常压室温等离子体结合紫外诱变筛选红霉素高产菌株

利用常压室温等离子体射流诱变(ARTP)和紫外照射对红霉素产生菌进行复合诱变,得到4株产量明显提高的突变菌株,4株菌的平均发酵效价较出发菌株提高25.2%;其中一株(12#菌株)经发酵摇瓶验证,红霉素发酵单位可达10029单位/mL,比出发菌株提高了28.6%,且遗传稳定性良好。实验证明ARTP-UV复合诱变是一种简单高效的筛选方法。     

复合诱变对枯草芽孢杆菌表面活性素抗病毒能力的影响

为了探究1株枯草芽孢杆菌复合诱变前后产生的表面活性素(surfactin,原始菌株YUAN.0和复合诱变菌株FHYB201030产生的表面活性素分别为YUAN.0S和FHYB201030S)对猪流行性腹泻病毒(Porcine epidemic diarrhea virus, PEDV)及猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)的作用,试验采用CCK-8试剂盒检测YUAN.0S和FHYB201030S对非洲绿猴肾(Vero)细胞及猴胚胎肾上皮(Marc-145)细胞的毒性,同时分析了不同质量浓度表面活性素对PEDV和PRRSV的直接灭活作用、吸附阻断作用及复制阻断作用。结果表明：YUAN.0S对Vero及Marc-145细胞的最大无毒性质量浓度均为7.81 mg/L,而FHYB201030S的最大无毒性质量浓度均提高至62.50 mg/L。YUAN.0S对PEDV和PRRSV基本上没有抑制作用,而FHYB201030S对PEDV和PRRSV有一定的直接灭活作用,FHYB201030S质量...     

一株高产中性蛋白酶菌株的筛选与诱变

通过牛奶平板初筛、摇瓶复筛，从土壤中筛选出产中性蛋白酶菌株L01，以紫外线和亚硝酸钠为诱变条件，分别利用单因素和复合诱变方法提高菌株的产酶能力，最后对遗传稳定性良好的菌株L01F进行鉴定。结果表明：菌株L01分别经过紫外线照射60 s和亚硝酸钠处理12 min时，其中性蛋白酶酶活力最高，为459.13 U/mL，是原始菌株的2.07倍。在先亚硝酸钠处理12 min后紫外线照射48 s的最适复合诱变条件下，筛选得到的高产中性蛋白酶菌株L01F，酶活力达到577.87 U/mL，是原始菌株L01的2.61倍，且该菌株传代10次后仍具有良好的遗传稳定性。最后经过形态学和分子生物学鉴定，确定筛选出的菌株L01F为枯草芽孢杆菌（Bacillus subtilis）。 [关键词] 中性蛋白酶|诱变|稳定性研究|枯草芽孢杆菌     

泰乐菌素高产菌株的推理选育

以弗氏链霉菌CK6-6为出发菌株,分别采用紫外(UV)、甲基磺酸乙酯(EMS)、紫外与氯化锂(LiCl)复合、甲基磺酸乙酯与紫外复合4种方法对其进行诱变。比较了4种方法的最大提高率、致死率、正变率及突变株的传代稳定性,结果发现诱变效果较好的为EMS与UV复合诱变,筛选出1株传代稳定菌株EU45-15,相对于出发菌株CK6-6,泰乐菌素产量的提高率为15.36%。经HPLC分析发现,它的最高活性组分A的含量高于对照,提高率为48.28%。同时利用豆油作为筛选物质对弗氏链霉菌进行定向筛选,筛选效果较好,筛出1株高产稳定菌株O3.6-8,效价提高率为12.15%。     

