大肠杆菌-长双歧杆菌穿梭载体的构建及PTEN在长双歧杆菌中的表达

长双歧杆菌可特异地定植于实体瘤低氧区,可用做肿瘤靶向性基因治疗的载体,而构建大肠杆菌-长双歧杆菌穿梭质粒则被证明是外源基因在长双歧杆菌中稳定表达的有效途径。为了构建能在长双歧杆菌中稳定表达外源基因的穿梭质粒并检测携带抑癌基因的工程菌对小鼠实体瘤的抑制效果,利用软件设计并合成了48条部分序列相互重叠的引物,通过PCR合成了长双歧杆菌质粒pMB1序列及长双歧杆菌HU启动子区序列,插入克隆载体pMD18-T,构建穿梭载体pMB-HU,该载体可在大肠杆菌DH5α及长双歧杆菌L17中稳定复制。PTEN基因编码具有蛋白质和酯类双重特异性磷酸酶活性的抑癌因子。将PTEN基因cDNA序列插入载体pMB-HU中HU启动子下游,构建重组质粒pMB-HU-PTEN,电击转化长双歧杆菌后,Western blot检测表明,表达产物中存在55kDa的PTEN蛋白特异条带。抑癌试验表明:与对照组相比,携带PTEN基因的长双歧杆菌可显著抑制小鼠实体瘤的生长。上述结果为以长双歧杆菌为载体的实体瘤靶向性基因治疗研究奠定了基础。

Construction of Escherichia coli-Bifidobacterium longum shuttle vector and expression of tumor suppressor gene PTEN in B. longum

It was reported that Bifidobacterium longum accumulated specifically in hypoxic solid tumors, therefore could be used as a delivery system for cancer-specific gene therapy. Furthermore, construction of E.coli-B. longum shuttle vectors was proved by other research to be an efficient way for stable gene expression in B. longum. To obtain a shuttle vector and analyze the inhibition on mice solid tumors by genetically engineered B. longum, 48 primers with mutual overlaps were designed, assisted by software package Oligo 6.0. By PCR with the above primers, a linear plasmid was synthesized, which consists of pMB1 and HU gene promoter, both from B. longum. pMB-HU was constructed by cloning the synthesized linear plasmid into E.coli vector pMD 18-T, and was proved to be stably replicated in both E.coli DH5alpha and B. longum L17. By inserting PTEN cDNA into pMB-HU, expression vector pMB-HU-PTEN was obtained, in which PTEN gene was reported as a major tumor suppressor gene encoding a dual-specificity phosphatase. pMB-HU-PTEN was then transferred into B. longum L17 by electroporation. After transformation, an effective expression of PTEN in B. longum L17 was confirmed by Western blot, and significant inhibition on growth of mice solid tumors was also observed with the above genetically engineered B. longum. Those obtained results have laid foundation for tumor-targeting gene therapy with B. longum.