XL1—blue菌株电穿孔转化方案的条件优化

目的：优化大肠杆菌ＸＬ１－ｂｌｕｅ菌株的电穿孔转化条件。方法：采用不同的条件组合转化大肠杆菌ＸＬ１－ｂｌｕｅ菌株，对转化条件进行优化。结果与讨论：用电穿孔方案转化大肠杆菌ＸＬ１－ｂｌｕｅ菌株，可得到明显高于其他转化方法的转化效率，外加电场强度和作用时间是决定转化效率的关键因素，其间存在一定的互补关系，转化效率和外源基因浓度之间呈线性关系，并与细菌浓度密切相关。     

乳杆菌电转化的研究进展

乳杆菌在食品、工业、微生物学等领域发挥着重要作用,作为益生菌,既可向体外生物反应器方向开发,又可作为潜在疫苗的抗原递送载体.由于乳杆菌属革兰阳性菌,细胞壁厚,重组质粒电转化效率低,甚至不能实现转化,影响了它作为基因工程受体菌的应用.笔者研究了近年来有关乳杆菌的电转化方案,总结了影响电转化效率的因素,如细胞壁预处理、电转缓冲液、电击参数、培养基、孵育时间以及菌体密度、质粒用量、热击条件等,并根据自己的试验结果提出优化方案,为乳杆菌在疫苗及生物反应器等领域的遗传开发研究提供较全面的借鉴.     

HeLa细胞去核条件的优化

细胞去核技术〔１〕的原理是：细胞在细胞松弛素Ｂ（ｃｙ－ｔｏｃｈａｌａｓｉｎＢ，ＣＢ）作用下，微丝聚集受到抑制，细胞核与细胞质间连接变得松散。在离心力的作用下，细胞核从细胞中脱出，从而得到去核细胞。这一技术主要应用于细胞重组，研究核、质间信息传递、ｍＲ...     

单链抗体的研究进展

抗体技术由细胞工程抗体(杂交瘤-单克隆抗体)发展到基因工程抗体,尤其是抗体库技术的出现,将抗体工程发展到了一个新阶段。本文从抗体库技术等方面介绍单链抗体(single-chain Fv antibody,sc Fv)的进展情况。     

基于CHO细胞定点整合技术的新型抗体呈现系统的构建

目的建立一个以CHO定点整合系统为基础的高通量抗体筛选平台,筛选获得抗VEGF高亲和力突变体抗汁。方法设计并构建了基于CHO细胞定点整合系统的通用minibody展示载体,将3株亲和力已知的抗VEGF抗体（A6,A63,A657）和1个抗VEGF轻链抗体库分别呈现在CHO细胞的表面,通过Western印迹、Bia-core3000、流式细胞分析等对该展示系统进行验证和评价,并通过流式细胞分选技术对抗体库进行了细胞筛选。结果成功实现了抗VEGF抗体在CHO细胞表面的展示,流式细胞分析抗体的亲和力与Biacore测定全抗体亲和力结果一致;通过筛选获得3株抗VEGF高亲和力突变体抗体。结论建立了基于CHO定点整合技术的新型抗体库展示系统,为抗体的亲和力成熟、分子改造等突变库的构建和筛选提供了新的技术支持。     

富含GC DNA PCR扩增条件的优化

目的 :探索富含GCDNA的PCR扩增条件 ,为大环内酯类化合物组合生物合成中模块和酶域的任意组合奠定基础。方法 :在PCR扩增体系中 ,添加甲酰胺、甘油、二甲亚砜 (DMSO)、Mg2 等增强剂 ,并选择合适的扩增体系 ,优化富含GCDNAPCR扩增条件。结果 :甲酰胺对富含GCDNAPCR扩增没有影响 ,而甘油和DMSO均可提高富含GCDNAPCR产物的特异性和产率 ,以 5 %～ 10 %DMSO效果最好。使用Roche长片段DNA扩增系统 ,在使用缓冲液 2反应体系中添加 10 %DMSO和提高 0 .2 5mmol LMg2 浓度时 ,可以扩增长度为 5kb、GC含量为 73%的DNA片段。结论 :应用优化的PCR扩增条件 ,可以扩增长达 5kb富含GC的DNA片段 ,可以满足大环内酯类组合生物合成模块和酶域随意组合的需求     

