人MC1R基因在杆状病毒-昆虫细胞表达系统中的表达

目的利用杆状病毒表达系统进行人恶性黑色素瘤A375细胞MC1R基因在Sf9昆虫细胞中的表达。方法以pMD18-T-MC1R为模板,利用PCR方法扩增人MC1R基因,将MC1R基因连接到pfastBac1质粒上,与穿梭载体DH10Bac转座,获得Bacmid-MC1R质粒后,通过脂质体介导,转染Sf9细胞,使其表达重组杆状病毒,经SDS-PAGE检测表达产物,放射受体分析法检测目的蛋白MC1R活性。结果Bacmid-MC1R质粒转染Sf9细胞后的SDS-PAGE图谱,在相对分子质量为35000处有一条新生蛋白带。放射受体分析结果显示表达的蛋白与标记配体特异亲和,具有生物学活性。结论MC1R基因成功在真核细胞中得到表达。     

新城疫病毒TL1株L基因的克隆及其真核表达载体的构建

目的对新城疫病毒TL1株L基因进行克隆、序列分析及真核表达载体构建。方法根据GenBank登录的新城疫病毒L基因序列,设计了一对引物,用RT-PCR对内蒙古分离株TL1的L基因进行整体扩增。将扩增产物提纯后,克隆入pGEM-Teasy载体,再亚克隆至真核表达载体pCI-neo上,通过酶切、PCR鉴定及测序进行验证。结果测序拼接得出L基因的全序列长度为6704bp,该基因的ORF总长为6615bp,编码2204个氨基酸。与GenBank登陆的12株参考毒株比较L基因编码区全核苷酸序列和氨基酸序列,发现TL1株与鹅源新城疫NA-1株、ZJ1株和SF02株的同源性极高,说明四者亲缘关系较近。结论已成功构建L基因真核表达载体,为对该株病毒进行反向遗传操作以及有关功能基因组研究奠定了基础。     

人血管生成素基因的克隆与表达

目的 为了获得人的血管生成素基因,并在原核表达系统中进行表达。方法 利用ＲＴ－ＰＣＲ技术 从人的肝脏组织中扩增出Ａｎｇ基因片段,并将其克隆到ｐＧＥＭ－Ｔ载体中进行序列分析,再亚克隆到原核表达到 ｐＥＴ２８ａ（＋）载体中。结果　构建了重组表达载体ｐＥＴＡ,转化宿主菌ＢＬ２１（ＤＥ３）后,经ＩＰＩＧ诱导,Ａｎｇ基因蛋白在 ＢＬ２１（ＤＥ３）中表达了相对分子质量约为１７０００的重组蛋白。ＳＤＳ－ＰＡＧＥ和薄层扫描分析表明,外源蛋白的表达量 占菌体蛋白总量的１０．６４％。结论 Ａｎｇ基因的克隆与表达为进一步研究其生物活性及应用奠定了基础。     

人Bid蛋白的原核表达及纯化

目的克隆人Bid基因,原核表达并纯化目的蛋白。方法用PCR方法从HeLa细胞cDNA文库中扩增人Bid基因,克隆至pGEX-6P-1表达载体,构建重组表达质粒pGEX-6P-1-Bid,转化大肠杆菌BL21(DE3),IPTG诱导表达。表达产物经GST亲和层析纯化。结果PCR扩增得到588bp的DNA片段,重组表达质粒pGEX-6P-1-Bid经双酶切鉴定和测序表明构建正确。表达的目的蛋白相对分子质量约48000,表达量约占菌体总蛋白的50%,纯化后纯度可达97%。结论已成功克隆并原核表达了人Bid基因,得到纯度较高的Bid蛋白,为进一步研究其结构和功能奠定了基础。     

大肠杆菌色氨酸操纵子基因的克隆与表达

目的克隆并表达大肠杆菌色氨酸操纵子基因,提高其酶活性。方法利用PCR方法,从大肠杆菌31884基因组中直接克隆出色氨酸操纵子,并将其连接到原核表达载体pET-22b(+)中,构建重组质粒pET-22b(+)-Trpoperon,转化大肠杆菌BL21,IPTG诱导重组蛋白表达,表达产物经SDS-PAGE分析,并测定酶活性。结果凝胶电泳可见PCR扩增产物大小约为7000bp,SDS-PAGE鉴定目的蛋白分别得到了表达,邻氨基苯甲酸合成酶和色氨酸合成酶的活性分别比对照提高了2.7和3.2倍。结论已成功构建了重组表达质粒pET-22b(+)-Trpoperon,邻氨基苯甲酸合成酶和色氨酸合成酶的活性在大肠杆菌中得到了提高,为高产色氨酸基因工程菌的构建奠定了基础。     

