转基因树突状细胞激活细胞毒性T细胞特异性杀伤淋巴瘤的体外实验

目的 探讨转基因树突状细胞激活细胞毒性T细胞产生抗淋巴瘤的特异性细胞免疫反应。方法 采用人骨髓来源的髓系前体细胞,在人细胞因子IL-4、GM-CSF和INF-α诱导下,在体外生成大量树突状细胞。将制备好的含有IgVH1核酸质粒,用脂质体法转染树突状细胞。转染成功的树突状细胞与外周血T淋巴细胞共培养,激活特异性CTL细胞,与阳性表达IgVH1的人淋巴瘤Namalwa细胞反应,用3H-TdR掺入法观察CLLs对瘤细胞的特异性杀伤效应。结果 树突状细胞能够用脂质体方法转染IgVH1核酸质粒,并且有效递呈给外周血T细胞,对表达IgVH1的人淋巴瘤Namalwa细胞产生特异性免疫杀伤活性,与对照组相比差异有显著意义（P<0.01）。结论 体外诱导扩增的 DC能够转染 IgVH1核酸质粒,体外激活T淋巴细胞,产生特异性细胞毒效应。     

重组质粒pMG36e-nisI-gfp在乳酸菌中的应用

本研究将带有nisI和咖（绿色荧光蛋白）基因的乳酸菌表达载体pMG36e-nisI-gfp转化至乳酸菌中,观察其在宿主内的稳定性．对重组菌进行了菌体形态、传代培养、质粒验证等研究,考察了该质粒的稳定性．结果显示：重组菌在不含抗生素的培养基中连续传代20代后,质粒丢失率低;菌体大小和形态基本不变;质粒经HindⅢ酶切后大小不变;GFP在第0、5、10、15和20代宿主菌中都可以表达,表达量没有明显差别,在SDS-PAGE中的带型一致;初步的动物实验验证了该质粒作为遗传标记的应用效果．说明该质粒在宿主菌中具有良好的稳定性．     

细菌质粒DNA的快速提取及检测

介绍一种质粒DNA的快速提取方法,所分离出的DNA纯度较好,全部操作仅在两只Eppendorf管中进行,含质粒DNA的上清液可直接点样走琼脂糖凝胶电泳。整个提取过程在较短时间内完成。该方法简便、快捷、效果好。     

pEgr-ssEndostatin的构建及其在体外的辐射诱导表达

为探讨pEgr-ssEndostatin重组质粒转染B16细胞后接受不同辐射剂量以及接受同一剂量不同时程endostatin表达的规律。采用RT-PCR方法从鼠肝脏中扩增出Endostatin cDNA,连入信号肽,构建了pEgr-ssEndostatin重组质粒,用脂质体介导的转染法转染小鼠B16细胞,用ELISA方法检测B16细胞上清中endostatin的表达水平。结果表明的分泌型Endostatin序列与报道完全一致,Egr-1启动子和Endostatin cDNA正确插入表达载体pcDNA3．1;pEfr-ssEndostatin具有辐射诱导表达特性。提示小鼠Endostatin基因成功地被克隆和表达,Egr-1启动子具有辐射激活和诱导下游基因表达增强的功能。     

两个重组质粒在快生型大豆根瘤菌中的稳定性的比较

将两个复制子相同的重组质粒导入快生型大豆根瘤菌中，考察了得到的转移接合子在培养和共生条件下的稳定性。发现带有质粒ＲＫ２ ｐａｒ基因的重组质粒ｐＴ２ＴＸ３Ｋ在快生型大豆根瘤菌中无论在培养还是共生条件下都可以稳定存在，而不带有该基因的重组质粒ｐＣＫ３在两种条件下都有一定程度的丢失。     

一种特异性检测单增李斯特菌的质粒DNA标准物质

研制了一种质粒DNA标准物质,包含目前单增李斯特菌检测常用的靶基因序列——内化素基因(Inl A)序列,可适用于扩增Inl A基因的单增李斯特菌PCR相关检测方法。采用紫外分光光度法(UV)和高分辨电感耦合等离子体质谱(HR-ICP-MS)两种方法,多家实验室联合定值的方法,对该套标准物质的均匀性、稳定性、量值以及不确定度进行详细的考察,质粒DNA标准物质的最终定值结果为94.9(±5.1)ng/μL。该质粒DNA标准物质可用于微生物实验室对单增李斯特菌PCR相关检测进行检测结果质量控制、开展方法比对和能力验证等。     

