A Stably Transgenic INA Enterobacter cloacae for Control of Insect Pests

Using the minitransposon pMini-Tn5 and the ice-nucleation active (INA) gene of iceA, a suicide recombinant plasmid pTnice1 was constructed, which has the ability of broad-host-range conjugal mobilization and integration of iceA into chromosomal DNA of many gram-negative bacteria by Tn5 transposition.We used this plasmid to integrate the iceA into chromosomal DNA of Ent. cloacae and obtained the transgentic strain Enc1. 2022ina. In this transgenic Ent. cloacae, iceA would never be transferred elsewhere through transposition, and constantly expressed high ice nucleation activity even in the absence of antibiotic pressure.The transgenic strain was ingested by corn borer larvae. Over the 7 d after ingestion, the mean supercooling points (SCPs) of the larvae was about 10℃ higher than those of larvae treated with distilled water (control).The maintenance of these high SCPs was related to the stable gut colonization of transgenic strain. At 6th day post ingestion, the larva was exposed at - 5 or - 7℃ for 12 h, the percentages of larvae frozen to death were 85and 100%, respectively. In contrast, none or a small proportion of control larvae was frozen to death under the same conditions. Further studies demonstrated that this transgenic strain bore weak epiphytic ability.Therefore, this genetically engineered strain may be a promising candidate for control of insect pests in agricultural fields.     

miR-144表达质粒的构建及其对肝癌细胞生物学行为的影响

【目的】构建microRNA-144(miR-144)的真核表达质粒,并探讨miR-144对人肝癌细胞株HepG2生物学行为的影响,为肝癌的生物学治疗提供依据。【方法】构建miR-144真核表达质粒并合成miR-144反义寡核苷酸,通过实时定量PCR检测miR-144的表达,采用MTT比色法及流式细胞仪技术,检测抑制与过表达miR-144对肝癌细胞增殖、凋亡和细胞周期的影响。【结果】成功构建了miR-144的真核表达质粒,过表达miR-144能够显著抑制肝癌细胞的增殖(P<0.05),促进细胞凋亡(P<0.05);而抑制miR-144的表达则得到相反的结果;抑制与过表达miR-144对肝癌细胞周期的影响都不大(P>0.05)。【结论】miR-144能影响人肝癌细胞株HepG2的生物学行为,具有抑制肝癌细胞增殖的作用。     

带有绿色荧光蛋白外源DNA转染绵羊成纤维细胞的研究

为了优化建立转染绵羊成纤维细胞系的外源基因转染体系,本研究利用带有绿色荧光蛋白（GFP）的外源基因,采用阳离子脂质体法,探讨了DNA用量、脂质体用量和转染时间等因素对转染绵羊成纤维细胞的影响。结果表明,在500 μL转染培养基中成纤维细胞达到85%汇合时,添加由50 μL DMEM中含有0.2 μg DNA和50 μL DMEM中含有2.0 μL LipofectamineTM 2000的两种溶液混合构成的转染液并转染12 h,可获得最高的转染效率。当转染12 h后,用基础培养液培养至第24小时,细胞核周区出现强绿色荧光,而当转染后48～64 h观察时,细胞核周区的荧光强度明显弱,而远离核周区的胞质中的荧光强度增加。     

不同来源嗜水气单胞菌的抗菌素耐药性及耐药机制分析

分离、收集不同时期来源于俄罗斯及国内各地的40株嗜水气单胞菌,常规生理生化鉴定结果表明这些菌株均为嗜水气单胞菌。9种抗菌素药敏纸片对全部菌株的测定结果显示,菌株的耐药强度与原分离地是否使用抗菌素高度相关。质粒与菌株耐药相关性分析结果表明,12个具有1个以上多拷贝质粒的菌株中,有11株呈现高度耐药性,提示耐药性与多拷贝质粒存在相关性;但无质粒或仅有低拷贝质粒的菌株,其耐药性强弱与质粒无相关性。菌株耐药稳定性试验结果证明,4℃条件下长时间保存会降低菌株的抗性,而连续多次冻存、复苏传代对菌株的抗性无影响。     

