变形假单胞菌2-酮基葡萄糖酸转运蛋白基因的克隆与分析

该研究旨在克隆变形假单胞菌JUIM01的2-酮基葡萄糖酸转运蛋白基因kgu T,并明确其基本的生物学信息。根据已报道的假单胞菌的基因组信息及其2-酮基葡萄糖酸操纵子的物理图谱设计简并引物,采用TD-PCR技术从变形假单胞菌中克隆到全长为1278 bp的kgu T,其核苷酸序列与Pseudomonas sp.CCOS191的编码2-酮基葡萄糖酸转运蛋白(Kgu T)的核苷酸序列的一致性为87%,编码一个由425个氨基酸残基组成的蛋白。该蛋白与Pseudomonas sp.M1的Kgu T在氨基酸序列上的一致性达90%,定位于细胞膜,是一个具有12个跨膜结构的疏水性的跨膜蛋白,无信号肽,其二级结构中α螺旋、延伸链和无规卷曲所占的比例分别为75.76%、2.12%和22.12%。本研究首次从2-酮基葡萄糖酸的工业生产用菌中克隆到基因kgu T,并对其进行了生物信息学分析,为变形假单胞菌的Kgu T的功能研究奠定了基础。     

Identification and characterisation of the major allergen of Chinese mitten crab (Eriocheir sinensis)

Tropomyosin (TM) from Chinese mitten crab (Eriocheir sinensis) was purified to homogeneity and TM genes were amplified from three species of crab (Chinese mitten crab, mud crab and swimming crab), respectively. Sequence analysis showed that all three cloned DNA fragments had open reading frames of 855 bp, predicted to encode proteins with 284 amino acid residues. Sequence alignment revealed that the three tropomyosins share high homology to tropomyosins from other crustaceans. Chinese mitten crab TM gene was further recombined with the vector of pGEX-4T-3 and expressed in Escherichia coli JM109. The expressed protein revealed a band of about 62 kDa on SDS–PAGE, suggesting the successful expression of glutathione S-transferase–tropomyosin (GST–TM) fusion protein. Immunoblotting analysis using sera from subjects with crustacean allergy confirmed that the expressed fusion protein reacted positively with these sera, indicating tropomyosin is a major allergen of Chinese mitten crab.     

Characterization of microbial community in the two-stage process for hydrogen and methane production from food waste

The structure of a microbial community in the two-stage process for H₂ and CH₄ production from food waste was investigated by a molecular biological approach. The process was a continuous combined thermophilic acidogenic hydrogenesis and mesophilic (RUN1) or thermophilic (RUN2) methanogenesis with recirculation of the digested sludge. A two-phase process suggested in this study effectively separate H₂-producing bacteria from methanogenic archaea by optimization of design parameters such as pH, hydraulic retention time (HRT) and temperature. Galore microbial diversity was found in the thermophilic acidogenic hydrogenesis, Clostridium sp. strain Z6 and Thermoanaerobacterium thermosaccharolyticum were considered to be the dominant thermophilic H₂-producing bacteria. The hydrogenotrophic methanogens were inhibited in thermophilic methanogenesis, whereas archaeal rDNAs were higher in the thermophilic methanogenesis than those in mesophilic methanogenesis. The yields of H₂ and CH₄ were in equal range depending on the characteristics of food waste, whereas effluent water quality indicators were different obviously in RUN1 and RUN2. The results indicated that hydrolysis and removal of food waste were higher in RUN2 than RUN1.     

木聚糖酶基因克隆和表达的研究进展

半纤维素分解微生物在自然界的物质循环过程中起着重要作用,半纤维素是植物多糖的重要成分之一,而木聚糖则是半纤维素的主要成分。木聚糖酶(xylanase)可催化木聚糖的水解,在各种生物体内均发现木聚糖酶。在过去几十年中,已有上百种木聚糖酶基因被克隆至同源或异源宿主内来表达木聚糖酶,以期改变宿主特性并适于商业应用。本文综述了木聚糖酶基因的克隆和表达,并对基因工程技术在木聚糖酶上的应用前景进行了展望。     

A study of the process control and hydrolytic characteristics in a thermophilic hydrogen fermentor fed with starch-rich kitchen waste by using molecular-biological methods and amylase assay

Starch-rich kitchen waste was chosen as the feedstock in this study, and a 3-L intermittent-continuous stirred tank reactor (I-CSTR) was established. Within 240 days, the maximum average hydrogen production rate of 2.2 L-H² L⁻¹ day⁻¹ and the highest average hydrogen yield of 2.1 mmol-H₂ g-COD⁻¹ were both observed in run 3-2, which was operated at an eight-day hydraulic retention time (HRT) and 39 g-COD L⁻¹ day⁻¹ of organic loading rate. According to the analyses of amylase and reducing sugar, the maximum average amylase activity was about 11 U mL⁻¹ in run 1, but the maximum solid carbohydrate hydrolysis rate was about 45% in run 3. Some Michaealis-Menton kinetic parameters, such as K_M (17 g L⁻¹) and the maximum activity (1.5 U mL⁻¹) of the amylase were obtained. The best amylase reacting temperature was 55 °C, and the best reacting pH was 4.4 tested with acetate buffer. Twenty-seven operational taxonomic units (OTUs) were selected from this reactor by using a cloning method. According to the data of terminal restricted fragment length polymorphism (T-RFLP) and amylase assay, the OTUs that were related to Thermoanaerobacterium thermosaccharolyticum and Clostridium sp. were in direct proportion to the amylase activity.     

