Construction,properties, and application of the pCB5 plasmid,a novel conjugative shuttle vector with a Cupriavidus basilensis origin of replication

Cupriavidus basilensis is a species with diverse metabolic capabilities, including degradation of xenobiotics and heavy metal resistance. Although the genomes of several strains of this species have been sequenced, no plasmid has yet been constructed for genetic engineering in this species. In this study, we identified a novel plasmid, designated pWS, from C. basilensis WS with a copy number of 1–3 per cell and a length of 2150 bp. pWS contained three protein-coding genes, among which only rep was required for plasmid replication. Rep showed no homology with known plasmid replication initiators. Unlike most plasmids, pWS did not have a cis-acting replication origin outside the region of rep. The minimal replicon of pWS was stable in C. basilensis WS without selection. A conjugative C. basilensis/Escherichia coli shuttle vector, pCB5, was constructed using the minimal replicon of pWS. Interestingly, the copy number of pCB5 was flexible and could be manipulated. Enhancing the expression level of Rep in pCB5 by either doubling the promoter or coding region of rep resulted in doubling of the plasmid copy number. Moreover, replacing the native promoter of rep with the lac promoter increased the copy number by over fivefold. Finally, using two different β-galactosidase reporting systems constructed with pCB5, we successfully demonstrated the different regulatory patterns of bph and dmp operons during diphenyl ether (DE) degradation in C. basilensis WS. Thus, this shuttle vector provided an efficient tool for DNA cloning and metabolic engineering in C. basilensis.

     

碱性磷酸酶标记链霉亲和素

碱性磷酸酶标记链霉亲和素（AP-SA）是酶放大时间分辨荧光免疫分析（EATRFIA）通用的、最关键的试剂.报告了AP-SA的戊二醛二步标记方法.用自行研制的荧光发展溶液和AP的底物5-氟水杨酸磷酸酯测定了标记物AP-SA的特性,AP的标记回收率为38.7%;AP-SA稀释200～12 800倍时,稀释倍数和Tb的信/噪比呈线性关系;至少在两个月内,AP-SA的酶活性是稳定的.     

源于Ⅳ型胶原α3、α6链的肿瘤新生血管生成抑制剂

肿瘤抑素（Tumstatin）和Hexastatin是分别源于Ⅳ型胶原α3、α6链的肿瘤新生血管生成抑制剂.肿瘤的发生、发展和转移都依赖于新生血管的生成.通过抑制肿瘤新生血管的生成,阻断肿瘤细胞赖以生存的氧气及营养供给,肿瘤细胞便会发生凋亡.这是肿瘤治疗的新策略.Tumstatin和Hexastatin是近年来研究比较多的两种肿瘤新生血管生成抑制剂,其中Tumstatin的结构、活性位点、抑制肿瘤新生血管形成机制研究得比较清晰;Hexastatin虽与Tumstatin有许多相似之处,但是还有很多问题需要进一步研究解决.该文就这两种肿瘤新生血管生成抑制剂等的最新研究进展做一综述.