质粒体

To study and know well the rudimentary knowledge and the basic techniques of modern Microbiology are demanded for Graduate students. The experiments including the principle and operation of phase contrast microscope and fluorescence microscope; Microphotography , the technique of photographic enlargement;Applications of polymerase chain reaction ; Construction, transformation and detection of plasmid in E.coli ; Infectivity of virus and its molecular detection ; The technique of protoplast fusion in filamentary fungi ; Induction and purification of β-galactosidase from E.coli and assay the enzyme activity; Detection of GC content and DNA-DNA hybridization in bacteria ; Production , extraction and detection of poly--hyduoxy butyrate from bacteria.

以实验教学为主，要求研究生学习和掌握一些现代微生物学研究所需的基本技术，内容包括：相差显微镜、荧光显微镜的使用和显微摄影技术，PCR技术的应用，细菌质粒的构建、转化及检测技术，病毒的侵染性及其分子检测技术，丝状真菌原生质体融合技术，大肠杆菌β-半乳糖苷酶的分离及纯化及其动力学测定技术，细菌G+C mol%的测定及细菌DNA-DNA杂交技术和聚羟基丁酸产生菌的发酵、产物提取及检测技术等。

Src l was an acidic cytolysin, which was reported forthe first time. The cDNA encoding the mature Src l was cloned into non-fusion expression vectorpBV220 and expressed successfully in E.coli DH5α in inclusion body. After washing anddenaturing-renaturing, the recombinant Src l protein was purified by Q Sepharose FastFlow ion exchange chromatograph and Phenyl Sepharose hydrophobic interactionchromatograph.

首先构建了Src I基因的非融合蛋白表达质粒pBV220-Src I，并成功地在原核细胞E.coli进行了重组表达，筛选出稳定表达的工程菌株DH5α，摸索了稳定表达的条件，摸索了包涵体的洗涤、变性和复性条件，摸索出了复性蛋白Src I的纯化工艺，纯化过程简单，经QSepharose Fast Flow阴离子交换层析和Phenyl Sepharose疏水层析两步纯化，就可获得了高纯度的重组蛋白样品(纯度达99％以上)，该纯化方法适合重组蛋白的大量纯化，为Src I的性质、结构和功能研究奠定了良好的基础。

By means of polymerase chain reactionand DNA recombinant techniques,the gene encoding mature ouse IL-4 was obtained from plasmid pCD-mIL4 and was inserted into expressionvector pBV220,which contains _RP_L double promoters,synthesized SD sequence and temperaturesensitive CIts857 gene,thus an expression lone eferred as pBV-mIL4 was constructed.

该质粒含有 P_RP_L 串联启动子，合成 SD 序列和温度敏感的CIts857基因，并且去除了完整小鼠 IL—4 cDNA 中3′侧翼非编码区，以原核系统高频使用的强终止密码子TAA 代替了原小鼠 IL-4基因的终止密码子 TAG，经 SDS-PAGE 及凝胶密度扫描分析，表达的重组小鼠 IL-4分子量约为14.2KDa，占总菌体蛋白量的25～30%。

The construction of the recombinant plasmid carrying shRNA to HPV6bL1 lays the basis for the study of its inhibitive effect on cauliflower excrescence.

经测序，测序结果均与目的序列相同，均符合设计要求。结论 HPV6bL1 shRNA质粒重组体的成功构建为研究尖锐湿疣基因靶向抗体打下基础。

A plastid that contains pigments other than chlorophyll, usually yellow or orange carotenoids.

有色体，色素体除了叶绿素外的含色素的质粒，通常为黄色或橙色的类胡萝卜素

CaN Aβ gene silencing can reduce myocardial hypertrophy in cultured cells, si1280 (21bp) of CaN Aβ gene is the most effective target site for siRNA. The method of intrapericardial injection of plasmid, microbubbles and erzymes can improve transfection efficiency of non-viral plasmid with satisfying targeted transfection. But the scope of transfected myocytes is still limited. CaN Aβ shRNA expressing plasmid transfection in vivo by pericardial injection results in decreased CaN Aβ protein expression of small part of myocytes, and CaN Aβ mRNA only shows decreased trend. The dosage of non-viral vector and the parameters of ultrasound energy should be optimized in further study.

结论RNAi技术抑制CaN Aβ基因表达可有效减轻Ald诱导的心肌细胞肥大程度；CaN Aβ基因中si1280(21bp)片段为实现RNAi的更有效片段；微泡+酶类心包腔内注射超声导入的方法可有效改善非病毒载体在体心肌的转染效率，同时具有一定的靶向性，但总的转染范围仍然有限；采用这一方法进一步进行&一肾一夹&心肌肥大模型大鼠在体心肌细胞的CaNAβ的RNAi干预，发现心肌肥大大鼠心外膜下局部心肌细胞CaN Aβ蛋白水平降低，CaN AβmRNA水平虽有下降趋势，但无统计学差异，提示质粒的用量及超声导入的参数有待进一步研究使其优化。

The results showed that about 3.18 x l0~7/ml of protoplasts were formed when the mycelia were incubated for 1 hour at 28 °C in TSB medium containing 2% glycin, and treated with 2 mg/ml lysozyme.

对东方拟无枝酸菌原生质体制备、再生及质粒DNA转化等条件进行了探索，考察了培养基组分、甘氨酸浓度、溶菌酶浓度等因素对原生质体的制备及其再生的影响。

The plasmid DNA remained intact and could be stored at 4℃ for at least one month in liposomes, it was also resistent to DNase I digestion.

重组质粒DNA在脂质体的制备过程中保持了稳定性，并能抵抗DNA酶的水解破坏作用。

AIM: To construct the eukaryotic expressing vector of human cbl and test its expression in African green monkey kidney cell line COS 7. METHODS: Human cbl gene tagged with flag was amplified from pEFHAcbl plasmid by PCR. The product was cloned into pGEM T easy, sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1.

以含有人全长cbl cDNA的质粒pEFHAcbl 为模板，采用PCR方法扩增cbl基因，并在其N 末端带上含24 bp的flag标签，克隆到pGEM T easy载体并测序，再亚克隆至真核表达载体pcDNA3 1，酶切鉴定正确后采用脂质体法瞬时转染COS 7细胞，Western blot检测flag cbl在细胞中的表达。

METHODS: Human cbl gene tagged with flag was amplified from pEFHAcbl plasmid by PCR. The product was cAIM: To construct the eukaryotic expressing vector of human cbl and test its expression in African green monkey kidney cell line COS7. METHODS: Human cbl gene tagged with flag was amplified from pEFHAcbl plasmid by PCR. The product was cloned into pGEMT easy, sequenced and then subcloned into eukaryotic expressing vector pcDNA3.1.

以含有人全长cbl cDNA的质粒pEFHAcbl 为模板，采用PCR方法扩增cbl基因，并在其N末端带上含24 bp的flag标签，克隆到pGEMT easy载体并测序，再亚克隆至真核表达载体pcDNA31，酶切鉴定正确后采用脂质体法瞬时转染COS7细胞，Western blot检测flagcbl在细胞中的表达。

She gently rebuff ed him, but agreed that they could be friends

她婉言拒绝了，但同意作为朋友相处。

If in the penal farm, you were sure to be criticized.

要是在劳改农场，你等着挨绳子吧！