抗A型产气荚膜梭菌α毒素单链抗体1A8的基因克隆及序列测定

应用ＲＴ－ＰＣＲ技术,从分泌具有中和活性的抗Ａ型产气荚膜梭菌α毒素单克隆抗体（ＭcＡb）的杂交瘤细胞１Ａ８中,扩增出抗体ＶＨ和ＶＬ基因,用linker（Ｇ１y４Ｓir３） ３基因,将ＶＨ和ＶＬ基因连接成ＳeＦＶ基因,并将其克隆至pＧＥＭ－Ｔ载体中。经核甘酸序列分析证实,ＶＨ和ＶＬ基因及linker基因拼接正确,基因全长７２９bp,经计算机分析,ＶＨ和ＶＬ基因均为新发现的基因序列,符合功能性重排的鼠抗体可变区基因特征。     

抗A型产气荚膜梭菌α毒素单链抗体基因的表达

将 2种抗 A型产气荚膜梭菌α毒素单链抗体 ( Sc Fv)基因 Sc Fv-2 E3和 Sc Fv-1 A8克隆至表达载体p UC1 1 9、p HOG2 1和 p ET2 0 b中 ,构建了重组质粒 p UC2 E3和 p UC1 A8、p HOG2 E3和 p HOG1 A8、p ET2 E3和 p ET1 A8后 ,分别转化至受体菌 JM1 0 5、JM1 0 9和 BL2 1 ( DE3 )中 ,得到重组菌株 JM1 0 5( p UC2 E3 )和JM1 0 5( p UC1 A8)、JM1 0 9( p HOG2 E3 )和 JM1 0 9( p HOG1 A8)及 BL2 1 ( DE3 ) ( p ET2 E3 )和 BL2 1 ( DE3 )( p ET1 A8)。 ELISA检测表明 ,经 IPTG诱导后所表达的目的蛋白存在于重组菌株 JM1 0 5( p UC2 E3 )和JM1 0 5( p UC1 A8)及 JM1 0 9( p HOG2 E3 )和 JM1 0 9( p HOG1 A8)的菌培养上清中 ,在重组菌株 BL 2 1 ( DE3 )( p ET2 E3 )和 BL2 1 ( DE3 ) ( p ET1 A8)的胞周质中检测到了 Sc Fv的存在。 SDS-PAGE和薄层扫描分析表明 ,重组菌株 BL2 1 ( DE3 ) ( p ET2 E3 )和 BL2 1 ( DE3 ) ( p ET1 A8)的蛋白表达产物分别占菌体可溶性蛋白的 0 .9%和 0 .7%,其大小约为 2 60 0 0 ,且表达的目的蛋白具有中和磷脂酶 C的活性。     

Effect of losartan on matrix metalloproteinases of myocardial extracellular matrix after infarction in the rat

AIM: To study the change of the activity of matrix metalloproteinases(MMPs) in the infarcted intestitium of rat heart and the effect of Losartan on them. METHODS: 80 Adult male Sprague-Dawley rats underwent the left descending coronary artery ligation, then being divided randomly into control, treated groups and sham operation group. After hemodynamic parameters were obtained,animals of 8 groups were killed at 4 timepoints ( day 3, day 7,day 14, day 42 after infarction ).Matrix metalloproteinases(MMPs) activity in infarct zone was measured by zymography,so did collagen concentration in both ventricles by the method of chloramine T. RESULTS: The activity of MMPs in the infarcted zone showed a transient increase, which raiesd by 4.5 folds at day 3 (compared with the sham-operated group killed at day 3), 6.5 folds at day 7, and declined thereafter,being 2 folds at day 14, 1.5 folds at day 42. Losaran treatment was associated with significant attenuation of MMPs activity. The activity of MMP-1 (54 kD band) decreased 33%, so did the MMP-2 (58 kD and 62 kD bands) 50%. Compared with control groups, the hypertrophy of the left ventricle was regressed, the rate of LVW/BW dropped significantly at day 14 and day 42 ( P< 0.01) .The collagen concentration decreased at treated day 42 group( P< 0.01).For the regression of heart dysfuncton in Losartan groups, LVEDP demonstrated a slightly raise at day 3, then decreased at day 7,14,42, all of three groups’ systolic function are higher than that in the control ( P< 0.05). CONCLUSION: The latent MMPs were activated after infarction, which resulted in the demage of the fibrillar network and dalitation of the left ventricle.Losartan not only attenuates the activity of MMPs but also prevents collagen synthesis, decreases the total collagen in the necrosis zone at the late stage after infaction, thus regressing the progress dysfuction of the infarcted heart and protecting the myocardium.     

