HCV E2蛋白诱导的体液免疫及CTL应答研究

应用PCR方法扩增出HCVE2基因编码417a．a-750a．a的DNA片段,克隆到原核表达载体pQE30 LacZ启动子下游,转化JM109菌株。在JM109菌株中诱导表达出N端含6个组氨酸的E2融合蛋白,用Ni-NTA-Superflow亲和层析柱纯化作为抗原免疫实验兔和BALB／c鼠。定期取兔血,采用间接ELISA方法检测兔子体内针对E2的抗体水平和维持规律。结果显示,距初次免疫14d兔子体内已有抗体产生,直至免疫第55d抗体水平持续上升,之后抗体水平保持稳定,抗体滴度达到1：3200。六周后,取鼠脾脏制各淋巴细胞,定向刺激扩增后与经过重组真核表达质粒pCE2转染的P815细胞作用,利用LDH释放试验检测作用效果。在E：T＝200：1的情况下,杀伤率超过30％。这些结果表明工程菌株表达的HCVE2蛋白具有良好的免疫原性,可以诱发免疫实验动物机体产生较高滴度的抗体及特异性CTL应答。由此我们认为E2蛋白是发展HCV预防工程蛋白疫苗的合适候选者。     

PNAS-4 expression and its relationship to p53 in colorectal cancer

PNAS-4 is a novel pro-apoptotic protein activated during the early response to DNA damage; however, the molecular mechanisms and pathways regulating PNAS-4 expression in tumors are not well understood. We hypothesized that PNAS-4 is a p53 down-stream target gene and designed this study. We searched online for putative p53-binding sites in the entire PNAS-4 gene and did not find any corresponding information. In HCT116 colon cancer cells, after being transfected with small interfering RNA to silence p53, the expressions of PNAS-4 and other known p53 target gene (Apaf1, Bax, Fas and Dr5) were determined by real-time PCR. We found that PNAS-4 was up-regulated while Apaf1, Bax, Fas and Dr5 were down-regulated. We then examined the expression of PNAS-4 and p53 mutation in colorectal cancer patients. PNAS-4 expressed both in colorectal cancers and normal tissues, but compared with paired control, PNAS-4 was up-regulated in cancers (P = 0.018). PNAS-4 overexpression ratios were correlated to the p53 mutant status (P = 0.001). The mean PNAS-4 expression levels of p53 mutant homozygote group and heterozygote group were higher than that of p53 wild type group (P = 0.013). The expression ratios of PNAS-4 (every sample in relative to its paired normal mucosa) were different between negative lymph node metastasis (66% up-regulated, 34% down-regulated) and positive metastasis (42% up-regulated, 58% down-regulated). Taken together, these findings suggested that PNAS-4 was not a p53 target, but overexpression of PNAS-4 was correlated to p53 inactivity in colorectal cancer.     

超嗜热酯酶EST2在不同宿主中的异源高效表达研究

来源于超嗜热古菌Alicyclobacillus acidocaldarius的酯酶EST2是目前报道的活性最高的超嗜热酯酶,具有极大的工业应用价值。为促进EST2的生产应用,将其分别在大肠杆菌及毕赤酵母中进行异源表达,并就不同宿主对表达情况和重组酶酶学性质的影响进行了分析。在大肠杆菌和毕赤酵母中重组表达的EST2酶学性质基本一致:最适温度分别为75℃和77.5℃,最适pH均为8.0,比活力分别为4656.6 U/mg和4078.3 U/mg,70℃水浴保温4.5 h,残余活力均在70%以上。在摇瓶发酵的基础上,于5 L发酵罐中进行了重组大肠杆菌及毕赤酵母的高密度发酵。毕赤酵母高密度发酵120 h菌体干重达68 g/L,最大表达酶活力为959.6 U/ml。大肠杆菌高密度发酵25 h菌体干重达60.8 g/L,最大酶活力14825.6 U/ml,表达量是毕赤酵母的15.4倍,单位时间产量是酵母的74.2倍。结果表明大肠杆菌发酵周期短、表达量高,更适合进行嗜热酯酶EST2的高效生产,这为促进嗜热酯酶在工业生物技术产业的应用奠定了基础。     

分子进化生物学中序列分析方法的新进展

简要介绍了分子进化生物学中序列分析方法的最新进展,特别强调了似然比检验和贝叶斯推论在分子进化和系统发育假说检验中的重要性,并介绍了新方法的一些成功应用,同时还给出了一些重要的信息资源。     

Genetic map of Triticum turgidum based on a hexaploid wheat population without genetic recombination for D genome

ABSTRACT: BACKGROUND: A synthetic doubled-haploid hexaploid wheat population, SynDH1, derived from the spontaneous chromosome doubling of triploid F1 hybrid plants obtained from the cross of hybrids Triticum turgidum ssp. durum line Langdon (LDN) and ssp. turgidum line AS313, with Aegilops tauschii ssp. tauschii accession AS60, was previously constructed. SynDH1 is a tetraploidization-hexaploid doubled haploid (DH) population because it contains recombinant A and B chromosomes from two different T. turgidum genotypes, while all the D chromosomes from Ae. tauschii are homogenous across the whole population. This paper reports the construction of a genetic map using this population. RESULTS: Of the 606 markers used to assemble the genetic map, 588 (97%) were assigned to linkage groups. These included 513 Diversity Arrays Technology (DArT) markers, 72 simple sequence repeat (SSR), one insertion site-based polymorphism (ISBP), and two high-molecular-weight glutenin subunit (HMW-GS) markers. These markers were assigned to the 14 chromosomes, covering 2048.79 cM, with a mean distance of 3.48 cM between adjacent markers. This map showed good coverage of the A and B genome chromosomes, apart from 3A, 5A, 6A, and 4B. Compared with previously reported maps, most shared markers showed highly consistent orders. This map was successfully used to identify five quantitative trait loci (QTL), including two for spikelet number on chromosomes 7A and 5B, two for spike length on 7A and 3B, and one for 1000-grain weight on 4B. However, differences in crossability QTL between the two T. turgidum parents may explain the segregation distortion regions on chromosomes 1A, 3B, and 6B. CONCLUSIONS: A genetic map of T. turgidum including 588 markers was constructed using a synthetic doubled haploid (SynDH) hexaploid wheat population. Five QTLs for three agronomic traits were identified from this population. However, more markers are needed to increase the density and resolution of this map in the future study.     

