一种具有G418抗性筛选标记的慢病毒RNA干扰表达载体的构建及应用

目的:构建具有新霉素抗性筛选标记的RNA干扰慢病毒表达载体,并检测它对乙型肝炎病毒x基因(HBx mRNA表达的抑制作用。方法:从pcDNA3.0载体中扩增新霉素抗性基因并插入删除嘌呤霉素编码序列的pLKO载体中;将改构的RNA干扰载体包装成慢病毒后感染肝癌细胞HepG2,并检测感染细胞对G418的抵抗作用;为验证改构RNA干扰载体的有效性,设计了2条针对HBx的RNA干扰序列以及针对编码萤光素酶cDNA的干扰序列并插入该载体,将含新霉素筛选标记的HBx的RNA干扰载体与辅助质粒在293T细胞中包装成慢病毒并感染过表达HBx的HepG2(嘌呤霉素抗性)细胞,运用G418筛选出稳定混合克隆,提取细胞总RNA,运用RT-qPCR检测其在HepG2细胞中对HBx RNA表达的抑制作用。结果:构建的载体与辅助质粒包装出的慢病毒感染肝癌细胞后,细胞获得G418抗性;HBx RNA干扰序列克隆入该载体可有效抑制肝癌细胞中过量表达的HBx mRNA。结论:构建了具有新霉素抗性筛选标记的RNA干扰慢病毒表达载体,运用该载体可有效筛选出抑制目的基因表达的G418抗性的稳定细胞株。     

硝磺草酮抗性菌株的筛选及抗性基因的克隆表达

【目的】从采集的土壤中筛选出硝磺草酮的抗性菌株,并从中克隆对羟苯基丙酮酸双加氧酶抗性基因。【方法】以酪氨酸为唯一碳源,采用富集培养法筛选分离硝磺草酮抗性菌株,利用16S rRNA基因序列分析对菌株进行初步鉴定。通过PCR扩增获得其HHPD基因序列,构建pETH4表达载体并在大肠杆菌Escherichia coli BL21(DE3)中进行异源表达。通过检测色素在440 nm处的吸收值分析菌株E. coli BL21(DE3)-pETH4对硝磺草酮的抗性特性。【结果】在含10 mmol/L硝磺草酮和1 g/L酪氨酸的选择培养基上,分离得到7株硝磺草酮抗性细菌,1株为不动杆菌属,2株为无色杆菌属,4株为假单胞菌属。从抗性最佳的Pseudomonas sp. AM-H4中扩增得到HPPD的基因片段为1 056 bp,其序列与Acinetobacter baumannii基因组中HPPD的基因序列相似性达到99%,341位点由天冬氨酸突变为丙氨酸。HPPD基因在大肠杆菌中实现异源表达,蛋白分子量大小约40 kD。菌株E. coli BL21(DE3)-pETH4在40 μmol/L硝磺草酮酪氨酸LB培养基中的色素吸收值显著降低,能够耐受高于200 μmol/L的硝磺草酮。【结论】克隆获得的HPPD具有良好的硝磺草酮抗性,将在新除草剂抗性作物选育中有一定的应用潜力。     

运用萎锈灵抗性基因构建香菇聚乙二醇介导的遗传转化方法

【背景】萎锈灵抗性基因作为筛选标记在植物和真菌中得到广泛应用。【目的】构建可以使用萎锈灵作为筛选标记的香菇遗传转化技术。【方法】利用溶壁酶消化培养4 d的香菇菌株411-4的菌丝体获得原生质体,加入适量pL-cbx质粒和聚乙二醇溶液,混合物涂布于再生筛选培养基上,培养后挑选菌落进行验证试验。【结果】在原生质体数目108个和添加4μg质粒DNA的情况下,得到了40个抗性转化子。利用PCR实验和转代实验对转化子进行验证,结果显示38个转化子的抗性可稳定遗传,表明萎锈灵抗性基因整合进入了供试菌株的基因组中。【结论】利用香菇411-4菌株建立了一套运用萎锈灵抗性基因作为分子标记的遗传转化技术体系。     