一株产抗菌脂肽芽孢杆菌筛选鉴定、产酶特性及其脂肽初步鉴定

为筛选可用于动物生产的优良枯草芽孢杆菌,试验通过抑菌性能筛选,从200余株分离自土壤、枯叶等材料的芽孢杆菌中筛选得到菌种S21,经分子生物学鉴定菌种S21为枯草芽孢杆菌(Bacillus subtilis);通过产酶定性分析,枯草芽孢杆菌S21产蛋白酶、脂肪酶、淀粉酶和纤维素酶;采用酸沉、醇提和半制备色谱对S21产抗菌脂肽纯化,利用ESI-TOF-MS对纯化出的抗菌脂肽进行鉴定,鉴定抗菌脂肽为surfactin家族抗菌脂肽;抑菌试验分析显示枯草芽孢杆菌S21产抗菌脂肽对革兰氏阳性、革兰氏阴性和霉菌都有抑制效果,热耐受性和酸碱耐受性好。综上所述,枯草芽孢杆菌S21有用于动物生产的潜力。     

利用梯度平板法结合紫外诱变、抗性筛选选育土霉素高产菌株

以龟裂链霉素TMSⅠ12-66为出发菌株,通过紫外-氯化锂诱变处理,并结合梯度平板法、氯化锂和四环素复合抗性平板上筛选土霉素高产菌株。通过对致死率的考察,确定了紫外的最佳诱变剂量为90 s。在分离培养基上层加入氯化锂和底层加入四环素进行正突变株的定向筛选。经过选育得到一株土霉素的高产菌株TMSⅠ14-180,摇瓶发酵验证效价达22985 mg/mL,较出发菌株提高22.36%,经传代试验考察,该菌株遗传性状稳定。     

不同诱变方法对TL-15028菌株产泰乐菌素相关性能的影响

为了选育出泰乐菌素高产菌株,以弗氏链霉菌(Streptomyces fradiae)TL-15028菌株作为出发菌株,利用化学诱变、物理诱变以及复合诱变的方法对菌株进行诱变处理,比较不同诱变方法的诱变效果,通过诱变筛选出1株遗传稳定性好、且具有泰乐菌素和豆油抗性的菌株TLEU-1503,其发酵效价比出发菌株提高了36.5%,达到14124μg/m L,对泰乐菌素高效生产、品质提升具有重要意义。     

不同诱变方法对TL-15028菌株产泰乐菌素相关性能的影响

为了选育出泰乐菌素高产菌株,以弗氏链霉菌(Streptomyces fradiae)TL-15028菌株作为出发菌株,利用化学诱变、物理诱变以及复合诱变的方法对菌株进行诱变处理,比较不同诱变方法的诱变效果,通过诱变筛选出1株遗传稳定性好、且具有泰乐菌素和豆油抗性的菌株TLEU-1503,其发酵效价比出发菌株提高了36.5%,达到14124μg/m L,对泰乐菌素高效生产、品质提升具有重要意义。     

快中子与DES复合诱变选育高产木聚糖酶黑曲霉菌株

为了提高木聚糖酶活力,以黑曲霉AS3.350为出发菌株,利用快中子和DES复合诱变的方法,筛选出一株高产木聚糖酶菌株DES20-4,该菌株经过液体发酵培养,产木聚糖酶活力为157.07 U/ml,是出发菌株的2倍。     

饲用高产丙酸菌的复合诱变

以费氏丙酸菌IFFI.10019为出发菌株,经2次紫外线、2次亚硝基胍、1次亚硝基胍-氯化锂多重复合诱变处理,选育获得饲用高产丙酸菌NL-3,其丙酸产量由原来的0.20g/l提高到1.23g/l,提高率达到515%;乙酸产量由原来的1.79g/l提高到2.61g/l。试验结果表明,经复合诱变处理得到的丙酸菌NL-3是一种较好的微生物饲料添加剂。     

具有万古霉素高产能的东方拟无枝酸菌诱变株的选育研究

为提高万古霉素(Vancomycin)发酵生产水平,选育出其发酵生产的优良菌株,以东方拟无枝酸菌(Amycolatopsis oriertalsis)菌株V1806作为原始菌株,利用EMS(甲基磺酸乙酯)、UV(紫外诱变)和ARTP(常压室温等离子体诱变)等单一诱变、复合诱变的方法,选育出1株遗传稳定性好、且对万古霉素耐受性强的菌株Vua-15,其中试发酵效价达到7863 mg/L,比原始菌株提高了74.3%。研究结果表明,复合诱变对东方拟无枝酸菌菌种选育的效果优于单一诱变,更易选育出符合预期的优良菌株。研究不仅对万古霉素工业化发酵生产具有重要意义,而且为其他产品发酵菌种的选育提供了一定借鉴。     