The terminal protein of the linear DNA plasmid pGKL2 shares an N-terminal domain of the plasmid-encoded DNA polymerase

The 36K protein attached at the 5′ end of the linear DNA plasmid pGKL2 from the yeast Kluyveromyces lactis was first purified and characterized. The terminal protein was purified from cells (1 kg wet weight) by ammonium sulphate precipitation and two rounds of centrifugation to equilibrium in CsCl gradients. The pGKL2 was present only in the post-microsomal supernatant. Approximately 10 mg of the purified pGKL2 was recovered and digested with DNase I. The terminal protein (final ca. 0·8 mg) was homogeneous by electrophoresis and we determined the N-terminal amino acid sequence up to ten residues, showing that it existed in the cryptic N-terminal domain of pGKL2-ORF2 (DNA polymerase) sequence.     

CAMBRIDGE PRIZE LECTURE DEVELOPING NEW STRAINS OF YEAST*

Genetic engineering or recombinant DNA technology is now routinely applied to the construction and development of new strains of brewing yeast. This is a direct consequence of the power of the technology which facilitates the modification, introduction and stable maintenance of specific genes in brewing yeast, without compromising the intrinsic brewing properties of the yeast itself. The way in which gene technology has been applied to the development of new strains of Bass yeast is briefly illustrated by the provision of plasmid-based systems for ensuring the stable maintenance of recombinant genes, the construction of amylolytic and β-glucanolytic yeast and the design and development of genetic systems for enhancing the value of waste brewers yeast. Commercial and regulatory issues are discussed.     

Novel episomal vectors and a highly efficient transformation procedure for the fission yeast Schizosaccharomyces japonicus

Schizosaccharomyces japonicus is a fission yeast for which new genetic tools have recently been developed. Here, we report novel plasmid vectors with high transformation efficiency and an electroporation method for Sz. japonicus. We isolated 44 replicating segments from 12 166 transformants of Sz. japonicus genomic fragments and found a chromosomal fragment, RS1, as a new replicating sequence that conferred high transformation activity to Sz. japonicus cells. This sequence was cloned into a pUC19 vector with ura4⁺ of Sz. pombe (pSJU11) or the kan gene on the kanMX6 module (pSJK11) as selection markers. These plasmids transformed Sz. japonicus cells in the early‐log phase by electroporation at a frequency of 123 cfu/µg for pSJK11 and 301 cfu/µg for pSJU11, which were higher than previously reported autonomously replicating sequences. Although a portion of plasmids remained in host cells by integration into the chromosome via RS1 segment, the plasmids could be recovered from transformants. The plasmid copy number was estimated to be 1.88 copies per cell by Southern blot analysis using a Sz. pombe ura4⁺ probe. The plasmid containing ade6⁺ suppressed the auxotrophic growth of the ade6‐domE mutant, indicating that the plasmid would be useful for suppressor screening and complementation assays in Sz. japonicus. Furthermore, pSJU11 transformed Sz. pombe cells with the same frequency as the pREP2 plasmid. This study is a report to demonstrate practical use of episomal plasmid vectors for genetic research in Sz. japonicus. RS1 has been submitted to the DDBJ/EMBL/GenBank database (Accession No. AB547343). Copyright © 2010 John Wiley & Sons, Ltd.     

乳酸菌食品级质粒及应用的研究进展

乳酸菌是食品发酵工业中重要的益生菌,利用分子生物学技术构建的菌种对食品产业发展及人类健康具有重要影响。本文主要通过介绍乳酸菌食品级系统的基本要求、食品级质粒的元件组成、食品级质粒构建策略及其应用的研究进展,展示了乳酸菌食品级分子操作系统的建立对乳酸菌的深层次开发利用所具有的重要意义。