紫云英AD-cDNA文库构建及与豆血红蛋白Lb相互作用靶蛋白的筛选

【目的】本文旨在构建紫云英酵母双杂交AD-cDNA文库和互作靶蛋白筛选平台,为深入研究共生固氮作用的分子机理奠定工作基础。【方法】以接种华癸中慢生根瘤菌7653R的豆科植物紫云英不同时期根部组织为材料,抽提和纯化RNA,构建了一个酵母AD-cDNA文库。库容量达到1.02×106/3μg pGADT7-RecDNA,插入片段大小1-1.5 kb左右。以紫云英豆血红蛋白基因AsB2510构建诱饵载体pGBKT7-AsB2510,利用酵母双杂交技术,筛选与诱饵蛋白相互作用的靶蛋白。【结果】在含有X-gal的SD四缺培养基上筛选得到26个克隆,经过质粒抽提、PCR鉴定、回转酵母验证获得10个阳性克隆。【结论】对阳性克隆的外源片段进行了测序和同源性分析,发现一个值得深入研究的含有tify domain和Divergent CCT motif的转录调控因子。     

毕赤酵母组成型表达脂肪酶及其高通量筛选方法

【目的】获得组成型表达脂肪酶毕赤酵母,建立利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因的有效方法。【方法】运用PCR技术从pGAPZαA表达载体上扩增得到GAP启动子片段,插入到表达载体pPIC9K-proRCL中,构建组成型表达载体pGAPK-proRCL。在保留含有同源双交换重组序列的诱导型启动子AOX1序列的基础上,电转化后华根霉Rhizopus chinensis CCTCC M201021脂肪酶基因proRCL表达盒在毕赤酵母基因组上发生双交换整合事件,从而组成型表达单拷贝的华根霉脂肪酶基因。【结果】重组菌发酵144 h后,脂肪酶最高酶活为130 U/mL。利用橄榄油罗丹明B平板高通量筛选组成型表达华根霉脂肪酶基因。【结论】该方法将初筛时间从12 d缩短为3 d,排除了多拷贝突变株的干扰,为后续脂肪酶的定向进化及筛选奠定了基础。     

米曲霉(Aspergillus oryzae) RIB40全长cDNA文库的构建

【目的】构建米曲霉RIB40的全长cDNA表达文库,为米曲霉功能基因的开发以及次生代谢产物合成途径相关基因的筛选与克隆奠定基础。【方法】采用RNAiso法从米曲霉RIB40菌体中提取总RNA。选用PolyATract mRNA Isolation System Ⅲ试剂盒分离纯化mRNA。以5μg mRNA为模板,按照ZAP-cDNA Synthesis Kit试剂盒说明书要求合成单、双链cDNA,使用CHROMA SPIN-400柱离心层析纯化后连接于Uni-ZAP XR表达载体上,体外包装后转染Escherichia coli XL1-Blue宿主菌。【结果】构建了米曲霉RIB40的全长cDNA文库,初级文库滴度约为2.96×106 CFU/mL,重组率约为97.8%,插入片段平均长度大于1.5 kb,达到一个高质量cDNA文库的要求。文库扩增后,滴度达到3.4×1010 CFU/mL。【结论】米曲霉RIB40全长cDNA表达文库的成功构建,将会对米曲霉基础生物学研究及相关基因的筛选与克隆奠定基础。     

