融合有酸性片段的抗菌肽串连重复基因的构建

为提高抗菌肽的表达,设计在抗菌肽基因的N端融合一段编码酸性肽的片段以及减轻表达产物对宿主的毒性,通过含有酶切位点的接头将该融合肽基因以同向串连的方式连接成多拷贝基因,克隆至pUC19载体。为此,分段设计合成了编码天蚕素A-蜂毒素杂合肽和酸性肽的DNA片段。首先将其连接成融合肽全基因,然后分别与含相同粘性末端的前后接头连接。通过控制基因和接头加入的量及次序,可得到两侧有EcoRI和SalI酶切位点的同向串连的多拷贝基因。选取合适拷贝数的基因,将其克隆至pUC19载体,PCR扩增和DNA测序证明多拷贝基因构建成功且基因方向相同。结果表明,该方法能简捷高效地获得所需的多拷贝基因,为提高表达产物的量打下基因。     

家蝇抗菌肽Defensin基因同向串联表达载体的构建和鉴定

目的:构建家蝇抗菌肽Defensin基因多拷贝串联体,并克隆到甲醇酵母分泌表达载体pPIC9K上。方法:PCR法扩增家蝇抗菌肽Defensin基因成熟肽片断,目的片断的上游5′端带有EcoRⅠ和NheⅠ位点,下游5′端带有NotⅠ和XbaⅠ位点,目的片断首先克隆入pMD18-T载体,利用pMD18-T载体的NdeⅠ位点和目的片断上的一对同尾酶(NheⅠ和XbaⅠ),多次酶切连接,串联成多拷贝的Defensin成熟肽基因,再用EcoRⅠ和NotⅠ双酶切,最后克隆入甲醇酵母分泌表达载体pPIC9K。结果:PCR鉴定、酶切鉴定和DNA测序证明多拷贝基因重组质粒构建成功。结论:该方法能方便高效地获得所需的多拷贝基因,为进一步进行高效表达打下基础。     

天蚕素A-蜂毒素杂合肽用pBV220载体的表达、纯化和活性测定

为提高抗菌肽的表达,在抗菌肽的N端融合了1段酸性小肽以中和表达产物对宿主的毒性;并将融合肽基因同向串连成多拷贝,在大肠杆菌中获得了较高的表达。用化学合成法分别合成了编码天蚕素A(1-8)-蜂毒素(1-10)杂合肽和酸性小肽的DNA片段,首先将其拼接成融合肽的完整基因,然后通过前后接头将融合肽基因连接成两侧具有EcoRI和SalI酶切位点的同向串连的多拷贝基因。将5份拷贝的基因克隆至pBV220表达载体,转化E.coliDH5α,温度诱导得到表达量为35%的融合蛋白。表达产物主要以包涵体形式存在,将包涵体溶解,经Ni²⁺-NTA琼脂糖亲和层析获得纯化的融合蛋白。融合蛋白再经CNBr切割和阳离子交换层析,得到纯化的抗菌肽,经蛋白质N端测序确认序列正确。琼脂糖扩散法和液相测定法证明了纯化的抗菌肽具有抗菌活性。     

pGEM-8Hepc克隆载体的构建与序列分析

hepcidin是一种由肝脏合成的富含半胱氨酸的小分子肽,它的主要生物学功能是维持机体铁稳态,并且具有抗菌活性.本实验从大鼠肝脏组织克隆出hepcidin基因,用同尾酶法构建多拷贝串联基因克隆载体pGEM-nHepc(n=1,2,4,8)(单个hepcidin基因之间用一段连接肽的DNA连接),分别含有不同拷贝首尾串连的hepcidin cDNA,并在大肠杆菌DH5 α中扩增.重组体经双酶切和PCR鉴定后进行序列测定,所得结果与genbank中的大鼠序列比对,结果完全正确,为后续进一步的表达纯化奠定了基础.     

