PCR条件对扩增SARS-CoV多聚酶部分基因的影响

目的：对该实验室已建立的检测SARS冠状病毒多聚酶基因的套式RT-PCR方法进行优化。方法：从SAPS病人的嗽口水标本中提取RNA,调整套式PCR的退火温度,扩增SARS冠状病毒多聚酶部分基因。扩增出的阳性片段连接入pGEM-T载体中,测序后比较其与已知SARS冠状病毒的同源性。结果：通过改变PCR条件,成功从一SARS病人的嗽口水中扩增出SARS冠状病毒多聚酶部分基因。结论：针对不同标本优化PCR反应条件非常重要。     

人噬菌体抗体库的有效表位多样性

噬菌体抗体库的有效表位是指用任何抗原都能从中筛同相应抗体片段,所以人源抗体库多样性的多少直接影响其实用性。本不目前提高抗体库多样性的几种途径及其进展进行综述和讨论。     

噬菌体抗体库的优化

噬菌体抗体组合文库技术作为噬菌体展示和抗体组合文库两种技术的集成,由于它具有库容量大、特异性高、和敏感性强的优点而被誉为抗体技术的第三次革命。但是由于一些技术上的原因,使得它无法得到广泛的应用,本文就其优化进行综述。     

抗体库的发展及未来

抗体库的出现为制备人源性单抗开辟了一条行之有效的途径．虽然该技术问世只有短短几年的时间,但其筛选系统已有了很大的改进;现已能够快速地从抗体库中筛选出各种具有不同用途的抗体．从而克服了传统杂交瘤技术的某些局限性．文章就抗体库的发展及其应用前景进行了概述.     

噬菌体抗体库构建技术在疾病防治中的应用

噬菌体抗体库技术(phage antibody library)是近年发展的一项分子生物学新技术,近年来越来越多的学者利用这项技术获得目标抗体以应用于医疗制药及安全检测等多个领域.噬菌体抗体库技术的发展和应用,为抗体技术领域带来了巨大的变化,极大地推动了各种性能优良基因工程抗体的开发和应用.针对噬菌体抗体库构建的原理、方法、构建过程中所需考虑的因素以及在不同领域的应用作了综述.     

噬菌体抗体库技术的研究进展及应用

噬菌体抗体库技术是继噬菌体展示技术发展而来的一项基因抗体工程新技术。它可将含不同物种全部抗体可变区基因的基因库转化成展示在噬菌体表面的蛋白库,不仅使单克隆抗体的生产更方便、快速、高效地在体外进行,还开辟了单克隆抗体人源化的新途径,促进了人类单克隆抗体生产的发展。就近年来噬菌体抗体库技术的基因来源、发展关键及抗体应用的研究作一综述。     

超大容量人源性抗体库的构建

构建大容量抗体库是获得高亲和力抗体的重要基础和保障,如何构建大容量抗体库一直是被关注的热点。本对比分析了天然库、半合成库及全合成库基因多样性的特点,指出获得多样的功能性抗体基因是建库的关键。构建大容量抗体库要从提高连接和转化效率、利用体内重组系统提高抗体基因组合多样性以及提高包装和呈现的效率等多个环节进行条件优化,提高库容量和多样性。     

抗整合素β3胞外区噬菌体抗体库的构建及筛选

用RT-PCR的方法从人胶质瘤BT-325细胞中扩增人整合素β3胞外区,并克隆到载体pET-24a中构建表达载体.表达的人整合素β3胞外区经变性、复性和纯化后免疫BALB/c小鼠,提取脾脏总RNA,用RT-PCR技术扩增小鼠抗体重链(VH)和轻链(VL)可变区基因,经重叠PCR(SOE-PCR)将VH和VL连接成单链抗体(scFv)基因,并克隆到噬菌粒载体pCANTAB5E中,电转化至大肠杆菌TGl,经辅助噬菌体M13KO7超感染,构建噬菌体单链抗体库.通过淘选从该抗体库中筛选特异性识别人整合素β3胞外区的噬菌体单链抗体.结果表明,成功构建了库容为2.6×106的抗人整合素β3胞外区的单链抗体库,初步筛选到了与人整合素β3胞外区特异性结合的单链抗体.     

噬菌体呈示单链抗体表达载体及小鼠非特异抗体库的构建

用大肠杆菌丝状噬菌体表面呈示技术构建抗体库的方法,为从抗原出发获得特异抗体提供了新的途径。报道的是,首先构建了一个用于噬菌体呈示抗体的噬粒表达载体ｐＦＵＷ８０,它具有既可以进行外分明表达,又可进行附着表达的特点。然后利用设计的一套扩增小鼠抗体重链和轻链可变区基因片段的ＰＣＲ引物,从未免疫小鼠脾细胞中扩增出了抗体重、轻链可变区基因,构建了一个１．２×１０６库容的小鼠非特异性单链抗体库。从这个抗体库中,筛选出了针对人ＩｇＧ的单链抗体噬菌体,并进行了ＥＬＩＳＡ检测和部分序列分析。这一初步结果为今后继续利用这一系统进行研究奠定了基础。     

