ABSTRACT
By employing the pUC19 as a backbone, the regulatory and signal sequences which encode kanamycin resistance, and mycobacterial plasmid origin of replication (oriM) were cloned into the pUC19. The recombinant E. Coli-mycobacteria shuttle expression plasmid pBCG-8000 was constructed. The pBCG-8000 was able to replicate in both E. Coli and mycobacteria (including BCG) systems, and to confer stable kanamycin resistance upon transformants. The study should facilitate the development of BCG and other mycobacteria into multivalent vaccine vectors.
Subject(s)
Escherichia coli/genetics , Genetic Vectors/genetics , Mycobacterium/genetics , Plasmids/genetics , Transformation, Bacterial , BCG Vaccine/genetics , Base Sequence , Cloning, Molecular , Molecular Sequence DataABSTRACT
The plasmid pTLIL-2, which only directed a low-level expression of human interleukin 2(IL-2) cDNA in E. coli, was reconstructed: a series of deletions were made in 3' non-coding region of human IL-2 cDNA, and 7 recombinant plasmids with different deletions were selected, on the other hand, a Tac promoter sequence from pDR540 was inserted to upstream of IL-2 cDNA in pTLIL-2 so that pTLIL-2DT, which contains double Tac promoters, was constructed. And then, E. coli JM103 was transformed with the above 8 recombinant plasmids respectively. The expression efficiency of IL-2 cDNA in E. coli transformed with different plasmids was evaluated by means of SDS-PAGE and measuring 3H-TdR incorporation of IL-2-dependent activated T lymphocytes in the presence of bacterial extracts. Three engineering strains with high efficiency of IL-2 expression were selected, and all of these strains could produce recombinant IL-2(rIL-2) 4 times more than E. coil JM103/pTLIL-2.
Subject(s)
Escherichia coli/genetics , Interleukin-2/biosynthesis , Recombinant Proteins/biosynthesis , DNA Transposable Elements , DNA, Complementary/biosynthesis , DNA, Complementary/genetics , Escherichia coli/metabolism , Gene Expression , Humans , Interleukin-2/genetics , Plasmids , Promoter Regions, Genetic , Species SpecificityABSTRACT
To observe the effect of Re-Du-Qing on the toxic effect of endotoxin, the rabbit model of general Shwartzman reaction was made by injection of endotoxin into ear vein and the H(+)-ATP synthase activities of rabbit liver mitochondria were measured. The results showed that the H(+)-ATP synthase activity of endotoxin group was significantly lower than that of the normal group and group which had been simultaneously injected Re-Du-Qing (P less than 0.05). The level of H(+)-ATP synthase activity was almost raised to normal value, when Re-Du-Qing or dexamethasone was injected simultaneously with endotoxin respectively. It suggested that Re-Du-Qing was able to alleviate the nocuous effect of endotoxin on H(+)-ATP synthase activity in the mitochondria of rabbit liver cells.
Subject(s)
Drugs, Chinese Herbal/pharmacology , Mitochondria, Liver/drug effects , Proton-Translocating ATPases/metabolism , Animals , Female , Male , Mitochondria, Liver/enzymology , RabbitsABSTRACT
In our experiment, lymphocyte membrane was labeled by DPH fluorescence probe. The rate of rotation of the probe can be measured from the value of fluorescence polarization (PDPH). With this method useful information could be provided about membrane fluidity of lymphocytes. It was found that the F value (unit of lipid fluidity of membrane) of leukemic lymphocytes was obviously higher than that of normal ones. Furthermore, the F value of cultured leukemic Ts lymphocytes was the highest. In contrast with normal spleen T-lymphocytes or mixed lymphocytes, the response of malignant lymphocytes to the stimulation with ConA or PHA was reflected in the decrease of PDPH value or the increase of F value. Unexpectedly, the F value of T-lymphocytes from "615" mouse not injected with tumour cells was also higher than that of the mixed. The possibility of using the membrane fluidity as a diagnostic criterion was also discussed.