一种用高拷贝质粒载体制备BAC载体基本功能基因的新方法

介绍一种改进的用普通高拷贝质粒pUC119来制备细菌人工杂色体（BAC）载体基本功能基因序列的新方法及其在构建分子克隆化病毒中的应用,该方法可使BAC载体基本功能基因序列的制备更为简易、高效。     

运用随机扩增多态性和质粒指纹图谱法进行大肠杆菌O157的分子多态性分析

为了解大肠杆菌O157分离菌株的分子特征,建立了随机扩增多态性和质粒指纹图谱2种分型方法,对10株从粪便和动物组织分离的大肠杆菌O157进行了分型研究,结合主要毒力基因的检测和药敏试验,以进一步验证分离菌株之间的亲缘关系.随机扩增多态性分析分别在遗传距离为0.89、0.973处将所有细菌分类:其中有2株菌株具有相似的扩增图谱,可以聚为同一类,亲缘关系较近,其余8株细菌类聚为两大类.质粒图谱分析在不同的进化距离处将所有细菌分类,形成具有复杂分枝的树状图.毒力因子检测结果显示:10株分离菌株均含有hly、eae、uid、flich7基因.药敏试验幸明:其中2株细菌具有不同于其他8株细菌的耐药谱,与随机扩增多态性分析和质粒图谱分析相一致.比较随机扩增多态性和质粒指纹图谱2种分型方法发现:它们虽具有相似的结果,但质粒图谱法分析更能反映出菌株之间的亲缘关系.     

含绿色荧光蛋白(gfp)基因的植物重组表达载体pBI121-GFP-62390和pBI121-GFP-51780的构建

[目的]构建含绿色荧光蛋白(gfp)基因的植物重组表达载体pBI121-GFP-62390和pBI121-GFP-51780。[方法]用PCR方法扩增psmGFP的GFP片段,BamHI和SacI双酶切PCR产物,同时用BamHI和SacI双酶切pBI121。从琼脂糖凝胶中回收纯化pBI121的大片段,经连接、转化、鉴定出改造后的质粒。[结果]用PCR方法扩增得到长度为740 bp的增强型的绿色荧光蛋白基因片段,克隆入pBI121表达载体后,获得了新的重组质粒pBI121-GFP。分别将编码拟南芥BAG7、BAG4蛋白的基因At5g62390和At3g51780通过PCR方法扩增后,克隆入pBI121-GFP,构建了用于超量表达BAG蛋白基因的GFP融合蛋白双元表达载体pBI121-GFP-62390和pBI121-GFP-51780。利用细胞感受态法将该植物表达载体分别导入根癌农杆菌GV3101中。[结论]为进一步研究BAG蛋白在拟南芥抗性胁迫中的功能及其在细胞内的动态分布奠定了基础。     

Effect of cell mediated immunity regulation of duck enhanced by duck IFN-α eukaryon expression plasmid and inoculated with DPV attenuated vaccine by gene-gun