贵州黔西县少数民族ABO血型分布及基因频率调查

对贵州黔西县1260例6个少数民族人群红细胞ABO表现型进行了检测;结果如下:贵州黔西县布依族、满族、苗族、白族这四个民族的ABO血型基因频率很相近,彝族和仡佬族与这4个民族的差别较大,布依族,苗族,满族,白族ABO血型分布为O>B>A>AB,彝族为O>A>B>AB,仡佬族为A>O>B>AB;经Hardy Weinberg吻合度检测可以证明贵州黔西县的ABO血型表现型分布状况及基因频率相对稳定,其分布符合hardy Weinberg平衡,获得了该地ABO血型系统群体遗传学数据,为群体遗传学的研究提供了一定的资料。     

Carbohydrate analysis by a phenol-sulfuric acid method in microplate format

Among many colorimetric methods for carbohydrate analysis, the phenol-sulfuric acid method is the easiest and most reliable method. It has been used for measuring neutral sugars in oligosaccharides, proteoglycans, glycoproteins, and glycolipids. This method is used widely because of its sensitivity and simplicity. In its original form, it required 50-450 nmol of monosaccharides or equivalent for analysis and thus is inadequate for precious samples. A scaled-down version requiring only 10-80 nmol of sugars was reported previously. We have now modified and optimized this method to use 96-well microplates for high throughput, to gain greater sensitivity, and to economize the reagents. This modified and optimized method allows longer linear range (1-150 nmol for Man) and excellent sensitivity. Moreover, our method is more convenient, requiring neither shaking nor covering, and takes less than 15 min to complete. The speed and simplicity of this method would make it most suitable for analyses of large numbers of samples such as chromatographic fractions.     

Water Supply Changes N and P Conservation in a Perennial Grass Leymus chinensis

Changes in precipitation can influence soil water and nutrient availability, and thus affect plant nutrient conservation strategies. Better understanding of how nutrient conservation changes with variations in water availability is crucial for predicting the potential influence of global climate change on plant nutrient-use strategy. Here, green-leaf nitrogen (N) and phosphorus (P) concentrations, N- and P-resorption proficiency (the terminal N and P concentration in senescent leaves, NRP and PRP, respectively), and N- and P-resorption efficiency (the proportional N and P withdrawn from senescent leaves prior to abscission, NRE and PRE, respectively) of Leymus chinensis (Trin.) Tzvel., a typical perennial grass species in northern China, were examined along a water supply gradient to explore how plant nutrient conservation responds to water change. Increasing water supply at low levels (< 9000 mL/year) increased NRP, PRP and PRE, but decreased green-leaf N concentration. It did not significantly affect green-leaf P concentration or NRE. By contrast, all N and P conservation indicators were not significantly influenced at high water supply levels (> 9000 mL/year). These results indicated that changes in water availability at low levels could affect leaf-level nutrient characteristics, especially for the species in semiarid ecosystems. Therefore, global changes in precipitation may pose effects on plant nutrient economy, and thus on nutrient cycling in the plant-soil systems.     

In vitro development and transfer of resistance to chlortetracycline in Bacillus subtilis

The present criteria and rules controlling the approval of the use of probiotics are limited to antibiotic resistance patterns and the presence of antibiotic resistance genes in bacteria. There is little information available in the literature regarding the risk of the usage of probiotics in the presence of antibiotic pressure. In this study we investigated the development and transfer of antibiotic resistance in Bacillus subtilis selected in vitro by chlortetracycline in a stepwise manner. Bacillus subtilis was exposed to increasing concentrations of chlortetracyclineto induce in vitro resistance to chlortetracycline, and the minimal inhibitory concentrations were determinedfor the mutants. Resistant B. subtilis were conjugated with Escherichia coli NK5449 and Enterococcus faecalis JH2-2 using the filter mating. Three B. subtilis tetracycline resistant mutants (namely, BS-1, BS-2, and BS-3) were derived in vitro. A tetracycline resistant gene, tet (K), was found in the plasmids of BS-1 and BS-2. Three conjugates (BS-1N, BS-2N, and BS-3N) were obtained when the resistant B. subtilis was conjugated with E. coli NK5449. The conjugation frequencies for the BS-1N, BS-2N, and BS-3N conjugates were 4.57×10^?7, 1.4×10^?7, and 1.3×10^?8, respectively. The tet(K) gene was found only in the plasmids of BS-1N. These results indicate that long-term use of probiotics under antibiotic selection pressure could cause antibiotic resistance, and the resistance gene could be transferred to other bacteria. The risk arising from the use of probiotics under antibiotic pressure should be considered in the criteria and rules for the safety assessment of probiotics.