猪霍乱沙门菌抗噬菌体菌株筛选及受体基因突变位点分析

【目的】 筛选抗噬菌体菌株并分析其受体基因突变位点,为解析猪霍乱沙门菌噬菌体抗性突变的产生机制提供理论依据。【方法】 以猪霍乱沙门菌CICC 21501及其裂解性噬菌体vB_SenS_S528（噬菌体S528）为研究对象,通过波动实验筛选自发突变的抗噬菌体菌株,观察菌落形态,并测定其对噬菌体的敏感性、吸附特性、生长曲线及对温度和pH的敏感性。通过全基因组重测序结合PCR验证定位耐受基因突变位点。【结果】 成功筛选出1株抗噬菌体S528的突变菌株,命名为B6-2。B6-2菌落边缘粗糙,与野生菌相比,生长曲线无显著差异,对pH表现出敏感性,在40、50 ℃时表现出温度耐受性。噬菌体S528对抗噬菌体菌株B6-2的吸附率减少60%（对野生菌吸附率为93%）。通过全基因组重测序及比对分析得知,B6-2菌株有3个位点发生了突变,分别为PROKKA_04510基因中2个位点和rfbC基因中的1个位点发生突变。突变基因片段的PCR产物电泳及测序结果均表明,PROKKA_04510基因上的2个位点并未发生突变,而真正发生突变的位点位于rfbC基因的350 bp处,碱基由C突变为T。【结论】 筛选到1株与受体改变有关的抗噬菌体菌株B6-2,其通过rfbC基因上的碱基突变来阻止噬菌体吸附。     

乳酸杆菌抑制致病菌及生物保鲜发酵剂特性评价

以15株乳酸杆菌为研究对象,动物双歧杆菌乳亚种BB-12作为对照,研究它们对肠道致病菌的抑菌特性.结果表明:9株菌对5株指示菌均有较好抑制作用,2株菌对金黄色葡萄球菌、肠炎沙门氏菌、大肠杆菌和蜡样芽孢杆菌均有抑制作用;以抑菌效果、凝乳状态、产酸性能为指标,筛选出4株菌,与嗜热链球菌ST-21组合制备生物保鲜发酵剂;通过...     

表达无毒性大肠杆菌ST1-LTB融合蛋白基因工程菌株的构建

利用基因突变技术，将形成ST1分子内二硫键的半胱氨酸碱基进行突变，使ST1失去本身毒性，进而将其与含有LTB基因的pET-28b( )连接，转化至受体菌BL21(DE3)，重组菌株BL21(DE3)(pXST3LTB)经IPTG诱导后，其表达产物免疫的小鼠能够抵抗大肠杆菌强毒菌的攻击并且消除了ST1的毒性，表明构建的工程菌株BL21(DE3)(pXST3LTB)可作为预防幼畜大肠杆菌性腹泻基因工程菌苗的候选菌株。     

分离自牧草根际四株促生菌株(PGPR)互作效应研究

利用钼蓝比色、显色和Salkowski比色等方法测定了筛选自苜蓿、白三叶和红三叶根际的4株优良PGPR菌株间的拮抗反应、单独和混合培养菌株的溶磷和分泌植物生长素(IAA)能力、培养液pH值和有机酸总量变化及其彼此之间的相关性。结果表明,各菌株间无拮抗反应,可以混合使用。无机磷培养液中,菌株Hsg+lhs11组合处理有效磷增量(536.2 mg/L)最大,Hsg+ls1-3+lhs11组合次之(475.6 mg/L)。Hsg+lhs11组合菌株处理有效磷增量呈现“1+1>2”的加成效应,Hsg+ls1-3+lhs11处理有效磷增量较单菌株处理时显著提高(P<0.01),但未表现“1+1+1>3”的溶磷效果。有机磷培养液中,lhs11处理有效磷增量(11.11 mg/L)最大,Hsg+ls1-3+lhs11组合次之(9.20 mg/L),Hsg+ls1-3+lhs11组合有效磷增量显著高于其他组合处理。各处理菌株培养液有效磷增量与pH值、有效磷增量与总有机酸量、pH值和总有机酸量之间存在线性相关。单菌株及菌株组合均具有分泌IAA的能力,分泌量在0.212~9.331 μg/mL,Hsg+lhs11组合菌株培养液IAA含量显著高于单菌株Hsg和lhs11(P<0.01),并呈现“1+1>2”的加成效果。综合各处理的促生特性,组合Hsg+lhs11和Hsg+ls1-3+lhs11菌株间互作效应较强,有望成为研制复合菌肥的最佳菌株组合。     

