基于细菌人工染色体的大基因质粒制备及应用研究

细菌人工染色体(BAC)最多可克隆300 kb的DNA片段且遗传特性稳定,是目前常用的克隆载体.但如何高效提取BAC装载的大基因及其大基因操作等方面还需不断的摸索完善.本研究采用超大基因质粒提取法从BAC中提取出165 kb的大基因,经Nanodrop测定浓度、酶切、脉冲场电泳,表明获得目标基因;将获得的大基因质粒转染猪胎儿成纤维细胞,经抗性筛选,获得细胞克隆;克隆细胞经PCR检测,证明大基因质粒被完整地导入猪胎儿成纤维细胞.本研究为构建细菌人工染色体提够基础数据.     

用酚-氯仿直接从克隆中提取质粒DNA

目的:对用酚-氯仿直接从克隆中提取质粒DNA的方法进行了研究。方法:采用不同次数的酚-氯仿抽提,在不同RNaseA作用温度及作用时间下提取了三种质粒。结果:用酚-氯仿抽提2次,RNase A45℃作用8min,质粒纯度(OD260/OD280)为1.85左右、产量大于0.8μg/克隆。结论:本法不但快速,且所提质粒均能满足后续实验如酶切鉴定、测序等方面的需要。对于大批量重组克隆的筛选尤为适合。     

口蹄疫病毒O/QYYS/s/06株感染性克隆的构建

本研究在完成FMDVO/QYYS/s/06株全基因组序列测定的基础上,分3段对全基因组进行克隆,其后将各片段克隆到载体P43中,从而获得携带O/QYYS/s/06株基因组全长cDNA的重组质粒P43C。将重组质粒P43C与表达RNA聚合酶的质粒T7共转染BHK-21细胞,48h后收获培养液接种2~3d乳鼠,取经乳鼠传代后的第4代病毒液,经反向间接血凝、中和试验和测序等方法证明拯救的病毒为O型FMDV。以上结果表明,O/QYYS/s/06株全长cDNA分子克隆的构建成功。     

一种大规模筛选单克隆及提取质粒的改进方法

利用α-互补(X-gal IPTG)从cDNA库中筛选特异克隆,以此去除含有未插入片段和插入片段小的克隆。运用展库的方法,通过辅助噬菌体对库中多个载体的切割,将载体以质粒的形式转化到大肠杆菌中;同时对质粒DNA碱裂解提取方法做了改进,建立了一种简单实用的大量提取质粒DNA的方法。该方法利用特定的蛋白质吸附膜吸附提取过程中的蛋白质,从而去除了使用酚-氯仿抽提蛋白质的复杂过程,减少了质粒DNA的损失,是一次性获得大规模质粒DNA的有效方法。获取的质粒DNA样品在纯度和浓度上都可以满足测序、PCR及Southern杂交等分子生物学实验的要求。     

AIV血凝素亚型同步检测基因芯片技术研究

对流感病毒14个血凝素亚型的基因芯片检测技术进行了初步研究。通过RT-PCR克隆禽流感病毒血凝素基因片段,获得重组质粒。从重组质粒扩增大约500bp的DNA片段,浓缩后点到氨基化玻璃载体上,制成芯片。待检病毒样品用TRIzolLS提取RNA,反转录过程中用Cy5标记样品cDNAs。将标记样品与芯片杂交,扫描芯片上待检样品与芯片上捕捉探针的结合位点,杂交信号与预期设想一致。结果显示,DNA芯片技术可以提供一种有效的AIV血凝素亚型鉴别诊断方法。     

重组真核质粒pCMV4-rZP3′构建及在小鼠体内的表达

选取兔透明带ZPC基因序列的部分片段（编码263－415位的氨基酸,rZP3′）作为靶抗原,构建兔ZP3′基因疫苗,研究其阻断受精的效果。提取成熟雌兔卵巢的总RNA,RT－PCR克隆兔透明带ZPC的cDNA,直接插入到克隆质粒PCRR2．1中,构建克隆质粒PCRR2．1－rZP3′,再将靶片段用HindⅢ、X-bal双酶切从克隆质粒PCRR2．1－rZP3′切下,1％的低熔点琼脂糖电泳回收,亚克隆到真核表达质粒pCMV4中,构建真核表达质粒pCMV4-rZP3′。将质粒直接注射到活体小鼠的大腿内侧肌肉中,在mRNA水平上观察到质粒能在肌肉细胞中正确表达,并且其表达可以维持12周以上。     