Neurturin基因的克隆及其在Vero细胞中的表达

目的克隆Neurturin(NTN)基因,并在Vero细胞中表达。方法用RT-PCR方法扩增hNTN cDNA,并克隆至pcDNA3真核表达载体中,经Lipofectamine2000转染Vero细胞,挑选稳定表达克隆,用RT-PCR及免疫荧光分析鉴定,并对转染后细胞的形态及生长状况进行分析。结果重组质粒pcDNA3/hNTN转染Vero细胞后获得了稳定表达克隆,且有目的蛋白表达,转染后细胞形态和生长特性发生了一定改变。结论获得了稳定表达NTN蛋白的Vero细胞株,为其移植并进行帕金森病猴基因治疗动物实验奠定了基础。     

Analysis of the bacterial community in a laboratory-scale nitrification reactor and a wastewater treatment plant by 454-pyrosequencing

For full understanding of the microbial community in the wastewater treatment bioreactors, one of the feasible and effective ways is to investigate the massive genetic information contained in the activated sludge. In this study, high-throughput pyrosequencing was applied to analyze the 16S rRNA gene of bacteria in a laboratory-scale nitrification reactor and a full-scale wastewater treatment plant. In total, 27,458 and 26,906 effective sequence reads of the 16S rRNA gene were obtained from the Reactor and the wastewater treatment plant activated sludge samples respectively. The taxonomic complexities in the two samples were compared at phylum and genus levels. According to the pyrosequencing results, even for a laboratory-scale reactor as simple as that in this study, a small size clone library is far from enough to reflect the whole profile of the bacterial community. In addition, it was found that the commonly used informatics tool “RDP classifier” may drastically assign Nitrosomonas sequences into a wrong taxonomic unit resulting in underestimation of ammonia-oxidizing bacteria in the bioreactors. In this paper the reasons for this mistakenly assignment were analyzed and correction methods were proposed.     

DRG2基因的克隆、表达及抑瘤活性研究

目的　克隆、表达抑癌基因NDRG2 ,纯化后考察其对肿瘤细胞的抑制作用。方法　将NDRG2基因克隆进pQE30 M15表达载体 ,经DNA测序 ,IPTG诱导表达 ,SDS PAGE测定表达量及相对分子质量 ,复性后浓缩 ,脱盐 ,亲和层析纯化 ,对纯化表达产物进行一系列检测 ,考察其抑瘤作用。结果　克隆构建的表达载体经DNA测序证明构建正确 ,表达产物相对分子质量为 39977,表达量为 4 0 0 5 % ,纯度为 94 % ,等电点为 6 3,表达蛋白N端氨基酸序列正确 ,NDRG2蛋白对肿瘤细胞有较强抑制作用。结论　构建了NDRG2的pQE30 M15表达载体并取得高表达 ,该蛋白对 790 1和HHCC肿瘤细胞均有抑制作用 ,为基因功能研究奠定了基础     

Towards more sustainable surimi? PCR-cloning approach for DNA barcoding reveals the use of species of low trophic level and aquaculture in Asian surimi

In recent years, authentication of commercialized seafood products has become a market priority. In this study, 29 Asian surimi products produced in China, India and Singapore, and commercialized in two Mediterranean countries (Egypt and Spain), were analyzed in order to authenticate species contained in surimi products. Due to the processing treatments, classical identification methods are not effective. Therefore, we conducted two molecular tracing methodologies for species identification: direct sequencing of 16S rDNA PCR products, and DNA mini-barcoding-based PCR cloning for Cytochrome oxidase subunit I (COI), with subsequent plasmid sequencing. In total, 10 fish species corresponding to 7 families were found. The Singaporean and Chinese surimi contained principally species of low trophic levels, like fringe scale sardines Sardinella fimbriata, and other, likely farmed, species such as striped catfish Pangasianodon hypophthalmus. The presence of low trophic level and aquaculture species suggests that current trends in surimi production are moving towards sustainability. The exception was a vulnerable shovelnose ray (Rhinobatos jimbaranensis) found in one product, which encourages further studies to detect the use of endangered species in such morphologically indistinct food items. We suggest PCR-cloning methodology for efficient species authentication in seafood controls, especially for mixed products.