里氏木霉分泌型表达载体的构建及绿色荧光蛋白的表达

【目的】构建里氏木霉分泌型表达载体,通过表达绿色荧光蛋白论证载体的可行性并初步观察绿色荧光蛋白在里氏木霉中的分泌过程。【方法】应用PCR及分子克隆技术将里氏木霉(Trichoderma reesei)纤维二糖水解酶(CBH1)的启动子及CBH1自身信号肽、终止子和潮霉素筛选基因依次插入骨架质粒pUC19中,构建出T.reesei表达载体Ppth15。将增强型绿色荧光蛋白(eGFP)基因装载入Ppth15中,获得eGFP表达载体Ppth15-eGFP。再将Ppth15-eGFP转化进T.reesei原生质体,通过潮霉素抗性筛选、基因组PCR检测等方法鉴定,获得阳性重组转化子。【结果】用PDA培养基培养阳性转化子2-3 d后,可在菌丝顶端、隔膜及培养基中清晰地观察到大量绿色荧光。【结论】表达载体构建成功且能够用于eGFP的表达,实验为进一步研究T.reesei表达其他基因提供了有效工具,同时为T.reesei胞外蛋白分泌的研究提供了参考。     

预防仔猪腹泻无抗性基因工程菌株的构建

以原来含热不稳定肠毒素B亚单位基因质粒DNA为载体,插入lacZ基因,构建了一个既能表达LT-B亚单位又能表达B-半乳糖苷酶的含四环索抗性基因的重组体。在该重组DNA的四环素抗性基因内再插入K 88粘附因子抗原基因,从而灭活了四环素抗性基因。经测定该重组体能表达LT－B和K88两种抗原,lacZ基因取代四环素抗性基因,成为良好筛选标记。所构建的重组质粒能稳定存在。     

以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍(MNNG)诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6~2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1~2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

以潮霉素B抗性为选择标记的深黄被孢霉原生质体转化

利用亚硝基胍（MNNG）诱变方法筛选了一株深黄被孢霉潮霉素B敏感型菌株M6-22-4。采用PEG介导的方法,将含有E.coli潮霉素B抗性标记的PD4质粒转入敏感株M6-22-4原生质体,并在潮霉素B浓度为400μg/mL的选择培养基上筛选转化子,获得了1.6～2.8个转化子/μg质粒DNA的转化频率。稳定性实验表明,质粒线性化后所获得的转化子在PDA培养基上传代10代以后,转接到选择平板上有31.6%仍具有HmB抗性;随机挑选了3个转化子,通过PCR方法检测到潮霉素抗性基因的存在,Southern杂交发现,潮霉素抗性基因已经以1～2拷贝数整合到深黄被孢霉M6-22-4染色体上,这是深黄被孢霉转化系统的首次报道。     

A general approach to high-yield biosynthesis of chimeric RNAs bearing various types of functional small RNAs for broad applications

RNA research and therapy relies primarily on synthetic RNAs. We employed recombinant RNA technology toward large-scale production of pre-miRNA agents in bacteria, but found the majority of target RNAs were not or negligibly expressed. We thus developed a novel strategy to achieve consistent high-yield biosynthesis of chimeric RNAs carrying various small RNAs (e.g. miRNAs, siRNAs and RNA aptamers), which was based upon an optimal noncoding RNA scaffold (OnRS) derived from tRNA fusion pre-miR-34a (tRNA/mir-34a). Multi-milligrams of chimeric RNAs (e.g. OnRS/miR-124, OnRS/GFP-siRNA, OnRS/Neg (scrambled RNA) and OnRS/MGA (malachite green aptamer)) were readily obtained from 1 l bacterial culture. Deep sequencing analyses revealed that mature miR-124 and target GFP-siRNA were selectively released from chimeric RNAs in human cells. Consequently, OnRS/miR-124 was active in suppressing miR-124 target gene expression and controlling cellular processes, and OnRS/GFP-siRNA was effective in knocking down GFP mRNA levels and fluorescent intensity in ES-2/GFP cells and GFP-transgenic mice. Furthermore, the OnRS/MGA sensor offered a specific strong fluorescence upon binding MG, which was utilized as label-free substrate to accurately determine serum RNase activities in pancreatic cancer patients. These results demonstrate that OnRS-based bioengineering is a common, robust and versatile strategy to assemble various types of small RNAs for broad applications.     

Saccharide-RNA recognition in a complex formed between neomycin B and an RNA aptamer.