全合成人源性噬菌体抗体库的构建

目的:构建全合成人源性噬菌体抗体库。方法:选择部分使用频率较高的人抗体胚系基因家族的框架区基因进行人工合成,同时人工设计合成半随机CDR3,通过重叠拼接延伸PCR的方法合成抗体基因。然后利用限制性内切酶SfiⅠ、NotⅠ分别双酶切抗体基因和噬菌体展示载体。连接后的重组噬粒通过电击转化的方法转入大肠杆菌TG1,构建抗体库。结果与结论:PCR结果显示,通过优化后的方法能在保证抗体库多样性的前提下,高效地获得抗体基因。经过数十次电击转化构建了库容量为2×109的全合成抗体库,菌落PCR、测序及筛选结果表明,抗体库质量优良,为筛选人源性治疗抗体奠定了基础。     

大肠癌噬菌体抗体库的构建及抗CEA抗体的筛选

目的构建及初步鉴定大肠癌噬菌体抗体Fab呈现库,筛选人CEA单克隆抗体,并进行序列分析。方法分离大肠癌患者外周血淋巴细胞,提取淋巴细胞总RNA,逆转录成cDNA。用PCR扩增人全套抗体基因片段,克隆于pComb3载体,再经电穿孔转化大肠杆菌XL1-Blue菌株,形成噬菌体抗体库。以固相化CEA抗原淘筛抗体库,ELISA鉴定噬菌体抗体。其中一个阳性克隆进行测序。结果逆转录PCR分别扩增出约680bp大小的κ、λ和Fd基因。PCR产物和载体经纯化、双酶切后进行连接转化,成功地构建了人源性Fab抗体基因库,库容量达2.1×107,Fab基因重组率为50%。以单抗捕获的CEA抗原淘洗4轮,出现特异性富集,阳性克隆经直接ELISA和交叉反应ELISA实验证实具有良好的抗CEA抗原特异性。DNA测序表明该抗体重链属IgG亚类并含有一条IgL亚类的轻链。结论成功构建了大肠癌患者自然致敏抗体Fab段噬菌体呈现库,从中获得可与CEA抗原结合的噬菌体抗体,由此为大肠癌早期诊断及基因治疗提供了一种新的思路和方法。     

外源性转化生长因子-β1抗体对周围神经慢性卡压后神经纤维化的缓解作用

目的 探讨外源性转化生长因子-β1(TGF-β1)抗体对周围神经慢性卡压后神经纤维化的缓解作用。 方法 将75只大鼠按抽签法随机分为假手术组(A组)、单纯卡压组(B组)和卡压+TGF-β1多克隆抗体注射组(C组),于术后2,4,6,8,10 周取神经行电镜、免疫组织化学、RT-PCR、Western-blot等检测,分别检测不同时段卡压神经组织形态学变化及Ⅰ型、Ⅲ型胶原蛋白含量。 结果 B组较A组TGF-β1、Ⅰ型、Ⅲ型胶原蛋白含量明显增多,Ⅰ型、Ⅲ型胶原蛋白于卡压损伤后6周达到高峰,后一段时间内处于平台期;C组神经形态学上胶原纤维组织明显减少,Ⅰ型、Ⅲ型胶原蛋白含量降低,与B组比较差异有统计学意义(P＜0.05)。 结论 周围神经慢性卡压后可致神经纤维化。TGF-β1抗体可有效抑制周围神经卡压后胶原合成,缓解慢性卡压后神经纤维化病变。     

抗双链 DNA 抗体与狼疮细胞检查的诊断价值比较

20 0 2年 ,我们对系统性红斑狼疮 (SLE) 7例 ,分别抽取双份血样进行血清抗双链DNA(ds DNA)抗体与狼疮细胞的检测。结果 :抗双链DNA抗体的阳性率明显高于狼疮细胞的阳性率 ,提示抗双链DNA抗体测定对SLE更具有诊断价值。1　对象和方法1 1　对　象　均为女性 ,年龄 2 7～ 4 8岁 ,平均 34 6岁。均为临床诊断明确的SLE。1 2　方　法　抽双份血送检 ,一份血用金标免疫斑点法测血清的抗双链DNA抗体 ,试剂盒由福建蓝波生物制品公司生产 ;另一份血行改良血块法做狼疮细胞检查 ,按全国临床检验操作规程 (第 2版 )操作。2　结　果7例中 ,抗双…     