In order to study the effect of cell mediated immunity regulation of duck IFN-α eukaryon expression plasmid (pcDNA-SDIFN-α) on duck plague virus (DPV) attenuated vaccine in ducks, pcDNA-SDIFN-α was administered to 28-day-old ducks at doses of 1, 3 and 6 μg per duck, respectively, by gene-gun. PBS and empty vector pcDNA were used as control. Fifteen days later, all ducks were injected with DPV attenuated vaccine and blood samples were collected at 3, 7, 14, 21, 28, 35, 49, 63 and 84 days after injection. T-lymphocyte proliferation tests (MTT) were used to detect the T-lymphocyte proliferation in the peripheral blood (PBL) of ducks. Blood samples collected at 7, 14, 21, 28, 35 and 49 days after injection were detected by fluorescence-activated cell sorter (FACS) for recording the number of CD₃ ⁺ T-lymphocytes of ducks. Results were as follows: (1) Reaction of T-lymphocytes in PBL to ConA (OD value) of ducks treated with pcDNA-SDIFN-α was higher than that of PBS and pcDNA control groups in 3–84 days. There were highly significant differences between the 1 μg per duck group and the two control groups in 3–84 days (P ⩽ (0.01), between the 3 μg per duck group and the two control groups in 3–84 days (P ⩽ 0.01, P ⩽ 0.05), and between the 6 μg per duck group and the two control groups in 7–49 days (P ⩽ 0.01, P ⩽ 0.05). The significant difference was also present between the groups of 1, 3 and 6 μg per duck in 3–35 days (P ⩽ 0.05). However, there was no significant difference between the 3 and 6 μg per duck groups (P ⩾ 0.05). The pcDNA control group was higher than PBS control group, but no difference was detected (P ⩾ 0.05). (2) Change of the number of CD₃ ⁺ T-lymphocytes in ducks administered with different doses of pcDNA-SDIFN-α was higher than that of PBS and pcDNA control groups in 7–49 days. The change in the 1 μg per duck group was significantly higher than that in PBS and pcDNA control groups in 14–49 days (P ⩽ 0.01). There were significant differences between the 3 μg per duck group and the two control groups in 21–49 days (P ⩽ 0.01, P ⩽ 0.05) and between the 6 μg per duck group and the two control groups in 7–49 days (P ⩽ 0.01, P ⩽ 0.05). However, no significant differences among the groups of 1, 3, and 6 μg per duck groups (P ⩾ 0.05) and between the two control groups (P ⩾ 0.05) were found. The results indicated that pcDNA-SDIFN-α administered 15 days before injection of DPV-attenuated vaccine could significantly enhance cellular immunity induced by DPV-attenuated vaccine. pcDNA-SDIFN-α is an excellent DPV-attenuated vaccine molecular adjuvant and the best result can be obtained with the dose of 1 μg per duck of pcDNA-SDIFN-α inoculated by gene-gun. __________ Translated from Acta Veterinaria et Zootechnica Sinica, 2007, 38 (10): 1066–1071 [译自: 畜牧兽医学报]     

猪瘟病毒囊膜蛋白E2基因真核分泌型表达载体的构建及其表达

【目的】构建猪瘟病毒囊膜蛋白(E2 protein)基因的真核表达载体,并将其在猪脐静脉血管内皮细胞中进行表达。【方法】采用PCR技术扩增出E2蛋白全长基因(E2qc)序列和去除跨膜基因(E2sh)序列,并将其克隆入真核表达载体pSecTag2(A)中,构建pSecTag-E2qc和pSecTag-E2sh表达载体,分别进行PCR、双酶切及测序鉴定。用脂质体法将阳性克隆瞬时转染猪脐静脉血管内皮细胞,对转染细胞进行RT-PCR检测目的基因的转录情况,同时对转染细胞及细胞上清液进行SDS-PAGE和Western-blot分析,以检测目的蛋白的表达情况。【结果】成功克隆了E2蛋白基因全长序列1119bp和去除E2蛋白跨膜基因序列999bp的目的基因。构建的表达载体经PCR、双酶切法及测序鉴定均无误。转染后细胞的RT-PCR结果显示,目的基因被成功转录。转染后细胞及细胞上清液的SDS-PAGE和Western-blot结果显示,目的基因被成功表达。【结论】成功构建了猪瘟病毒E2蛋白的真核表达载体,转染猪脐静脉血管内皮细胞后,分泌表达了猪瘟病毒E2重组蛋白。     

大肠杆菌耐药基因定位及耐药质粒消除

对采集的9株野生型大肠杆菌在药敏试验的基础上,进行了耐药基因定位。结果表明,4个菌株的氨苄青霉素耐药基因位于质粒上,不同畜群来源菌株之间耐氨苄青霉素质粒存在差异。用中草药艾叶的乙醇提取物和蒸馏提取物对大肠杆菌庆大霉素耐药性及耐药质粒进行了消除试验,结果艾叶乙醇提取物的消除率最高可达69.4%。