Extraction,Purification and Activity Analysis of Lipopolysaccharides from Escherichia coli Isolated from Swine

In order to obtain higher purity and high yield of LPS isolated from Escherichia coli, and evaluate its virulence,excrement of diarrhea piglet was collected as pathological samples, of which, some strains of Escherichia coli were isolated and identified. And furthermore, a strain of Escherichia coli was acquired by 16S rDNA identification. The LPS from this strain of Escherichia coli was isolated with the hot-phenol water method and purified by DNaseⅠ, RNaseA, proteinase K treatment and alcohol precipitation. The polysaccharide, protein and nucleic acid content of purified LPS were detected by phenol-sulfuric acid method, Bradford method and UV points spectrophotometric method. And its bioactivity and acute median lethal dose was determinated by tachypleus amebocyte lysate (TAL) method and acute toxicity experiment method, respectively. The results showed that a strain of highly pathogenic Escherichia coli was successfully isolated. The average yield of purified LPS was 3.74%, the proportion of the polysaccharide was 13.40%, the protein was 0.48%, and the nucleic acid was 0.06%. SDS-PAGE electrophoresis and silver stain showed that the bands were mainly concentrated in the range of 14 to 160 ku. The LAL bioactivity of the prepared LPS was 1.21×10⁷ EU/mg, LD₅₀ of the mouse abdominal cavity was 31.86 mg/kg. The results revealed that the diarrhea piglets in the farm was caused by pathogenic Escherichia coli,purified LPS from this strain of Escherichia coli had the advantages of higher purity, high yield and strong virulence, which would provide theoretical data for clinical prevention and treatment of diarrhea piglets.     

1株黄曲霉颉颃菌的鉴定及其抑菌活性成分分析

【目的】 筛选黄曲霉颉颃菌,探究其最佳培养条件及有效抗菌成分,为实际生产中应用生物防治方法抑制黄曲霉污染提供新的理论依据。【方法】 通过平板对峙法及分子生物学鉴定方法对待测菌株进行筛选鉴定。以抑菌率作为衡量标准,判断所得黄曲霉颉颃菌的最佳培养温度、培养时间、接种量、转速、pH及装液量。通过硫酸铵沉淀法从颉颃菌无菌发酵上清液中提取蛋白,观测粗提物对黄曲霉菌丝生长的影响。通过离子交换色谱和凝胶过滤色谱进一步纯化粗提蛋白,并进行质谱测序及序列对比分析,筛选鉴定颉颃菌产生的有效抑菌成分。【结果】 筛选到1株解淀粉芽孢杆菌B10(菌种保藏号CCTCCNO:M2018353)。最佳培养条件为温度40 ℃,培养时间36 h,接种量3%,转速120 r/min,pH 6.0,装液量30 mL,最大抑菌率可达46.56%。其粗提蛋白能有效抑制黄曲霉生长,破坏菌丝正常形态,并从中筛选出几丁质结合蛋白、壳聚糖酶、葡聚糖酶等8种可能的有效抑菌成分。【结论】 本试验分离鉴定出1株抑制黄曲霉正常生长发育的解淀粉芽孢杆菌B10,其产生的多种蛋白成分对黄曲霉有显著抑菌活性。     

葡萄糖氧化酶的提纯方法研究

主要研究葡萄糖氧化酶从废菌丝中的提取方法。由于葡萄糖氧化酶是胞内酶,需要先将菌丝破壁,使其中的葡萄糖氧化酶释放出来,而且要采取措施保证酶的活力损失最小。获得的初酶液,再经过初步纯化,即可制成酶制剂。最终确定了破壁的方法为超声破壁,超声破壁的最优条件为:超声时间50min,超声温度60℃,超声功率200W。破壁后选取65%乙醇一次沉淀葡萄糖氧化酶,然后用16%的PEG二次沉淀酶。     

A simple and rapid method for isolation of caliciviruses from liver of infected rabbits

Rabbit Haemorrhagic Disease Virus (RHDV), a member of the Caliciviridae family, is the etiologic agent of Rabbit Haemorrhagic Disease (RHD); this viral disease is highly contagious and kills more than 90% of infected adult rabbits. Research on experimental calicivirus infection uses inocula obtained from livers of rabbits dying from calicivirus infection. This implies that caliciviruses have to be purified from liver homogenates. Current methods to isolate caliciviruses from rabbit livers are time consuming. We propose here a new procedure for fast purification of rabbit caliciviruses from liver homogenates that uses centrifugation through an iodixanol gradient. This method offers in approximately 2 h a sample with a high degree of calicivirus purity, as shown by its biochemical and immunocytochemistry analysis, which is also able to kill adult rabbits from RHD within 48 h of inoculation.     

利用胰酶粉提取胰激肽释放酶静态条件的初探

研究利用胰酶粉提取胰激肽释放酶的静态提取工艺.分别考察了浸提pH值、平衡液pH值、吸附过程中的NaCl添加量、洗脱过程中pH值、NaCl添加量以及醋酸钠正交试验对利用胰酶粉静态提取胰激肽释放酶的影响.利用胰酶粉静态提取胰激肽释放酶的最佳条件为:浸提pH值为6.0,平衡液pH值为6.5,吸附过程中添加0.02mol/L NaCl,洗脱优化条件控制在pH值为6.5,NaCl0.35 mol/L以及不添加醋酸钠,提取率达到76%以上,该静态条件的考察为胰激肽释放酶的进一步分离纯化提供了理论依据.     