玉米浆发酵产生物丁醇的氨基酸代谢动力学模拟

为了深入挖掘利用丙酮丁醇梭菌产生物丁醇过程中氨基酸代谢的动态过程,探究利用廉价氮源玉米浆中的氨基酸用于丙酮丁醇梭菌产生物丁醇的生产策略,寻找生产丁醇的高效率廉价氮源来降低发酵生产成本。该研究首先利用高通量测序技术对玉米浆中微生物多样性进行分析;同时基于丙酮丁醇梭菌（Clostridium acetobutylicum）生产丙酮-丁醇-乙醇（Acetone-Butanol-Ethanol,ABE）碳代谢动态模型的基础上,构建氨基酸代谢模型,以此模拟15种氨基酸在利用木糖为碳源发酵生产ABE中的氨基酸代谢过程,并对氨基酸的代谢与丙酮丁醇梭菌的生物量以及ABE的合成相关性关系进行冗余分析;通过模型预测实际生产中利用玉米浆发酵时氨基酸的消耗过程。结果表明,梭状芽胞杆菌属（Clostridium）占细菌总数的68.76%,是玉米浆中的优势菌群;最佳参数校正后构建了氨基酸代谢模型,模拟值与试验值有较好拟合度;11种氨基酸（苯丙氨酸、苏氨酸、异亮氨酸、亮氨酸、蛋氨酸、缬氨酸、酪氨酸、甘氨酸、丝氨酸、精氨酸、天冬酰胺）在培养过程中迅速消耗用于细胞生长和溶剂生成,3种氨基酸（脯氨酸、组织胺、天冬氨酸）保持稳定状态,同时发酵过程中谷氨酰胺积累;冗余分析表明其中5种氨基酸对发酵产物及生物量影响具有相关性（P<0.05）,相关性排序从大到小依次为丝氨酸、甘氨酸、亮氨酸、缬氨酸、天冬酰胺;模拟预测玉米浆中缬氨酸、甘氨酸、丝氨酸在发酵过程中基本被消耗,推测其为发酵后期的营养限制性因子。该结论可证实玉米浆可作为丙酮丁醇梭菌发酵丁醇的优势氮源,为丙酮丁醇梭菌的氨基酸代谢调控及下一步利用并优化玉米浆作为氮源生产生物丁醇提供一定的理论参考和数据支撑。     

饲料中添加酪酸菌对宝石鲈生长性能的影响

为了研究在饲料中添加不同浓度的酪酸菌(Clostridium butyricum)对集装箱循环水养殖的宝石鲈(Scorturm barcoo)生长性能、肠道消化酶活性及肝脏抗氧化性的影响,分别向基础饲料里添加浓度为0.1%,0.5%和1%酪酸菌饲喂宝石鲈。结果显示:添加浓度0.5%酪酸菌试验组终末体质量显著高于对照组(P<0.05),肠道胰蛋白酶,淀粉酶活性最高,显著高于对照组(P<0.05),且该试验组宝石鲈总抗氧化能力(T-AOC)、超氧化物歧化酶(SOD)和过氧化物酶(CAT)等抗氧化指标显著高于对照组(P<0.05),丙二醛(MDA)含量显著低于对照组(P<0.05)。标明酪酸菌最适宜添加浓度为0.5%。     

产气荚膜梭菌ε毒素及其疫苗研究进展

产气荚膜梭菌病是由产气荚膜梭菌引起的一种重要的人兽共患传染病,可导致山羊、绵羊等动物的肠毒血症或坏死性肠炎,并且可引起动物脑、心、肺和肾组织的水肿.B型和D 型产气荚膜梭菌所产生的ε毒素是引起动物上述病理变化和死亡的重要因素之一.虽然甲醛灭活的毒素疫苗能对动物产生保护性抗体,但是,灭活苗潜在的安全性原因使其在应用上受到限制.因此,基于ε毒素基因的重组疫苗和弱毒疫苗就成为人们研究的目标.     