苦瓜MAP30基因原核表达载体的构建和PCR快速检测重组子的研究

根据已报道的序列,把苦瓜(Momordica charantia) MAP30基因成功克隆到原核表达载体pET28a( )中,并对PCR快速检测阳性克隆进行了研究.结果表明,直接用菌落、菌液、酚氯仿处理过的菌液,以及提取的质粒进行PCR都可以成功地筛选阳性菌落.其中,酚氯仿处理过的菌液PCR与质粒PCR的结果最接近,而且比质粒PCR简单,因此可作为方便可靠的阳性克隆筛选的新方法.     

粘质沙雷氏菌几丁质酶chiB基因的克隆与序列分析

采用改进的方法提取粘质沙雷氏菌基因组DNA,通过PCR扩增,得到大小为1 500 bp特异性DNA片段(chiB基因),以pUC18质粒构建了pUC-ch iB克隆载体,转化至感受态细胞E.coliDH5α培养,并筛选出重组质粒。经测序分析,证明克隆片段与文献报道相一致。     

里氏木霉半纤维素酶基因的克隆

通过PCR技术从里氏木霉基因组中扩增出基因xyn I,把基因与大肠杆菌质粒相连接,再把重组的质粒转入到感受态的Top10大肠杆菌中,待Top10大肠杆菌产生大量的重组质粒后,用中量制备质粒DNA方法把重组的质粒提取出来,再用双酶切方法检验重组质粒。实验结果显示,已经得到含有重组质粒的大肠杆菌菌落,完成了xyn I基因的克隆。     

质粒提取

这里所介绍的方法已成功地用来提取不同菌株中的各种质粒。一般来说,质粒愈小,效果愈好。因为随着质粒分子量的增加,其性质就愈来愈接近宿主DNA的性质。对于大于25kb的质粒分子,用此方法分离是非常困难的,而且得率较低。然而,通常用于克隆的所有质粒相对来说是较小的,因而此方法可获得良好的结果。     

Rapid preparation of total nucleic acids from E. coli for multi-purpose applications

Separate protocols are commonly used to prepare plasmid DNA, chromosomal DNA, or total RNA from E. coli cells. Various methods for the rapid preparation of plasmid DNA have been developed previously, but the preparation of the chromosomal DNA and total RNA are usually laborious. We report here a simple, fast, reliable, and cost-effective method to extract total nucleic acids from E. coli by direct lysis of the cells with phenol. Five distinct and sharp bands, which correspond to chromosomal DNA, plasmid DNA, 23S rRNA, 16S rRNA, and a mixture of small RNA, were observed when analyzing the prepared total nucleic acids on a regular 1-2% agarose gel. The simple and high-quality preparation of the total nucleic acids in a single tube allowed us to rapidly screen the recombinant plasmid, as well as to simultaneously monitor the change of the plasmid copy number and rRNA levels during the growth of E. coli in the liquid medium.     

生物可降解微球作为乙型肝炎基因免疫佐剂的研究

探讨生物可降解微球对基因免疫的增强作用。采用有机溶剂蒸发法制备聚乳酸聚乙醇酸共聚 物（ＰＬＧＡ）微球,构建含有乙型肝炎病毒表面抗原Ｓ基因的ｐＲＣ－ＣＭＶ真核表达载体,用微球与基因 载体共孵育法制备其混合物。肌肉注射免疫Ｂａｌｂ／ｃ小鼠。结果表明：微球注射组的血清抗体滴度达到 ｌ：１６００,其效果与乙型肝炎病毒表面抗原加铝佐剂注射组相近,而裸ＤＮＡ注射组没有反应。说明了 生物可降解微球可显著的提高基因免疫的免疫反应。     

A QuikChange-like method to realize efficient blunt-ended DNA directional cloning and site-directed mutagenesis simultaneously

Here we present a QuikChange-like method to efficiently realize blunt-ended DNA cloning and conveniently introduce a site-directed mutation to recombinant plasmid at the same time. After blunt-ended DNA ligation and transformation, the plasmid DNA mixture is extracted from pooled transformants and directly used as template for PCR amplification with a pair of complementary mutagenic primers. With this method, sam1 gene was inserted into pUC19 vector by blunt-end ligation, and a unique restriction site Spe I was introduced to the recombinant plasmid at the same time. The randomly selected transformants were analyzed by DNA sequencing, and most of the clones were found to have correct sequences. However, no correct construct was found from randomly selected transformants after traditional blunt-ended DNA ligation and transformation.     