超滤膜分离纯化花生壳中水溶性膳食纤维

为探索花生壳中水溶性膳食纤维(SDF)的有效分离纯化方法和效果,选择适宜的膜组件,采用不同截留分子量的超滤膜以及优化膜分离过程的操作条件,对SDF提取液进行超滤分离,试验结果表明:选用PS-30聚砜膜,在压力为0.08MPa、料液比为1:75g/mL、温度为30℃时,分离纯化效果最为显著。其中,膜通量达到127.2L/(m2·h)、SDF的得率达到67.56%,非淀粉多糖(NSP)的质量分数由49.85%提高到92.36%,蛋白质量分数从5.53%降到0.92%。与传统的提取法比较,超滤膜分离纯化花生壳水溶性膳食纤维具有生产周期短,成本低,产品纯度高等优点。     

重组海参溶菌酶C端基因(SjLys-C)的可溶性表达和抑菌活性分析

为进一步研究海参(Stichopus japonicus)溶菌酶基因(Sjys)(Genbank登录号:EF036468)中不司片段表达产物的生物特性,本研究通过对其cDNA片段的分析,发现C端基因区域所对应的蛋白质序列中含有非酶活性.根据已知的海参溶菌酶的cDNA序列,设计山含有Nco Ⅰ和EcoR Ⅰ酶切位点的特异性引物,从新鲜的海参肠中提取总RNA,以其为模板利用RT-PCR扩增出长度为259 bp的溶菌酶C端(SjLys-C)基因.将该目的基因连接到pET-32a(+)载体上,构建重组质粒pET-32a(+)-SjLys-C,再转化至大肠杆菌(Escherichia coli)Rosetta(DE3)pLysS,成功地构建了重组蛋白SjLys-C的基因工程菌.利用该工程菌诱导发酵,结果显示它能高效表达出26 kD左右的重组蚩白SjLys-C.经过Western blot分析,该重组蛋白在26 kD左右能够与Penta-His抗体发生特异性免疫反应.对纯化的重组蛋白SjLys-C进行了抑菌特性的分析,结果发现它对溶壁微球菌(Micrococcus lysodeikticus)和副溶血弧菌(Vibrio parahae molytic us)有较高的抑菌活性.此外,将该重组蛋白经100℃、40 min处理后,其抑菌能力提高了5％～21％.研究结果表明,重组蛋白SjLys-C基因工程菌能够制备出具有可溶性的、并具有抑菌活性的重组蛋白SjLys-C,在农业和医药等行业中有潜在应用和开发价值.     

秋茄人工湿地净化循环海水养殖废水效果

为了深入研究采用人工湿地方法处理循环海水养殖废水的可行性及其运行特性,以红树林植物秋茄(Kandelia candel)为湿地植物构建耐盐人工湿地,对循环海水养殖废水进行生物处理。该文对比研究了秋茄人工湿地与无植物人工湿地的处理效果,在系统启动17d后的稳定运行阶段,秋茄人工湿地对化学需氧量(chemical oxygen demand,COD)和氨氮(NH4+-N)的去除率分别在66.4%～73.8%和64.3%～72.4%,明显高于无植物人工湿地对COD56.7%～62.4%的去除率和对NH4+-N51.2%～57.5%的去除率;在总磷(total phosphorus,TP)去除方面,秋茄人工湿地和无植物人工湿地的去除率分别为55.7%～61.7%和54.3%～60.4%,两者并无明显差别。对人工湿地基质酶活性的分析发现,秋茄人工湿地的基质脲酶活性和磷酸酶活性均明显高于无植物人工湿地;同时在秋茄人工湿地基质床中,根系分布最多的表层0～5cm区域基质酶活性明显高于其他区域,表明秋茄的根部对于提高人工湿地基质床的生物活性及其去污能力发挥了显著的作用。对秋茄人工湿地水力条件的研究表明,表面水力负荷对系统去除有机物和氮有较明显的影响,表面水力负荷控制在0.1m3/(m2·d)及以下时,出水水质可达到国家渔业水质标准的要求。该文研究结果为循环海水养殖废水提供了一种生物处理途径。     

上样量和洗脱液温度影响磁珠法纯化mRNA效果

对总RNA上样量和洗脱液温度的比较试验表明,用Promega公司生产的磁珠法试剂盒PolyAT-tract^R mRNA Isolation Systems Ⅲ Z5300纯化mRNA,每管总RNA上样量以550μg适中。将洗脱液加热至70℃,有利于提高洗脱率。550μg上样量与70℃洗脱液温度配合运用,既可保证mRNA纯化质量,充分利用总RNA样品,又可避免磁珠浪费。     

植物收割对人工湿地基质酶活性的影响

研究了7月份植物收割对人工湿地基质磷酸酶、脲酶、蛋白酶、脱氢酶活性及养殖废水中总氮(TN)、总磷(TP)、COD去除率的影响。结果显示:植物收割后磷酸酶、脲酶、蛋白酶活性开始增高,分别于第20天(13.7 mg/100 g)、第16天(1.9 mg/100 g)、第12天(61.7 mg/100 g)达到最大值,均于第28天(10.1 mg/100 g、1.1 mg/100 g、43.2 mg/100 g)恢复到收割前水平。脱氢酶活性在植物收割后第4天(142.3μL/100 g)降到最小值,此后开始增高,第16天(249.5μL/100 g)达到最大值,于第24天(195.6μL/100 g)恢复到收割前水平。净化效果研究表明,植物收割后TP、TN去除率开始增高,分别在植物收割后第20天(75.1%)、16天(60.6%)达到最大值,均于第28天(51.1%、34.1%)恢复到收割前水平。COD去除率在收割后第4天(25.3%)降到最小值,此后开始增高,第16天(70.2%)达到最高值,第24天(46.5%)恢复到收割前水平。