BACKGROUND: Aminoglycoside antibiotics can target RNA folds with micromolar affinity and inhibit biological processes ranging from protein biosynthesis to ribozyme action and viral replication. Specific features of aminoglycoside antibiotic-RNA recognition have been probed using chemical, biochemical, spectroscopic and computational approaches on both natural RNA targets and RNA aptamers identified through in vitro selection. Our previous studies on tobramycin-RNA aptamer complexes are extended to neomycin B bound to its selected RNA aptamer with 100 nM affinity. RESULTS: The neamine moiety (rings I and II) of neomycin B is sandwiched between the major groove floor of a 'zippered-up' G.U mismatch aligned segment and a looped-out purine base that flaps over the bound antibiotic. Specific intermolecular hydrogen bonds are observed between the charged amines of neomycin B and base mismatch edges and backbone phosphates. These interactions anchor 2-deoxystreptamine ring I and pyranose ring II within the RNA-binding pocket. CONCLUSIONS: The RNA aptamer complexes with tobramycin and neomycin B utilize common architectural principles to generate RNA-binding pockets for the bound aminoglycoside antibiotics. In each case, the 2-deoxystreptamine ring I and an attached pyranose ring are encapsulated within the major groove binding pocket, which is lined with mismatch pairs. The bound antibiotic within the pocket is capped over by a looped-out base and anchored in place through intermolecular hydrogen bonds involving charged amine groups of the antibiotic.     

以neo作选择标记富集和筛选阳性重组杆状病毒

将苜蓿银纹夜蛾(AcNPV)极早期基因IEI启动子控制的neo基因表达盒插入p10型转载体pAcEP106中得到pAcPIneo,将它和野生型AcNPV DNA共转染Sf细胞得到neo^+重组病毒.因为neo基因的表达使得neo^+基因的重组病毒可以在G418的存在下复制,而野生型则不能.将共转染得到的上清液以很低的感染复数连续传代,通过G418的选择作用,使重组病毒得到富集,三代以后重组病毒比例可达90%以上,如此之高的重组比例使得重组病毒的筛选不须经空斑纯化只须有限稀释即可得到.利用杆状病毒早期基因启动子表达neo基因为用早期基因表达外源毒素基因作重组病毒杀虫剂打下了基础,同时也建立了一种简便有效的筛选、富集阳性重组病毒的方法.     

稳定表达禽β防御素6的DF-1细胞系的建立及其抑菌活性

【背景】禽β防御素6是禽体内分泌的一类抗菌肽,在抵抗病原入侵和免疫调节中发挥着重要作用,但其常规表达方式效率较低,难以在产业化生产中加以应用。【目的】建立稳定表达AvBD6的细胞系,并检测其表达产物对耐药大肠杆菌的抗菌活性,为其他防御素表达提供参考。【方法】利用显微镜观察构建真核重组表达载体pLOV-eGFP-AvBD6转染至293T细胞后的转染效率;收集293T细胞上清液并感染DF-1细胞,通过嘌呤霉素加压筛选稳定表达株;利用RT-PCR和Western Blot分别检测目的基因在转录水平和蛋白水平的表达情况;利用扫描电镜观察细胞培养上清液对耐药大肠杆菌的抗菌效果及其对菌体的损伤。【结果】成功构建重组表达载体pLOV-eGFP-AvBD6,筛选出稳定表达AvBD6的DF-1细胞系,而且目的基因在转录水平和蛋白水平均有表达;细胞培养上清显著降低大肠杆菌和副伤寒沙门菌存活率,对金黄色葡萄球菌的抗菌活性较低。【结论】建立了稳定表达AvBD6的DF-1细胞系,其表达产物对耐药大肠杆菌具有良好的抗菌效果,对推动防御素的应用提供技术支持。     

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes

We examined the suppression of virus expression by cleaveage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.     

Effective suppression of HIV-1 gene expression by a mammalian tRNA 3' processing endoribonuclease and external guide sequence oligozymes.