一种构建多拷贝串联小分子多肽基因的方法

目的:构建蛇毒锯鳞蝰血抑环肽(Ecs)基因串联多拷贝重组质粒。方法:采用重叠延伸PCR克隆Ecs基因,将第28位Met的密码子突变为Leu,利用其序列特点及酶切位点,在表达载体pET30a上将Ecs基因以同向串联方式连接,在E.coliBL21(DE3)中表达产物。结果:工程菌表达的串联Ecs与预期结果相符,实现了Ecs基因的串联表达。结论:为活性小肽的体外表达提供了新的思路和方法。     

双拷贝人α型心钠素基因的组建及大肠杆菌分泌型克隆与表达

将单拷贝人α心钠素基因3′端用Ban Ⅱ酶解除去包括终止密码在内的36个碱基对,代之以人工合成的含Glu-Lys-Phe-Glu连接片段与另一单拷贝人α心钠素基因的5′端串连成编码60肽的双拷贝心钠素基因,克隆于大肠杆菌分泌型表达载体pIN-Ⅲ-OmpA_2质粒中,表达生成60肽的双拷贝人α型心钠素衍生物,在信号肽的作用下分泌至胞膜间质并自动切割为60肽的外源基因产物。分子量约8K的表达产物用分子筛或超滤膜分离后再经HPLC纯化,表达产物具有明显的心钠素放免活性和舒张血管活性。     

具有除草剂抗性的TA克隆植物表达载体的构建

TA克隆载体由于能够用于PCR产物的快速克隆且操作简单,因此被广泛应用。p CXSN是以TA克隆为连接方式的植物表达载体,T-DNA区筛选标记为潮霉素抗性基因。本实验利用抗除草剂基因bar、gdh A替换载体上的潮霉素基因,构建了两种可以直接连接PCR产物的TA克隆植物表达载体,获得的含有除草剂抗性基因的p CXSN表达载体适合用于基因的正义和反义表达,不需要添加酶切位点,可以直接连接PCR产物,节省载体构建的步骤和时间。     

鲑鱼降钙素串联基因的克隆、表达及重组蛋白的生物学活性

目的：将鲑鱼降钙素(salmon calcitoni,sCT)基因以同向串联方式连接,构建串联多拷贝基因的表达质粒pHis-nCT(n≤3),并在原核中表达,研究表达产物的降钙活性。方法：采用半化学半酶促法合成sCT基因,利用基因的特点及特殊的酶切位点NdeI和SamI在表达载体pTrcHisC中进行sCT基因与载体基因的融合及sCT基因的串联,并在TOP10中表达串联多拷贝基因。表达产物形成包含体,对包含体变性、复性后,以Ni-Chelating Sepharose亲和纯化。用血清钙浓度测定法研究串联表达产物及裂解产物的降钙活性。结果：重组菌表达的串联融合蛋白经过变性、复性及亲合层析,纯度达到90％以上。活性试验表明,串联融合蛋白及其裂解产物可以抑制破骨细胞,降低血清钙浓度,且呈剂量效应关系。结论：原核表达质粒pTreHisC可以有效表达串联的sCT基因,重组的串联蛋白及裂解产物均有降钙活性。     

克隆木糖还原酶XYL1基因的工程菌YT发酵玉米芯水解液的研究

克隆毕赤氏酵母(Pichia stipitis)木糖还原酶基因XYL1,将其连接到适用于酿酒酵母工业菌株的多拷贝载体pYMIKP中,构建得到表达质粒pYMIKY-XYL1,转化酿酒酵母工业菌株Saccharomyces cerevisiae 6508.利用G418筛选转化子,得到含高拷贝木糖还原酶基因的酿酒酵母重组菌YT,以YT发酵玉米芯工业水解液生产木糖醇,研究其发酵特性和规律,为工业上生物转化法生产木糖醇提供参考.     

鞘蕊苏贝壳杉烯酸氧化酶基因的克隆及表达载体的构建

为提高鞘蕊苏有效成分的含量,开展Coleus Ent-kaurenoic acid oxidase(KAO)基因的克隆以及表达载体构建方面的研究。提取鞘蕊苏叶片的总RNA,利用套式PCR技术克隆获得KAO基因全长,将该基因连接到克隆载体p MD-18T,经测序鉴定正确后,再将该基因连接到p ET-28a表达载体中。结果显示,成功克隆出的鞘蕊苏KAO基因大小为1 500 bp,对克隆基因进行测序及酶切鉴定,均得到正确大小的DNA片段,说明表达载体构建成功。本研究成功克隆得到鞘蕊苏贝壳杉烯酸氧化酶基因并构建其表达载体,为提高鞘蕊苏有效成分含量提供了基础。     

Structural bias in T4 RNA ligase-mediated 3'-adapter ligation

T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to RNA 3'-ends. After analyzing biases in RNA sequence, secondary structure and RNA-adapter cofold structure, we conclude that T4 RNA ligases do not show significant primary sequence preference in RNA substrates, but are biased against structural features within RNAs and adapters. Specifically, RNAs with less than three unstructured nucleotides at the 3'-end and RNAs that are predicted to cofold with an adapter in unfavorable structures are likely to be poorly ligated. The effect of RNA-adapter cofold structures on ligation is supported by experiments where the ligation efficiency of specific miRNAs was changed by designing adapters to alter cofold structure. In addition, we show that using adapters with randomized regions results in higher ligation efficiency and reduced ligation bias. We propose that using randomized adapters may improve RNA representation in experiments that include a 3'-adapter ligation step.     