STR genotyping of exogenous hair shaft DNA

Most hairs found at crime scenes yield low quality and/or low quantities of nuclear DNA. This DNA is further depleted when stringent hair cleaning procedures are applied in the laboratory, suggesting that detectable DNA exists exogenously. The phenomenon of exogenous hair DNA is the subject of this study. DNA was extracted from washed and unwashed hairs and the resulting Profiler? Plus STR genotypes were compared with those of reference (buccal) swabs from the hair donors. The DNA extraction procedure involved no prior cleaning of the hair sample and no dissolution of the hair during digestion, in contrast to standard procedures. The STR genotyping success was measured by recording the two dominant alleles at each locus and comparing them with the reference DNA profile. The effect of hair cleanliness was examined by leaving donors’ hair unwashed for periods of 1, 3 and 7 days before sampling. It was found that the genotyping success for unwashed hair was significantly higher than that for freshly washed hair, with the majority of clean hair samples producing little or no DNA. Genotyping success was also lower for donors with cosmetically treated hair compared with those having untreated hair. Although the quality of STR profiles (i.e. allele dropout, differential amplification) from hair shafts or telogen hair clubs is reduced compared with those from other biological sources, the genotypes obtained in this study may be usable and are certainly discriminating if alternative interpretational methods are applied.     

A comparative study for the isolation of exogenous trace DNA from fingernails

Often fingernails from a victim or suspect involved in a physical assault, such as murder or sexual assault, are submitted to crime laboratories for DNA testing of foreign/exogenous biological material; however, very few studies have been conducted comparing the effectiveness of different sampling methods on the removal of foreign/exogenous DNA while minimizing the fingernail endogenous DNA. In this study three different sampling methods (swabbing, PBS soak, and PrepFiler® lysis buffer soak) were compared in order to identify one that minimizes the amount of endogenous DNA removed and maximizes the amount of foreign/exogenous male DNA removed. The samples were processed using the Tecan HIDEVO150 robot in order to reduce analyst time and the DNA mixtures were interpreted using the probabilistic genotyping software STRmix™. For each sampling method the quantity of male DNA, the mixture proportions, the number of foreign/exogenous male alleles detected, the amount of DNA degradation, and the discrimination power via the likelihood ratio obtained for the foreign/exogenous male DNA donor were determined and compared. The PrepFiler® lysis buffer soak and swabbing sampling methods appear to be equally effective at removing foreign/exogenous DNA from fingernails; however, the lysis buffer soak sampling method extracts more female endogenous DNA from the fingernail and the female DNA is degraded. Marginally higher likelihood ratios were obtained for the swab samples versus the PrepFiler® lysis buffer soak samples; therefore, it was determined that the swabbing sampling method was the best sampling method for the recovery of foreign exogenous DNA from fingernails while minimizing the amount of endogenous DNA removed.     

Oxidation of exogenous carbohydrate during prolonged exercise in fed and fasted conditions

The oxidation of glucose and fructose ingested during moderate exercise performed on a cycle ergometer (120 min, 52% VO2max) was compared in ten young males fasted (n = 5) or fed (n = 5) before exercise. The subjects ingested randomly 1.33 g/kg body weight (approximately 96 +/- 9 g) of either enriched 13C-glucose (G), 13C-fructose (F), or water only (W); the solutions were evenly distributed over the exercise period. The fasted subjects began the three exercises with a lower blood glucose (P less than or equal to 0.05 for F only) and insulin (P less than or equal to 0.05) levels and a higher free fatty acid (FFA) concentration (P less than or equal to 0.05) than the fed ones. Throughout the exercise period, blood glucose level was maintained in fasted as well as in fed group for G and F ingestions, while it decreased (P less than or equal to 0.05 at the 100th min in fasted subjects) with water ingestion. Insulin level was similar in both fed and fasted conditions with F and W ingestions and lower than G trials for the fed subjects. For the three ingestions, FFA was lower (P less than or equal to 0.05) in the fasted than in the fed group over the exercise period. Over the 2-h period of exercise, a greater (P less than or equal to 0.05) amount of exogenous F was oxidized in the fasted (49 +/- 6 g) than in the fed (36 +/- 5 g) group, which represent 31% and 20% of the total carbohydrate energy supply, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Optimization irradiation conditions for determination of LD50 in pigs.