Wistar大鼠热休克蛋白70基因重组腺病毒质粒的构建及病毒制备

目的构建Wistar大鼠热休克蛋白(Heat shock protein 70,HSP70)基因重组腺病毒质粒,并制备重组腺病毒。方法利用RT-PCR技术从Wistar大鼠脾脏组织中扩增HSP70基因序列,并克隆至pMD18-T载体中,测序鉴定后,将克隆的HSP70基因编码阅读框亚克隆至穿梭载体pShuttle-CMV中,并利用Ⅰ-CeuⅠ与Ⅰ-SceⅠ将重组穿梭质粒上的HSP70基因的表达框切下,连接到携带腺病毒骨架载体pAdxsi上。重组腺病毒质粒经PacⅠ酶切线性化后,转染至293(R)细胞进行病毒的包装,获得重组腺病毒pAd-CMV-HSP70,收集病毒上清,感染BRL细胞,Western blot检测BRL细胞中HSP70的表达。病毒颗粒经氯化铯密度梯度离心纯化后,检测重组腺病毒纯度、颗粒滴度及感染性滴度。结果克隆质粒、穿梭质粒经PCR及酶切鉴定,均可见2 269 bp的目的基因条带,测序结果与GenBank登录的序列一致;XhoⅠ酶切结果显示,HSP70基因已正确克隆至腺病毒质粒中;重组腺病毒感染BRL细胞后,细胞中HSP70的表达量显著升高(P<0.01);重组腺病毒纯化后纯度为1.29,颗粒滴度为5.31×1012VP/ml,感染性滴度达1×1011pfu/ml。结论已成功构建Wistar大鼠HSP70基因重组腺病毒质粒,并制备出高滴度的重组腺病毒颗粒,为进一步研究HSP70的生物学活性及作用机制奠定了基础。     

牛乳腺炎金黄色葡萄球菌肠毒素A基因的克隆及序列分析

根据Genbank上公布的金黄色葡萄球菌肠毒素A的全序列,利用生物学软件Primer 5.0和oligo 6.0设计了一对特异性引物来扩增靶序列片段,经克隆预测序,结果表明扩增片段长度为101 bp,锦州分离株与标准菌株的基因片段序列相似性为100%,与Genbank上公布的金黄色葡萄球菌菌株(EF520720.1)SEA基因相似性达到99.14%。高度相似性结果为进一步研究建立分子检测技术奠定了实验基础。     

烟草牻牛儿基牻牛儿基焦磷酸合成酶基因的克隆及分析

采用RACE技术,从烟叶中克隆了一个新的编码GGPPS蛋白的基因,命名为Ntggpps3,并对其进行了生物信息学分析。Ntggpps3的cDNA序列为1251 bp,包含一个长度为1092 bp的开放阅读框,编码363个氨基酸。NtGGPPS3蛋白与其他植物GGPPSs高度保守。生物信息学预测结果表明,NtGGPPS3的分子量为39.5 kD,等电点为6.28,为亲水性蛋白,N端有叶绿体信号肽。二级结构主要由螺旋和无规则卷曲组成,三级结构含有两个富含天冬氨酸的区域,并通过同源建模构建了NtGGPPS3的3D模型。进化分析表明,植物的GGPPS分为大亚基和小亚基两个分支,而且NtGGPPS3属于大亚基。     

弓形虫亲环蛋白基因的克隆及原核表达

目的克隆并原核表达刚地弓形虫(Toxoplasma gondii)亲环蛋白(Cyclophilin,CyP)基因。方法收集、纯化Rh株弓形虫速殖子,提取总RNA,应用RT-PCR技术扩增TgCyP基因,插入原核表达载体pET-28a中,构建重组表达质粒pET-28a-TgCyP,转化E.coliBL21(DE3),经IPTG诱导后,进行SDS-PAGE和Western blot分析。结果测序结果与GenBank中登录的基因序列同源性为100%,重组表达质粒pET-28a-TgCyP经PCR及双酶切鉴定证明构建正确。SDS-PAGE分析表明,重组蛋白的相对分子质量约为26000,表达量约占菌体总蛋白的40%,Western blot显示其能被鼠抗弓形虫免疫血清识别。结论已在E.coliBL21(DE3)中表达了Rh株TgCyP,其有望作为弓形虫疫苗的候选抗原。