We examined the suppression of virus expression by cleavage of the HIV-1 RNA gene using a mammalian tRNA 3' processing endoribonuclease and an External Guide Sequence Oligozyme (EGS) in vivo. We constructed an EGS expression vector that used the tRNA(met) promoter as an expression cassette for EGS. The EGS expression vector was targeted to the upstream region of gag, region. The EGS expression vector was co-transfected into COS cells with the HIV-1 gene plasmid vector. As compared with the EGS non-expressing cells and the EGS expressing cells, the EGS expressing cells with the targeted gag start codon had a clearly decreased amount of the HIV-1 gag p24 protein. The EGS expressing cells with the targeted gag start codon showed effective suppression of HIV-1 gene expression. Thus, these studies describe novel gene targeting agents for the inhibition of gene expression and antiviral activity.     

以杆状病毒为载体在鸡原代骨骼肌细胞中表达IBDV VP2基因

摘要：【目的】本研究旨在构建在鸡原代骨骼肌细胞中表达IBDV病毒VP2基因的重组杆状病毒。【方法】从IBDV适应细胞毒中提取RNA,用RT-PCR技术扩增VP2基因,将其克隆到自主构建的杆状病毒转移载体的CMV启动子之下,通过Bac-to-Bac系统获得VP2重组Bacmid,并将其转染Sf9昆虫 细胞,获得了VP2重组杆状病毒。重组病毒经扩增后以50个MOI感染鸡原代骨骼肌细胞,接种72h后裂解细胞收获蛋白。【结果】蛋白样品经SDS-PAGE和Western blot证实VP2蛋白获得表达,分子量约48kDa,与预测蛋白大小一致,且能被IBDV阳性血清所识别。【结论】重组杆状病毒可以有效地将VP2基因导入鸡原代细胞,并在CMV的启动下表达具有抗原性的VP2蛋白,本研究为研制IBDV及其他重要禽类传染病的杆状病毒载体疫苗奠定了基础。     

Cloning of the nptII gene of Escherichia coli and construction of a recombinant strain harboring functional recA and nptII antibiotic resistance

In an attempt to clone the ORF of the nptII gene of Escherichia coli K12 (ATCC 10798), two degenerate primers were designed based on the nptII sequence of its Tn5 transposon. The nptII ORF was placed under the control of the E. coli hybrid trc promoter, in the pKK388-1 vector, transformed into E. coli DH5α ΔrecA (recombinant, deficient strain). Transferred cells were tested for ampicillin, tetracycline, kanamycin, neomycin, geneticin, paromomycin, penicillin, and UV resistance. The neomycin phosphotransferase gene of E. coli was cloned successfully and conferred kanamycin, neomycin, geneticin, and paromomycin resistance to recombinant DH5α; this did not inhibit insertion of additional antibiotic resistance against ampicillin and tetracycline, meaning the trc promoter can express two different genes carried by two different plasmids harbored in the same cell. This resistance conferral process could be considered as an emulation of horizontal gene transfer occurring in nature and would be a useful tool for understanding mechanisms of evolution of multidrug-resistant strains.     

Recognition of a cognate RNA aptamer by neomycin B: quantitative evaluation of hydrogen bonding and electrostatic interactions

Aminoglycosides are an important class of antibiotic that selectively target RNA structural motifs. Recently we have demonstrated copper derivatives of amino-glycosides to be efficient cleavage agents for cognate RNA motifs. To fully develop their potential as pharmaceutical agents it is necessary to understand both the structural mechanisms used by aminoglycosides to target RNA, and the relative contributions of hydrogen bonding and electrostatic interactions to recognition selectivity. Herein we report results from a calorimetric analysis of a stem-loop 23mer RNA aptamer complexed to the aminoglycoside neomycin B. Key thermodynamic parameters for complex formation have been determined by isothermal titration calorimetry, and from the metal-ion dependence of these binding parameters the relative contributions of electrostatics and hydrogen bonding toward binding affinity have been assessed. The principal mechanism for recognition and binding of neomycin B to the RNA major groove is mediated by hydrogen bonding.