The Manufacture Of Mis-matched DsRNA And Its Biological Evaluation

Polynucleotide phosphorylase (PNPase) is a key enzyme to synthesize poly I:C₁₂U. In our researches, we used a 5-litre hollow fiber reactor to carried out a continuous fermentation with cell recycling for Escherichia coli AS. 1.183. The growth inhibitors of cell were overcome successfully. At the end of the fermentation, the concentration of E. coli cell reached 250 (g/l wet wt, 70% moisture content), with polynucleotide phosphorylase activity maintained above 80 (u/g wet wt). The volumetric productivity in a hollow fiber reactor was found to be ten times higher than that in conventional batch culture.For the PNPase purification, the cells were best broken by ultrasonic waves, and a method of desalination through ultra filtration membrane was used instead of dialysis. The desalination time is sharply shortened and the buffer quantity used is declined by applying this method in purification. This method would produce a considerable economic benefit in the large-scale production.PNPase and nucleoside diphosphates were used to synthesize Poly I:C₁₂U at suitable condition, the ratio of cytidine monophosphate (C) and uridine monophosphate (U) can be controlled at 12:1, and the experimental injection was made for its biological evaluation.For the quality control, the chains of poly C₁₂U were reverse-transcripted into cDNA after PCR amplification, the PCR products were cloned into T vector and sequenced, the sequence indicated that the ratio of the C and U in the poly C₁₂U neared 12:1, and U was inserted in the chain of poly C separately. The reverse phase high performance liquid chromatography method was used to analysis the base ratio in synthesis of poly I:C₁₂U. The sample had to be treated by alkali andalkaline phosphorylase before the test. The result show that the rate of C to U in poly I:Ci2U was kept near to 12:1, which was the ratio designed.The acute toxicity experiments of poly I-.C^U were tested by mice, rats, and Beagle dogs. The results showed that the toxicity symptoms in the animals were invertible reactions, which had no any side effect to animals. The pyrogenic effects in rabbit caused by double stranded RNA (poly I:C) and its mismatched analogues (poly I:Ci2U) were tested. Poly I:Ci2U have not appeared any toxicity and other side effects, but poly I:C caused rabbit fever and even death.The biological effects of poly LC12U against the human malignant cancers was studied. In vitro, thirteen tumor lines were used to evaluate the antitumor activity of poly I:Ci2U, the IC50 values in all test were over lOug/ml, that meaning the poly I:Ci2U had no antitumor effect. In contrast, poly I:Ci2U appeared potential antitumor activity in vivo comparing with antitumor drug taxol and cyclophosphamide. Cancer cell lines included Sarcoma S-180, liver cancer H22, Bel-7402, melanoma B16, lung cancer L795, Lewis LC, PPA lymphocytic leukemia were tested in KM, C57BL, B/C, DBA, T739 mice and nude mice. The antitumor activity in most of tests were over 40% inhibitory ratio, some reached 70%. These results indicated that the mechanisms of poly LC12U was to stimulate host immunomodulatory effects, which can inhibit the growth of the tumors.The Hela cells were cultured with or without poly I:Ci2U and its toxic analogue poly I:C for 6 hr at 37°C before and after Coxsackievirus B3(CVB3) attacking. The results showed that poly LC^U could protect the cells infected by CVB3, and had no protection to the cells which treated by poly I:Ci2U and poly I:C before CVB3 attacking. The result was almost same between the poly I:C|2Uand poly I:C.