Construction of T7 RNA polymerase gene expression system in Anabaena sp. PCC 7120 for the expression of hG-CSF / 生物工程学报

The low expression rate of exogenous genes in cyanobacteria is one of the bottlenecks of cyanobacteria genetic engineering. The T7 RNA polymerase expression system has achieved the efficient expression of exogenous genes in Escherichia coli. Cyanobacteria and E. coli are both Gram-negative bacteria with high genetic homology. The construction of T7 RNA polymerase expression system in cyanobacteria may improve the expression of foreign genes. In order to construct the T7 RNA polymerase expression system in Anabaena sp. PCC 7120, methods such as overlapping extension PCR and digestion-ligation technique were used to construct a site-specific integration vector pEASY-T1-F1-TacT7RNAPCmR-F2 and a shuttle expression vector pRL-T7-hG-CSF. The site-specific integration vector is capable of expressing T7 RNA polymerase, and the shuttle expression vector expresses hG-CSF driven by the T7 promoter. Then we introduced the site-specific integration vector into the wild type cyanobacteria by electroporation and transferred the shuttle expression vector into the site-integrated transgenic cyanobacteria by triparental conjugative transfer. In the end, we identified the presence of foreign genes in cyanobacteria by PCR, tested the transcription level of foreign genes in cyanobacteria by RT-PCR, and detected the protein expression of foreign genes in cyanobacteria by Western blotting. The two vectors were successfully constructed, the T7 RNA polymerase gene and hG-CSF gene were transferred into cyanobacteria well, and both genes were also expressed in cyanobacteria. In summary, the T7 RNA polymerase expression system was successfully constructed in cyanobacteria, and the expression rate of hG-CSF gene was doubled than the traditional cyanobacteria expression systems. This expression system will provide a better tool for the application of cyanobacteria genetic engineering and will promote the development of cyanobacteria as a chassis cell in the fields of synthetic biology in the future.

Establishment of IBRS-2 Cell Line Stably Expressing T7 RNA Polymerase and Recovery of SVDV From IBRST7 Cells / 生物化学与生物物理进展

The bacteriophage T7 RNAP gene was amplified via PCR from -lysogen DE3, and the gene was cloned into pBABEpuro retrovial vector, a recombinant plasmid named as pT7BABEpuro was constructed and sequenced. Then the pT7BABEpuro and pVSV-G plasmids were cotransfected into GP2-293 packaging cells by liposomese, some pseudotype viruses were ingathered and transfected into IBRS-2 cell under polybrene. The IBRS-2 cell was propagated in DMEM with puromycin. The genome extraction from the cells transfected different times, the T7 RNAP gene was amplified from the genome by PCR, the mRNA of T7 RNAP protein expressed in IBRST7 cells was analyzed by RT-PCR, respectively, the results showed the T7 RNAP gene had been integrated into the chromosome of IBRS-2 cell and expressed stably at high level. To study whether T7 RNAP is of transcriptional activity in the established IBRST7 cell line, a plasmid pIERS-EGFP-ET with a reporter gene (EGFP) under control of the T7 promoter was constructed. IRES element from FMDV (for CAP-independent translation) was cloned into plasmid pET-43.1a-c(+) downstream of the T7 promoter sequence, then EGFP gene was cloned in frame downstream of the AUG codon of the FMDV IRES, resulting in the plasmid. IBRST7 cells were transfected with plasmid pIERS-EGFP-ET using lipfection, EGFP was expressed, the results showed the T7 RNAP in IBRST7 cells has transcriptional activity. IBRST7 cell line was directly transfected with linearized full-length cDNA of swine vesicular disease virus (SVDV) HK/70, infectious SVDV was efficiently recovered from the cDNA. The reverse genetic procedure is simplified to a faster, one step protocol to recover RNA virus and will be useful to understand the mechanisms of molecular pathology of RNA virus and develop effective vaccines.