重组大肠杆菌碱裂解方法的改进

为了降低质粒DNA的生产成本,对经典碱裂解法中的溶液III进行了改进,以表达溶菌酶基因的pcDNKLYZ重组质粒转化的大肠杆菌DH5α为指示菌,用标准碱裂解和改进碱裂解法提取质粒pcDNKLYZ,以提取的质粒产量和质量为指标,判断优化碱性裂解法的性价比,结果显示,用改进后的碱裂解法裂解重组菌,提取的pcDNKLYZ质粒产量和质量等指标与标准方法接近,而成本仅为标准方法的1/4,可用于重组质粒的大规模制备。     

Use of a 'Miniprep' for rapid extraction of plasmids from vancomycin- and gentamicin-resistant Enterococcus faecium

Conventional methods of plasmid extraction are largely unsuited to diagnostic laboratories. The 'Miniprep' is a rapid method that utilises a centrifugable column to separate plasmid DNA from chromosomal DNA. We have modified this technique to extract plasmid DNA from seven strains of vancomycin- and gentamicin-resistant Enterococcus faecium (VGREF): 1% mannitol was added to the growth medium and cell lysis was achieved by incubation in 10 mg of lysozyme/ml in 10 mM Tris, 1 mM EDTA and 25% sucrose at pH 8.0. RNase A was added to plasmid eluate rather than at the lytic step. In comparison to a standard phenol/chloroform method, Miniprep completely eliminated chromosomal interference in gel electrophoresis but otherwise produced identical plasmid profiles. Plasmids obtained from the VGREF ranged from 42 to 1.3 Md. Band densities on a single elution from the Miniprep varied from 8.3 to 106.3 relative units. Double elution increased band densities from the same preparation from 30.4 to 196 relative units; mean percentage increases per track between 7.0 and 34.6%. This method is suitable to achieve plasmid DNA extraction from VGREF within 1 h, making the process more suitable for diagnostic laboratories.     

A PCR-after-ligation method for cloning of multiple DNA inserts

Here we present a novel and simple PCR-after-ligation method for efficient assembly of multiple DNA inserts. After initial ligation of multiple inserts and vector, the ligation mixture is used as template for a PCR using a pair of primers flanking the cloning sites on the vector. The fragment with correct size is gel purified and inserted into the vector by conventional two-way ligation. With this method, a recombinant plasmid containing four DNA inserts was correctly constructed. As a control, all of the constructs obtained directly from DNA ligation were found to be self-ligation of the vector.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples

A method based on three-DNA-component, sandwich hybridization has been designed for the detection and quantitation of nucleic acids in crude samples using adenovirus DNA as a model. Two non-overlapping restriction fragments of adenovirus type 2 (Ad2) DNA were cloned into two vectors, the pBR322 plasmid and M13 phage. The recombinant plasmid DNA was immobilized onto nitrocellulose filters and the single-stranded recombinant phage DNA was labeled with 125I and used as a probe. When these two reagents were incubated under annealing conditions no radioactivity became filter-bound; only if denatured adenovirus DNA was added as the third reagent, it mediated the attachment of the radioactive probe to the filters. Hybridization efficiency was shown to be dependent on both the filter and probe DNA concentrations and on the hybridization conditions. When standardized, the assay is quantitative, and under the conditions used 0.2 ng of adenovirus DNA (8 X 10(-6) pmol) could be detected by an overnight incubation. The test is suitable for crude samples, e.g., solubilized cell extracts, without any purification steps. Less than 100 cells infected with Ad2 can be detected, implying that the assay could be applicable to virus diagnostics.     

三种快速制备含重复序列质粒PCR模板的方法

为了探索含重复序列质粒PCR模板的快速简易制备方法,分别利用高速离心法、TE煮沸法和TE/SDS煮沸法处理过夜培养的含重组质粒大肠杆菌菌体,制备模板后进行PCR扩增发现,均能得到比较清晰的目的条带,可以用于含重复序列质粒的初步鉴定。相比较而言,高速离心5 min、TE煮沸3 min、TE/SDS煮沸2.5 min制备的样品PCR扩增后效果较好。这3种方法快速、简便、费用低、无污染,简化了PCR模板制备的过程,为重组质粒大量筛选鉴定的研究奠定了基础。