Effect of primer mismatch, annealing temperature and PCR cycle number on 16S rRNA gene-targetting bacterial community analysis

In the attempt to explore complex bacterial communities of environmental samples, primers hybridizing to phylogenetically highly conserved regions of 16S rRNA genes are widely used, but differential amplification is a recognized problem. The biases associated with preferential amplification of multitemplate PCR were investigated using 'universal' bacteria-specific primers, focusing on the effect of primer mismatch, annealing temperature and PCR cycle number. The distortion of the template-to-product ratio was measured using predefined template mixtures and environmental samples by terminal restriction fragment length polymorphism analysis. When a 1 : 1 genomic DNA template mixture of two strains was used, primer mismatches inherent in the 63F primer presented a serious bias, showing preferential amplification of the template containing the perfectly matching sequence. The extent of the preferential amplification showed an almost exponential relation with increasing annealing temperature from 47 to 61 degrees C. No negative effect of the various annealing temperatures was observed with the 27F primer, with no mismatches with the target sequences. The number of PCR cycles had little influence on the template-to-product ratios. As a result of additional tests on environmental samples, the use of a low annealing temperature is recommended in order to significantly reduce preferential amplification while maintaining the specificity of PCR.     

人抗E．Coli J5噬菌体抗体制备的初步研究

以E．Coli J5株对合人Ig基因的噬菌体抗体库进行淘筛富集,免疫印迹筛选,以及ELISA检测,结果获得4株能与E．Coli J5株结合的阳性克隆,且阳性克隆结合抗原的活性可分别被E．Coli J5株、E．Coli Rc-LPS和抗E．Coli J5株核心糖脂域MAb抑制．PCR检测表明,4株阳性克隆均分别带有约660bp大小的重链和轻链基因片段．SDS－PAGE与蛋白质印迹的结果显示,经IPTG诱导的阳性克隆能表达分子量约为50000大小的蛋白,提示该4株阳性克隆能够表达具有一定抗原结合活性的人源Fab片段．     

猕猴巨细胞病毒(RhCMV)nested PCR检测方法的建立与初步应用

目的建立猴巨细胞(RhCMV)病毒的nested PCR检测方法,并初步应用。方法根据GenBank中报道的RhCMV全基因序列,针对其中的保守区域Rh85设计两对引物进行nested PCR反应,利用此方法对20份猕猴全血标本进行检测,将检测到的猕猴阳性标本扩增片段进行克隆测序。结果利用保守区域Rh85设计的引物可对人HCMV阳性对照进行扩增。用此方法检测的20份猕猴全血标本,出现2例阳性。其中一例扩增片段经纯化、回收克隆测序后用BLAST软件进行同源性对比,与GenBank中报道的RhCMV序列基本相同。结论建立了从猴全血中直接检测猴RhCMV病毒DNA的敏感、特异的nested PCR方法。     

双退火温度PCR扩增DNA

【目的】与设置单一退火温度的常规PCR(S-T_m PCR)不同,本研究探讨双退火温度PCR(D-T_m PCR)由高到低设置2条引物各自退火温度。【方法】以PxF61和VPel为正/反向引物,用Q5 DNA聚合酶扩增4.3 kb的模式DNA pET20b-Xyn(黑曲霉木聚糖酶基因)。PCR程序为:98°C预变性3 min,30次循环{98°C变性30 s,设置双退火[T_(m1) 70°C(Px F61)退火15 s、T_(m2) 62°C(VPel)退火15 s],72°C延伸130 s}。【结果】与S-T_m PCR(61°C)相比,D-T_m PCR扩增4.3 kb的目的条带亮度更高,减少2条杂带;经25次循环目的 DNA产物量最高。D-T_m PCR用于长片段引物扩增5.3 kb重组质粒DNA条带更明显。【结论】D-T_m PCR直接扩增目的条带,避免了探讨T_m的麻烦,不要求2条引物T_m相近,从理论上更加清晰地认识引物与各自模板分步退火过程。     

Nested real-time PCR for hepatitis A detection

Aims:  The hepatitis A virus (HAV) is one of the most important human foodborne pathogens causing a number of worldwide outbreaks each year. The detection of HAV in food samples remains a complex issue, because commonly used detection tools, such as conventional or even real-time PCR assays, are often unable to detect HAV with sufficient sensitivity. The aims of this study were to develop highly sensitive and specific nested real-time PCR (NRT-PCR)-based method for HAV detection in food and to compare it with currently available methods.
Methods and Results:  By combining conventional PCR, nested PCR and real-time PCR techniques, we have developed a specific NRT-PCR assay for the detection of HAV. The procedure involves two consecutive PCRs, the first of which is performed as a conventional RT-PCR using primers specific for HAV 5' noncoding region. The second reaction involves a real-time PCR using a nested primer pair specific for the first PCR product and a TaqMan probe.
Conclusions:  We have developed a novel NRT-PCR method capable of detecting as little as 0·2 PFU of HAV, which is significantly more sensitive than any other PCR technique tested in our system.
Significance and Impact of the Study:  NRT-PCR provides a potentially useful method for detecting HAV at extremely low levels, as frequently found in food samples, and can be potentially adopted as a regulatory method to ensure food safety.     