一种致命的隐形杀手:肉毒杆菌与肉毒毒素

2013年8月,因肉毒杆菌污染,问题奶粉再次进入公众视野。肉毒杆菌(Clostridium botulinum)是一种革兰阳性粗短杆菌,严格厌氧,有A～G 7个亚型,每个亚型都可产生一种剧毒的大分子外毒素,即肉毒毒素(botulinum toxin)。这种毒素可引起人和动物发生以松弛性麻痹为主症的肉毒中毒(botulism),虽然并不常见,但却是一种致命的中毒性疾病。本文综述了肉毒杆菌生物学特征、肉毒毒素结构与毒性、肉毒中毒临床表现以及检验鉴定等,并介绍了食品安全生产中肉毒杆菌与肉毒毒素的安全风险与控制措施。     

互助藏羊梭菌病病原体的分离鉴定与分析

青海省互助县某羊场藏系绵羊患病死亡,伴有腹泻、神经症状,为快速确诊发病原因,及时防控和治疗,采集病羊样品5份,通过镜检、细菌分离培养、分子生物学试验及致病性试验进行病原鉴定分析。结果证明此次病原菌为羊致病性D型魏氏梭菌,毒素基因分析显示该菌同时含有α和ε两种毒素基因,动物致病性试验结果表明该菌对昆明系小鼠具有较强的致病性,并从试验致死的小鼠中分离到与藏羊病原相一致的细菌。三种基因的遗传进化树显示,该菌具有较强的多样性。研究确定了本次藏羊致死的主要病原为D型魏氏梭菌,结果可为该羊场梭菌病的治疗和预防提供科学依据。     

鸡传染性支气管炎病毒血凝抑制试验抗原的研制

为应用血凝抑制(HI)试验检验鸡传染性支气管炎疫苗免疫效力(血清、卵黄抗体水平),建立了鸡传染性支气管炎病毒HI试验抗原的制备方法。该方法是通过选取抗原谱最广的鸡传染性支气管炎病毒(IBV)M41株,经SPF鸡胚增殖培养36h后无菌收取鸡胚尿囊液,4℃、12 000r/min离心10min,上清用聚乙二醇(PEG)20000浓缩100倍;兔源A型产气荚膜梭菌中国标准株(C57-1株)37℃增殖培养18h,4℃、12 000r/min离心10min取上清,经PEG 20000透析袋浓缩5倍后通过0.20μm滤膜过滤除菌,然后将IBV液和A型产气荚膜梭菌菌液(含α毒素)二者按一定比例混合后经37℃恒温振荡感作2h,4℃经48h后制成。经大量试验表明,制备的IBV HI试验抗原效价高、稳定性好,可替代进口抗原应用于鸡群IBV疫苗免疫后血清抗体及卵黄抗体的HI效价检测。     

Clostridium Infection (Jisizheng) in Yaks in Qinghai,China

Since the mid-1980s, outbreaks of a disease characterized by a sudden onset, acute deaths and extensive haemorrhages in the viscera and digestive tract of yaks have been prevalent in Qilian, Qinghai, China. The disease is known as jisizheng by local people. Virulent Clostridium perfringens type A and Clostridium haemolytica were isolated from yaks that had died of jisizheng. In 1996 and 1997, yaks were immunized with a polyvalent inactivated vaccine against C. perfringens and with an inactivated vaccine against C. haemolyticum, and this prevented the occurrence of jisizheng.