Large Protein Production Through Split Intein-Mediated Trans-Splicing / 中国生物工程杂志

Npu DnaE intein was used to produce some large proteins,which were difficult to obtain through conventional expression systems.A T7 expression system was described,by which the gene of T7 RNA polymerase is split into two pieces,and each piece fuses with Npu DnaE N-and C-terminal sequences respectively.Functional T7 RNA polymerase is created by mixing the two kinds of fusion constructs in vitro.The approach of split intein-mediated production of large proteins,in theory,readily generalizable to the purification of other large,cytotoxic or membrane proteins.

Construction of specific short hairpin RNA (shRNA) for Nucleostemin by T7 RNA polymerase promoter in vitro / 中国药理学通报

Aim In order to further explore the feasibility of gene therapy with RNA interference(RNAi) for acute leukemia,we synthesized short hairpin RNA(shRNA)for nucleostemin(NS)in vitro.Methods The designed DNA template consists of the sequence complementary to target mRNA,which was screened out consensus motif of three variants of NS gene,using 5′-UCUCUUGAA-3′ as loop sequence,The sequence of 5′-GGATCCTAATACGACTCACTATA-3′ acts as T7 promoter.Two shRNA were produced by T7 RNA polymerase and named as shRNA-NS-1 and shRNA-NS-2,respectively.The purified shRNA was quantified by gel electrophoresis.The interference effect of shRNA-NS transfected into HL-60 cell was examined by resultant cell morphology and inhibition rate of NS-mRNA expression.Results The concentrations of two shRNA-NS without degradation and diffusion were 5.24 ?mol?L~(-1)and 3.35 ?mol?L~(-1),respectively.There were significant decline in density and aggregation of cells and significant differences in size between cells after interfering HL-60 cell for 48 h.Furthermore,some of HL-60 cells treated by shRNA-NS-2 were changed from round to fusiform shape even with pseudopod.Compared with control group,the inhibition rates of shRNA-NS-1 and shRNA-NS-2 to NS-mRNA expression were 37.82% and 71.88%,respectively.Conclusion The two shRNA for NS gene were successfully constructed,which suppress NS gene expression significantly.shRNA-NS-2 also can chang HL-60 cell′s morphology and might be a useful tool to explore NS gene function and possible therapy for acute leukemia.

Technique and its significance of detecting colorectal cancer cells by fluorescent amplification catalyzed with T7 RNA polymerase / 解放军医学杂志

Objective To develop a new method for detecting colorectal cancer cells in peripheral blood using a hybrid technology referred to as fluorescent amplification catalyzed with T7 polymerase technique (FACTT). Method A model of colorectal cancer circulating tumor cells in peripheral blood was reproduced by mixing serial dilutions of HT29 cells with 1?107 peripheral blood monocytes from healthy subjects. After all nucleated cells were isolated from the model, biotinylated monoclonal antibody CEA against the Gold epitopes Ⅳ, which was expressed on the cell surface of human colorectal cancer cells but not on the normal cells, was added followed by the adding of avidin-biotinylated-DNA. Then T7 RNA polymerase and nucleoside triphosphates (NTPs) were added to perform RNA amplification at room temperature for 3h. Finally, RNA products were quantified by adding the RNA intercalating dye RiboGreen. RT-PCR was used to detect the expression of CEA mRNA as a control to assess the sensitivity of fluorescent amplification catalyzed with T7 polymerase technique. Results A method of detecting circulating colorectal cancer cells in peripheral blood by fluorescent amplification catalyzed with T7 polymerase technique was established. It was shown that the technique had a detection level of 5 HT29 cells among 1?107 monocytes, and it was more sensitive than RT-PCR technique, by which the level of detection was 1?102 cells among 1? 107 monocytes. Conclusion Fluorsescent amplification catalyzed with T7 polymerase technique is a new and sensitive technology for detecting circulating colorectal cancer cells in peripheral blood.