Universal and blocking primer mismatches limit the use of high‐throughput DNA sequencing for the quantitative metabarcoding of arthropods

The quantification of the biological diversity in environmental samples using high‐throughput DNA sequencing is hindered by the PCR bias caused by variable primer–template mismatches of the individual species. In some dietary studies, there is the added problem that samples are enriched with predator DNA, so often a predator‐specific blocking oligonucleotide is used to alleviate the problem. However, specific blocking oligonucleotides could coblock nontarget species to some degree. Here, we accurately estimate the extent of the PCR biases induced by universal and blocking primers on a mock community prepared with DNA of twelve species of terrestrial arthropods. We also compare universal and blocking primer biases with those induced by variable annealing temperature and number of PCR cycles. The results show that reads of all species were recovered after PCR enrichment at our control conditions (no blocking oligonucleotide, 45 °C annealing temperature and 40 cycles) and high‐throughput sequencing. They also show that the four factors considered biased the final proportions of the species to some degree. Among these factors, the number of primer–template mismatches of each species had a disproportionate effect (up to five orders of magnitude) on the amplification efficiency. In particular, the number of primer–template mismatches explained most of the variation (~3/4) in the amplification efficiency of the species. The effect of blocking oligonucleotide concentration on nontarget species relative abundance was also significant, but less important (below one order of magnitude). Considering the results reported here, the quantitative potential of the technique is limited, and only qualitative results (the species list) are reliable, at least when targeting the barcoding COI region.     

Synthetic approach to the generation of antibody diversity

The in vitro antibody discovery technologies revolutionized the generation of target-specific antibodies that traditionally relied on the humoral response of immunized animals. An antibody library, a large collection of diverse, pre-constructed antibodies, can be rapidly screened using in vitro display technologies such as phage display. One of the keys to successful in vitro antibody discovery is the quality of the library diversity. Antibody diversity can be obtained either from natural B-cell sources or by the synthetic methods that combinatorially generate random nucleotide sequences. While the functionality of a natural antibody library depends largely upon the library size, various other factors can affect the quality of a synthetic antibody library, making the design and construction of synthetic antibody libraries complicated and challenging. In this review, we present various library designs and diversification methods for synthetic antibody library. From simple degenerate oligonucleotide synthesis to trinucleotide synthesis to physicochemically optimized library design, the synthetic approach is evolving beyond the simple emulation of natural antibodies, into a highly sophisticated method that is capable of producing high quality antibodies suitable for therapeutic, diagnostic, and other demanding applications. [BMB Reports 2015; 48(9): 489-494]     

A focused antibody library for improved hapten recognition

The topography of the antigen-binding site as well as the number and the positioning of the antigen contact residues are strongly correlated with the size of the antigen with which the antibody interacts. On the basis of these considerations, we have designed a focused scFv repertoire biased for haptens, designated the cavity library. The hapten-specific scFv, FITC8, was used as a scaffold for library construction. FITC8, like other hapten binders, displays a characteristic cavity in its paratope into which the hapten binds. In five of the six complementarity-determining regions, diversity-carrying residues were selected rationally on the basis of a model structure of FITC8 and on known antibody structure-function relationships, resulting in variation of 11 centrally located, cavity-lining residues. L3 was allowed to carry a more complex type of diversity. In addition, length variation was introduced into H2, as longer versions of this loop have been shown to correlate with increased hapten binding. The library was screened, using phage display, against a panel of five different haptens, yielding diverse and highly specific binders to four of the antigens. Parallel selections were performed with a library having diversity spread onto a greater area, including more peripherally located residues. This resulted in the isolation of binders, which, in contrast to the clones selected from the cavity library, were not able to bind to the soluble hapten in the absence of the carrier protein. Thus, we have shown that by focusing diversity to the hotspots of interaction a library with improved hapten-binding ability can be created. The study supports the notion that it is possible to create antibody libraries that are biased for the recognition of antigens of pre-defined size.     

工程抗体酶的研究进展

本文评述了工程抗体酶的研究现状和抗体酶在防化医学研究中的应用。抗体库技术的出现使抗体酶的研究呈现了新的生机。不仅使不具备细胞工程实验条件的实验室能够制备抗体酶,而且实验周期大大缩短,比杂交瘤技术提供多20倍的结合性抗体作为潜在的抗体酶,使抗体酶更易获得。抗体库技术能够容易地评价编码抗体酶的基因,因而可以在分子水平上认识和改造抗体酶。噬菌体抗体库可以不经免疫即可制备抗体的特点,使通过人源性噬菌体抗体库生产的抗体酶能够直接用于临床治疗。随着工程抗体酶研究的不断深入,新颖实用的抗体酶将会被应用于包括军事医学在内的多种领域中。