Construction and evaluation of a delta cya delta crp Salmonella typhimurium strain expressing avian pathogenic Escherichia coli O78 LPS as a vaccine to prevent airsacculitis in chickens.

Avian pathogenic strains of Escherichia coli cause a number of extraintestinal diseases in poultry, including airsacculitis and colisepticemia. Expression of O78 lipopolysaccharide (LPS) is frequently associated with pathogenic isolates. Salmonella, a common poultry contaminant, is a major public health concern. The purpose of this work was to develop an E. coli vaccine for poultry with the use of an attenuated Salmonella typhimurium carrier that would benefit both the bird and the consumer. Orally administered attenuated S. typhimurium delta cya delta crp strains have been shown to provide excellent protection against wild-type Salmonella challenge in chickens. This work describes the construction of a delta cya delta crp derivative of an avian pathogenic S. typhimurium that expresses both the homologous group B determinants (O1,4,5,12) and the heterologous E. coli O78 LPS O antigens. This was accomplished by inserting the E. coli rfb region, which encodes the genes required for O78 expression, into the chromosomal cya gene of S. typhimurium, creating a defined deletion/insertion mutation. A delta crp mutation was introduced in a subsequent step. Expression of both O antigens was stable in vitro and in vivo. Vaccination of white leghorn chicks at day of hatch and 14 days with the recombinant vaccine strain induced serum immune responses against both S. typhimurium and E. coli LPS and protected the birds against subsequent challenge with an avian pathogenic E. coli O78 strain. Introduction of a mutation in rfc, which encodes the O antigen polymerase, reduced the chain length of the S. typhimurium LPS without affecting the expression of O78. The rfc mutation further enhanced the ability of the vaccine strain to protect chickens against E. coli challenge.     

Demonstration of polymorphism among Brucella ovis field isolates by pulsed-field gel electrophoresis

Brucella ovis is recognized worldwide as an important pathogen of sheep, and has also been identified in farmed deer in New Zealand. Previously, only one strain type of B. ovis has been identified. The objective of this paper was to perform pulsed-field gel electrophoresis (PFGE) on field isolates of B. ovis to determine whether strain variations exist, whether sheep and deer are affected by the same strains, and to compare the performance of the rare-cutting restriction enzymes XbaI and SwaI. Ten B. ovis isolates from sheep and two from deer in New Zealand, as well as the type strain, were subjected to PFGE analysis using both XbaI and SwaI. PFGE of XbaI restriction fragments produced two banding patterns consisting of 27-28 bands, which were found to be 98% similar by cluster analysis, and were named X1 and X1a. PFGE of SwaI restriction fragments resulted in three banding patterns consisting of 13-15 bands each. Ten of the isolates had identical banding patterns and were named S1. One isolate differed by one band, representing a subtype named S1a. Two isolates differed by six bands, representing a different strain type of B. ovis and this was named S2. Cluster analysis showed S2 to be 78% similar to the S1/S1a cluster. Both strain types were isolated from both sheep and deer. Thus, two distinct strain types of B. ovis were identified in New Zealand, which is the first report of more than one strain type being identified worldwide. Neither strain was species-specific for sheep or deer. The restriction endonuclease SwaI was found to be more discriminatory than the enzyme XbaI, which has been used in previous studies.     

