     

副鸡嗜血杆菌的分离与鉴定

[目的]对1例鸡传染性鼻炎疑似病例进行病原菌的分离与鉴定。[方法]从安徽省某鸡场发生的以脸面肿胀、流泪和产蛋下降为临床特征的病鸡上呼吸道分泌物中分离细菌,并进行染色、镜检、菌落形态观察,并利用"卫星现象"和PCR等方法进行鉴定。[结果]成功分离到1株副鸡嗜血杆菌,经鉴定为C型副鸡嗜血杆菌。动物回归试验表明,该分离株能引起典型的鸡传染性鼻炎,对鸡具有较强的致病力。[结论]该研究可为鸡传染性鼻炎的预防与控制提供依据。