Radiation LD50/30 values were determined in 36 twelve-week-old pigs (with a mean body weigth of 21 kg) exposed to whole-body X-ray irradiation on a revolvable table rotated at a rate of 2.5 rpm using the following conditions: 180 kV, 15 mA, focal distance 79 cm, HVT 0.9 mm Cu, dose rate 2.42 X 10(-3) to 2.68 X 10(-3) C kg-1 min-1 (9.4 to 10.4 R/min) depending upon the animal size. The coefficient of mean irradiation uniformity was 1.4. Under these conditions the LD50/30 for pigs was found to be 5.89 X 10(-2) C kg-1 min-1 (228.3 R) with the biological range of effectiveness being 5.22 X 10(-2) to 6.90 X 10-(2) C kg-1 (202.4 to 267.6 R). Furthermore experiments on 77 pigs showed that LD50 determined in this study had actually the median lethal effect.     

Optimization of exposure conditions for amorphous selenium direct conversion DR-based mammography system

A new direct-conversion detector for DR mammography has improved the detectability of microcalcifications and masses. Each optimized exposure condition (target/filter combination and tube voltage) was defined through comparison of physical values and visual evaluation on breast specimens using the innovative DR mammography. The contrast-to-noise-ratios (CNRs) of PMMA phantoms of various thicknesses were obtained under a variety of exposure conditions whose average glandular doses (AGDs) were made consistent. Fifty breast specimens were irradiated under these combinations. Visual evaluation was conducted on the images, whose histograms were controlled for consistency. In the phantoms with thicknesses of 20 mm or more, tungsten/rhodium had the highest CNRs of the targets/filters such as molybdenum/molybdenum and molybdenum/rhodium. For visualizing microcalcifications and masses on breast specimens of thicknesses of 35 mm and below, molybdenum/molybdenum was the best. Nevertheless, to obtain better image quality, molybdenum/rhodium was superior for 35-55 mm thickness, and tungsten/rhodium was superior for 55 mm and above under the same AGD, enabling accurate and efficient diagnosis. The study showed that the exposure conditions differ for obtaining the highest CNR using phantoms and those under which breast specimen images allow the most accurate and efficient diagnosis. In addition, image evaluations of the breast specimens allowed optimization of exposure conditions that are closer to those of the actual diagnosis using mammography.     

CUL4A-DDB1细胞稳定株筛选及DNA双链断裂模型构建

目的为了鉴定并验证CUL4A-DDB1泛素连接酶复合体中参与DNA损伤修复反应过程的1～2种关键成分在损伤识别、早期及晚期修复中的动态变化,拟构建含有串联亲和纯化（TAP）标签载体并筛选高表达CUL4A/DDB1的细胞株,建立DNA双链断裂模型。方法利用PCR获得CUL4A/DDB1基因,构建重组表达载体pNTAP-A-CUL4A/DDB1;使用顺铂和电离辐射刺激等外界刺激建立合适的DNA双链断裂细胞模型,使用G418筛选稳定表达CUL4A/DDB1的细胞株。结果与结论成功构建pNTAP-A-CUL4A/DDB1表达载体,建立合适的DNA双链断裂细胞模型,筛选得到稳定表达CUL4A/DDB1的细胞稳定株,为下一步质谱分析CUL4A-DDB1泛素连接酶在DNA损伤修复过程中的新功能打下基础。     

Optimization of recovery of human DNA from envelope flaps using DNA IQ™ System for STR genotyping

An optimized protocol based on the DNA IQ™ System has been tested for the extraction of DNA from envelope flaps. DNA is extracted directly without the need for opening and swabbing the flaps. The method is repeatable with <10% R.S.D. (relative standard deviation). The results of a systematic study show that it is an equilibrium extraction, and a small sample volume as well as high lysis buffer content in sample contribute to high extraction efficiency. The extracted DNA requires no further purification steps following its extraction with the DNA IQ™ System. Complete but skewed 15-locus short tandem repeat (STR) profiles, which is typical of degraded of DNA, have been generated from the DNA extracted from 6 to 9 years old casework envelope samples.