牛冠状病毒S基因的序列分析及原核表达

为了解牛冠状病毒（bovine coronavirus,BCoV）的S基因变异情况并建立ELISA检测方法,本研究对采自不同牛场的新生犊牛腹泻（CD）和成年牛冬痢（WD）腹泻样本提取总RNA,反转录合成cDNA,利用PCR扩增S全基因和S1基因。将S1基因目的片段连接表达载体pET-32a （+）,并转化大肠杆菌BL21（DE3）感受态细胞,经PCR、双酶切及测序验证正确后,进行IPTG诱导表达。结果显示,CD与WD分离株S基因核苷酸为98.4%,CD和WD分离株与参考毒株BCoV-ENT株核苷酸同源性最高,分别为98.4%和98.5%,CD分离株与参考毒株SUN5株的同源性最低,为97.5%,WD分离株与FRA/EPI/Caen/2003/13同源性最低,为97.3%。通过比对可知,分离株与已知毒株之间存在较大差异,为疫苗候选毒株筛选提供依据。本试验同时构建了pET-32a-S1表达载体,在0.2 mmol/L IPTG诱导5 h时,重组菌能在大肠杆菌BL21感受态细胞中产生大量的S1融合蛋白,获得约58 ku表达产物。本试验成功表达了S1蛋白,并对BCoV进行了核苷酸进化分析,为疫苗免疫效果评价方法的建立奠定了基础。     

Phenotypic expression of K88 adhesin alone or simultaneously with K99 and/or F41 adhesins in the bovine enterotoxigenic Escherichia coli strain B41

F41-positive and F41-negative derivatives of bovine enterotoxigenic Escherichia coli strain B41 carrying K88 or K88 and K99 plasmids were investigated for stability and expression of genes for their fimbrial antigens. Either K88 plasmid alone or both K88 and K99 plasmids could be maintained in these strains though stability could depend on culture medium. K99 antigen could be detected in each strain bearing K99 plasmid. Clones that produced K88 antigen or clones that did not produce this antigen could be isolated from each strain, except from the strain that possessed K99 plasmid in the strain that did not possess the ability to produce F41 antigen. Strains possessing K88 plasmid in the strain able to produce F41 antigen produced clones expressing either both K88 and F41 antigens, (also F41 appeared strongly expressed in some clones) or clones that produced only F41 antigen or no antigen at all. Clones that produced only K88 antigen or others that did not produce this antigen could be produced from a strain bearing only K88 plasmid and that did not possess the ability to produce F41 antigen. None of these strains bearing K88 plasmid alone or additionally K99 plasmid produced mannose-resistant hemagglutination of horse or sheep erythrocytes at 20°C as found for K99 and F41 ETEC natural strains, respectively. These results suggested that the structures of pili when several genetic determinants were present simultaneously may not be identical to those of original strains. In this study, clones expressing either one, two or three adhesin bearing antigens could be obtained from the strain B41.     

耐氟喹诺酮类药物禽源大肠埃希菌gyrA基因的检测分析

探讨不同禽源大肠埃希菌中喹诺酮类药物的耐药情况及耐药基因gyrA的分布和突变特征。采用K-B药敏纸片法、gyrA基因的PCR扩增,对9株大肠埃希菌进行喹诺酮类药物试验,并将gyrA基因的PCR产物测序,对测序结果采用DNA MAN、DNA Star、MEGA6等软件分析。药敏试验结果表明,C1、C2、C3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星敏感,D1、D2、D3、B1、B2和B3菌株对左氧沙星、氧氟沙星、环丙沙星、诺氟沙星均表现为耐药和中介;gyrA基因的测序结果表明,除B1菌株有1处核苷酸突变位点和B2菌株有14处核苷酸突变位点;B2菌株gyrA基因的氨基酸突变发生在87位Ile→Val替代、101位Leu→Met替代、102位Ala→Ser替代、129位Lys→Gln替代。9株禽源大肠埃希菌的同源性和进化树分析表明,不同禽源耐氟喹诺酮类药物的大肠埃希菌菌株中B2菌株gyrA基因与其他9株菌株相比,同源性在90%左右,进化树不在一个分支上,研究中的B2菌株将为大肠埃希菌的氟喹诺酮类耐药机制的研究提供候选菌株。