泥鳅抗菌肽在毕赤酵母SMD1168中的高效表达及活性检测

【背景】泥鳅抗菌肽Misgurin是泥鳅非特异性免疫防御系统的重要组成部分,具有广谱和较强的抗菌能力,所以获得大量抗菌肽是很有必要的。【目的】为了实现高效表达泥鳅抗菌肽Misgurin。【方法】将泥鳅抗菌肽的目的基因与p PIC9K表达载体连接,构建重组表达质粒p PIC9K-misgurin,Sal I酶切线性化,再通过电击法将其整合到毕赤酵母SMD1168染色体上。在MD固体培养基挑选阳性克隆子到MD液体培养基中,30°C、200 r/min摇瓶培养96 h,转接到BMMY液体培养基进行诱导表达,每隔24 h加入5%的甲醇。通过比较抑制大肠杆菌和金黄色葡萄球菌抑菌圈直径的大小,筛选出活性较高的菌株p PIC9K-misgurin-22。将该菌株在100 L的发酵罐中诱导表达,48 h后进行活性检测。【结果】经Tricine-SDS-PAGE蛋白胶检测和质谱分析鉴定,p PIC9K-misgurin-22菌株诱导表达的活性物质为抗菌肽Misgurin。发酵罐发酵48 h相对于摇瓶发酵48 h,抗大肠杆菌的生物效价提高了1.47倍,抗金黄色葡萄球菌的生物效价提高了1.43倍;抗菌肽Misgurin对鲍曼不动杆菌、沙门氏菌有较弱的抗菌活性,对致病菌产气荚膜梭菌和益生菌如枯草芽孢杆菌、粪肠球菌、乳酸菌没有抗菌活性,无明显溶血活性;当温度达到90°C时发酵液的抗菌活性明显减弱,调发酵液的p H值在1.0-12.0之间都有抗菌活性;加入胰蛋白酶、胃蛋白酶、蛋白酶K后抗菌活性减弱。【结论】获得了一株在毕赤酵母中表达量较高、有工业化生产潜力的产抗菌肽Misgurin的菌株。     

Analysis on folding of misgurin using two-dimensional HP model

Misgurin is an antimicrobial peptide from the loach, while the hydrophobic-polar (HP) model is a way to study the folding conformations and native states in peptide and protein although several amino acids cannot be classified either hydrophobic or polar. Practically, the HP model requires extremely intensive computations, thus it has yet to be used widely. In this study, we use the two-dimensional HP model to analyze all possible folding conformations and native states of misgurin with conversion of natural amino acids according to the normalized amino acid hydrophobicity index as well as the shortest benchmark HP sequence. The results show that the conversion of misgurin into HP sequence with glycine as hydrophobic amino acid at pH 2 has 1212 folding conformations with the same native state of minimal energy -6; the conversion of glycine as polar amino acid at pH 2 has 13,386 folding conformations with three native states of minimal energy -5; the conversion of glycine as hydrophobic amino acid at pH 7 has 2538 folding conformations with three native states of minimal energy -5; and the conversion of glycine as polar amino acid at pH 7 has 12,852 folding conformations with three native states of minimal energy -4. Those native states can be ranked according to the normalized amino acid hydrophobicity index. The detailed discussions suggest two ways to modify misgurin.     

Use of a new retrieving adaptor in the cloning of a synthetic human insulin A-chain gene

A DNA duplex encoding the A-chain of human insulin was constructed from eight chemically synthesized oligomers by enzymatic ligation to form a partial duplex followed by repair synthesis to complete the complementary strands. After sequential addition of translation start and stop signal adaptors the assembly was cloned in pBR322. To regenerate the end of the coding sequence by precise removal of extraneous nucleotides a new method using a synthetic retrieval adaptor was developed. The procedure included filling in the cohesive ends of the EcoRI site by repair synthesis, ligating a symmetrical adaptor having an MboII recognition sequence to the resulting blunt end, cutting with MboII and removing the single protruding 3′-nucleotide using the 3′ exonuclease activity of DNA polymerase I. Synthetic oligomers useful for ligation to a synthetic insulin C-chain gene were added to the retrieved end of the gene. Sequence analysis established that retrieval adaptors of this type may be used for precise excision of up to eight nucleotides from the end of a cloned DNA fragment.     

