Study On The Gene Reconstruction Of BCG--Construction Of The Shuttle Plasmids With HIFN-α-2B Or GFP And Their Stable Expression In BCG

Bladder cancer is the most common urological malignant disease in China. Although intravesical administration of Bacillus Calmette-Guerin (BCG) has been accepted as the most effective therapy for superficial bladder cancer and carcinoma in situ of the bladder, 25-40% of patients never respond to BCG therapy. Furthermore, long-term remission (>5yr) is only achieved in 50%of patients. Toxicity associated with the BCG therapy is frequent with occurrence of severe adverse effects in 5% of patients and life-threatening symptoms in 0.5% of patients.Other than BCG, interferon-alpha (IFN-a) has also been used as an intravesical agent for treating superficial bladder cancer. Although its 40% response rate is clearly inferior to that of BCG therapy, a proportion of BCG non-responders have been shown to benefit from IFN-a monotherapy. In addition, intravesical IFN-a produces only minimal local and systemic toxicity. However, it is very costly and requires repeated applications.In an attempt to obtain an improved remedy for bladder cancer immunotherapy, an alternative schedule with concomitant administration of low-dose BCG plus IFN-a has been proposed. A number of pilot clinical trialshave shown that combination therapy is well tolerated and can yield a high complete response rate. These limited clinical trials have shown that adding IFN-a to BCG bladder cancer immunotherapy could lower BCG toxicity due to the reduced BCG dose while at the same time preserving or enhancing BCG activity against tumours.Base on the current understanding of the combination therapy in bladder cancer, in this study we intended to make a new strain of BCG thatconstitutively expresses rhIFN-a-2B and evaluated the newly constructedrBCG-hIFN-a-2B.Although extensive studies have suggested that intravesical BCG acts upon TCC by inducing a complex localized immune response leading to the destruction of bladder cancer, the exact mechanism by which BCG mediates the antitumor activity remains unclear. Understanding of the trafficking of intravesical BCG during the treatment of TCC is important for exploring the mechanisms by which this intracellular pathogen mediates antitumor activity. To date, little is known about the fate of intravesical BCG after intraluminal attachment. By electron microscopy, both human (T-24) and mouse (MBT-2) transitional epithelial cells were demonstrated to be capable of internalizing BCG in vitro. Internalized and degraded BCG organisms in urothelial cells from bladder washes of intravesical BCG-treated patients were also identified by electron microscopic observation. By use of fluorescence microscopy and fluorescein isothiocyanate (FITC)-labeled BCG, the ability of T-24 cells to take up the pathogen was also observed. Although both electron microscopy and FITC labeling are useful approaches to the internalization study, they require either a tedious and time-consuming sampling process or treatment of the pathogen prior to infection. In order to provide a better approach to monitoring BCG trafficking, we initiated the present study to genetically engineer novelBCG strains to efficiently express green fluorescent protein (GFP), an in vivo real-time marker molecule from the jellyfish Aequorea victoria. We demonstrated that the green recombinant BCG (rBCG) organisms were clearly visualizable by UV microscopy.Part1 The construction of phIFN-a-2B shuttle plasmid andits expression in BCG1. The construction of phIFN-a-2B shuttle plasmidThe inserted sequence was derived from polymerase chain reaction (PCR) amplification of plasmid including human IFN-a-2B using a pair of primers for the mature form of the cytokine. The sequence of the sense primer was CAAGggatccTGTGATCTGCCTCAAACCCACAG (BamHl site in lower cases) and that of antisense primer was GCCGgaattcTCATTCCTTACTTAAACTTTCT TG (EcoRI site in lower cases). The upstream primer overlapped the first one to eight codons of human IFN-a-2B encoding sequence, whereas the downstream primer terminated at the stop codon UGA of the sequence. Th...