ABSTRACT
Hepatitis B Virus [HBV] DNA polymerase transactivated protein 1 [HBVDNAPTP1] is a novel protein transactivated by HBV DNA polymerase, screened by suppression subtractive hybridization technique [GenBank accession no: AY450389]. The biological function of HBVDNAPTP1 was investigated in this study. We constructed a vector pcDNA3.1 [-]/myc-His A-HBVDNAPTP1 and used it to transfect acute monocytic leukemia cell line THP-1. HBVDNAPTP1 expression was detected by western blot analysis in the cells. A cDNA library of genes transactivated by HBVDNAPTP1 in THP-1 cells was made in pGEM-T Easy using suppression subtractive hybridization [SSH]. The cDNAs were sequenced and analyzed with BLAST search against the sequences in GenBank. Some sequences, such as CIP4, might be involved in apoptosis development. mRNA and protein expression of CIP4 was identified by Real time RT-PCR and western blot in THP-1 cells. HBVDNAPTP1 could down-regulate the expression of CIP4 at both transcription and translation levels. HBVDNAPTP1 may be involved in the positive regulation on the initiation of monocyte apoptosis. The result contribute to reveal the HBVDNAPTP1 biological functions and provide new evidences for further exploration of the regulatory mechanism of HBVDNAPTP1
ABSTRACT
Objective To establish a recombinant Mycobacterium (M.) smegmatis secreting interliukin-2 (IL-2) for the prevention and treatment of human bladder cancer. Methods The M. tuberculosis HSP70 promotor, ?-antigen signal peptide gene, and human IL-2 cDNA were amplified from plasmid pY6013, pIJK-1, and pHIG53 respectively by PCR. The products were cloned into plasmid pRR3 to construct a mycobacterial shuttle-expressing plasmid pR-a-IL-2 secreting human IL-2. After confirming the construction was correct by enzyme digestion, plasmid pR-?-IL-2 was transduced into M. smegmatis mc 2 155 by electroporation. The stability of the recombinant mycobacteria was evaluated and the activity of IL-2 secreted by the bacteria was assayed. Results Structure of the pR-a-IL-2 was correct and it was effectively transduced into M. smegatis mc 2 155. The recombinant mycobacteria stably expressed IL-2. The IL-2 activity in the medium was 118.5 U/ml. Conclusion The successful establishment of recombinant M. smegmatis can provide the basis for the research of biotherapy and prevention of the recurrence of bladder cancer.
ABSTRACT
Alpha-toxin gene was amplified from chromosomal DNA of Clostridium perfringens type C by polymerase chain reaction(PCR). PCR product was inserted into vector pGEM-T directively. The cloned recombinant plasmid pXCPA1 possesses positive nucleotide sequence of alpha-toxin. A 1.2 kb alpha-toxin gene fragment was cleaved with restriction endonucleases NcoI/EcoRI from plasmid pXCPA1, and then inserted into an expression vector pET-28c which cleaved with NcoI/EcoRI by blunt-end ligation. The recombinant expression plasmid pETXA1 was studied in detail by restriction endonucleases analysis and nucleotide sequencing. The results showed that the recombinant expression pETXA1 possessed a positive alpha-toxin gene sequence and reading frame. BL21(DE3) (pETXA1) could produce alpha-toxin and the expressed products were recognized by alpha-toxin monoclonal antibodies, and the expression level of the alpha-toxin proteins were about 16.28% of total cellular protein by SDS-PAGE and thin-layer gel scanning analysis.