蛋白质多肽文库构建中限制性显示技术的接头设计

为了探讨限制性显示(RD)技术在构建蛋白质多肽文库中灵活的接头设计,分别根据原核表达载体pET22b以及酵母表达载体pNMT-TOPO设计了三套接头,三套接头依次增加一个碱基以保证与之连接的片段总有可能表达正确的开放阅读框.然后以HIV-1 B亚型代表株U26942全基因质粒DNA为对象,利用RD技术分别建立了相应的蛋白质多肽文库.从每个库中各随机挑选12个克隆进行测序分析并进行蛋白质表达预测.结果从原核表达文库中获得了一个可以表达HIV Pol多肽的克隆,SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)结果显示该克隆在细菌BL21(DE3)中有较高的表达,蛋白质印迹为阳性,与理论预测相符.这些结果提示,RD技术是一种建立基因组随机多肽文库的新方法,该方法灵活的接头设计可以满足不同的表达载体需求.     

SPLICE: a technique for generating in vitro spliced coding sequences from genomic DNA

We describe a rapid and cost-effective technique for the in vitro removal of introns and other unwanted regions from genomic DNA to generate a single sequence of continuous coding capacity, where tissues required for RNA extraction and complementary DNA synthesis are unavailable. Based on an overlapping fusion-PCR strategy, we name this procedure SPLICE (for swift PCR for ligating in vitro constructed exons). As proof-of-principle, we used SPLICE successfully to generate a single piece of DNA containing the coding region of a five-exon gene, the short-wavelength-sensitive 1 (SWS1) opsin gene, from genomic DNA extracted from the brown lemur, Eulemur fulvus, in only two short rounds of PCR. Where the genomic structure and sequence is known, this technique may be universally applied to any gene expressed in any organism to generate a practical unit for investigating the function of a particular gene of interest. In this report, we provide a detailed protocol, experimental considerations, and suggestions for troubleshooting.     

TE-AFLP: combining rapidity and robustness in DNA fingerprinting

A new type of fingerprinting technique is presented, based on amplified fragment length polymorphism (AFLP). Rather than two endonucleases as in AFLP, we propose the use of three enzymes, hence the method is called three endonuclease (TE)-AFLP. Genomic DNA is digested and two sets of adapters are selectively ligated onto the restriction fragments in a single reaction volume. No adapters complementary to the ends generated by a frequent cutter are added. Due to the addition of a third endonuclease, the TE-AFLP method provides a high discriminatory power and a reduction in the number of bands. The latter makes it especially suitable for the analysis of complex genomes. TE-AFLP fingerprints are suitable for detection by automatic fluorescent sequencers and are obtained in less than half the time and at reduced costs compared to a typical AFLP. The reliability of this method was investigated by determining the influence of varying digestion, ligation and PCR components on the fingerprint. Moreover, cross-experiments to study inheritance of loci were performed with a primitive insect and with tomato strains. The features of TE-AFLP are discussed in comparison with conventional AFLP.     

Adapters, shims, and glue--service interoperability for in silico experiments

MOTIVATION: Computationally, in silico experiments in biology are workflows describing the collaboration of people, data and methods. The Grid and Web services are proposed to be the next generation infrastructure supporting the deployment of bioinformatics workflows. But the growing number of autonomous and heterogeneous services pose challenges to the used middleware w.r.t. composition, i.e. discovery and interoperability of services required within in silico experiments. In the IRIS project, we handle the problem of service interoperability by a semi-automatic procedure for identifying and placing customizable adapters into workflows built by service composition. RESULTS: We show the effectiveness and robustness of the software-aided composition procedure by a case study in the field of life science. In this study we combine different database services with different analysis services with the objective of discovering required adapters. Our experiments show that we can identify relevant adapters with high precision and recall.     

Adapter-tagged competitive PCR (ATAC-PCR) – a high-throughput quantitative PCR method for microarray validation

Adapter-tagged competitive PCR (ATAC-PCR) is an advanced version of competitive quantitative PCR that is characterized by the addition of unique adapters to cDNA derived from each sample RNA. Using multiple adapters, we can accurately measure the relative expression ratios of many samples, with a calibration curve obtained from internal standards included in the same reaction. ATAC-PCR can identify differences in gene expression as small as twofold, even from very small amounts of sample RNA. This technique is suitable for confirming results obtained with cDNA microarrays or differential display, and it can process more than a thousand of genes per day when used in conjunction with a capillary DNA sequencer.