海枣曲霉木聚糖酶的纯化及末端序列研究

海枣曲霉麸曲经水浸提、硫酸铵盐析、凝胶过滤、离子交换层析及ＨＰＬＣ分子排阻层析制备了ＰＡＧＥ,ＳＤＳ－ＰＡＧＥ、ＰＡＧＥ酶谱及ＨＰＬＣ纯的木聚糖酶。     

A Comprehensive Study of Neutralizing Antigenic Sites on the Hepatitis E Virus (HEV) Capsid by Constructing,Clustering, and Characterizing a Tool Box

The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459–606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process.     

Classification of human and zoonotic group hepatitis E virus (HEV) using antigen detection

Hepatitis E virus (HEV) is one of the major pathogens that cause acute viral hepatitis. The human (genotypes 1 and 2) and zoonotic (genotypes 3 and 4) groups of HEV present different epidemiology and clinical features. In this study, we developed a classification method for rapidly classifying HEV into human or zoonotic groups that combines a general antigen test with a zoonotic group-specific antigen test. Evaluation of serial samples from HEV-infected rhesus monkeys indicated that HEV antigen-positive samples can be classified using the antigen-based classification method. The antigen-based classification method was evaluated further on 55 genotyped samples from acute hepatitis E patients, including 9 human and 46 zoonotic groups. The novel method was completely consistent with the sequencing results: 9/9 for the human groups (100%, 95% confidence interval [CI] 66.4–100%) and 46/46 for the zoonotic groups (100%, 95% CI 92.3–100%). This method was also successfully used for the clustering of some samples that could not be clustered by sequencing. Compared with the sequencing-based method, this method is less time-consuming, less expensive, and less technically complex and is therefore ideal for large numbers of samples. In conclusion, this study provides a convenient and sensitive method for classifying different groups of HEV, and it has potentially important public health applications, especially in underdeveloped areas that cannot afford the high cost of nucleic acid testing.

     

抑瘤素McDNA的克隆及表达研究

抑瘤素M（onecostatinM,OSM）是一种多功能的细胞生长调节因子．从PMA刺激后的U937细胞系提取总RNA,采用逆转录-PCR方法分离到了抑瘤素M的cDNA;将抑瘤素M的cDNA克隆到质粒pUC19中,筛选三个阳性克隆进行序列分析,与国外报导序列完全一致;将抑癌素M的cDNA克隆到质粒pBV220后再转化DH5a进行模拟表达,SDS-PAGE分析表明有OSM表达,表达量约占细菌总蛋白5％,经过初步纯化的OSM能明显抑制A375细胞的生长．     

CS3菌毛可以作为异源抗原决定簇的表达载体

CS3是肠毒素源性大肠杆菌的菌毛蛋白 ,是一种很强的免疫原 .利用CS3菌毛作为异源抗原决定簇载体 ,在计算机分析预测CS3亚基的抗原表位区、二级结构的基础上 ,运用PCR定点突变在CS3亚基结构基因中引入了SacⅡ酶切位点序列 ,插入霍乱毒素B亚基抗原表位CTP3的DNA序列 ,构建了表达CS3/CTP3的重组菌株 .电镜和免疫电镜观察证明 ,CS3/CTP3以杂合菌毛的形式存在于菌体表面 .SDS聚丙烯酰胺凝胶电泳显示了CS3/CTP3杂合蛋白的存在 .口服和腹腔注射免疫Balb/c小鼠 ,该重组菌株可诱发抗CS3和抗CTP3的双重免疫应答 .结果表明CS3可以作为表达异源抗原决定簇的表达载体 ,可望成为研制口服粘膜免疫多价疫苗的新型系统     

海枣曲霉木聚糖酶降解寡聚木糖的特性

利用滤纸层析或ＡｃｒｙｌｅｘＰ－２凝胶过滤从落叶松木聚糖硫酸水解液中分离纯化子木二糖至木五糖。采用硅胶薄层层析分析底物和产物的方法研究了海枣霉木聚糖酶降解寡聚木糖的特点。此酶作用于寡糖的最适ＰＨ为５．０,终产物为Ｘ和Ｘ２。酶作用于Ｘ３、Ｘ４及Ｘ５的相对初速度分别为１、３４和４００,Ｘ２几乎不被酶解,推断该酶的底物结合部位至少具有５个亚位点,在高底物浓度,低酶量,远离最适ＰＨ以及在反应初期都能检测到     

产碱菌麦芽四糖淀粉酶的化学修饰

不同蛋白质侧链修饰剂对麦芽四糖淀粉酶进行修饰。在一定条件下,分别用ＩＡＡ、ＮＥＭ、ＥＤＣ和ＮＡＩ处理后,酶活力不受影响,仍为１００％,说明巯基、羧基和酪氨酸残基与酶活力无关。用ＤＥＰ、ＮＢＳ和ＨＮＢＢ修饰后,酶活力大幅度下降,说明组氨酸和色氨酸基为酶活力所必需。     

戊型肝炎病毒ORF1多聚蛋白的不同功能域在细胞内的定位

为了探讨戊型肝炎病毒多聚蛋白ORF1的多个功能域在宿主细胞中的表达和定位情况,我们首先将psk-HEV重组载体上的ORF1各功能域的编码序列克隆到绿色荧光蛋白载体pcDNA3.1-GFP上,构建成融合表达的重组质粒,并测序和酶切鉴定其构建成功。再通过Western-Blot验证各融合蛋白在细胞中正确表达,并用激光扫描共聚焦显微镜观察融合蛋白在细胞内的分布和定位。在Huh7细胞中,RdRp蛋白主要分布于细胞核内,HEL蛋白以囊泡状分布于细胞核周,MET蛋白以颗粒状存在于细胞核和细胞质中,PLP蛋白呈极性分布于细胞核周,X蛋白在细胞核和细胞质中均存在。各融合蛋白在细胞中的不同定位印证了对这些蛋白质的功能预测和体外研究结果,这为进一步研究HEV不同蛋白功能提供了支持。     

Alteration of heat sensitivity by treatment with nonpermissive temperature or cycloheximide in temperature-sensitive CHO mutant tsH1 cell

1. 1.|The temperature-sensitive mutant CHO-tsH1 and wild type (CHO-SC) cells became thermal resistant when cells were treated for either 2 h at 39.5°C before heating at 43°C or 2 h with 10 μg/ml cycloheximide (CHM) before and during heating at 43°C.

2. 2.|There was a 2000-fold increase in survival after 2.5 h at 43°C by preincubation at 39.5°C in both cell types. There was also a 200- or 700-fold increase in survival after 2.5 h at 43°C by treatment with CHM in tsH1 or SC cell type respectively.

3. 3.|In contrast to the effects at 43°C, at 41.8°C these protective effects were not evident in tsH1 cells. In wild type, however, there was an 800- or 1800-fold increase in survival after 8 h at 41.8°C by preincubation at the temperature of 39.5°C or treatment with CHM, respectively.

4. 4.|Therefore, these results suggest that killing of tsH1 at low temperature hyperthermia (41.8°C) is probably due to denaturation of thermolabile leucyl-tRNA synthetase.

5. 5.|The denaturation of this enzyme may not be protected by inhibition of protein synthesis by preincubation at the nonpermissive temperature of 39.5°C or by CHM.