INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivat...INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes展开更多
Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,...Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.展开更多
The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the...The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication and nucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully.Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.展开更多
基金Projects of the Science Development Foundation of Shanghai (994919033)Tackling Key Problems in Science and Technology from the State Science and Technology Ministry(TJ99LA01)
文摘INTRODUCTIONHepatitis B virus (HBV) is regarded as one of themain etiologic factors involved in the developmentof human hepatocellular carcinoma (HCC).The open reading frame (orf)of X gene of HBVencoded a transactivating factor is the evidence thatstrongly supported the notion that the X gene ofHBV DNA integrated in HCC genomic DNA couldcontribute to the carcinogenesis of liver cells byactivation of some related cellular genes
文摘Human β-defensin 3(hBD-3) is a short polypeptide with a wide range of antimicrobial activity,which was purified from human lesional psoriatic scales in 2001.To obtain high level expression in E.coli of β-defensin 3,four pairs of oligonucleotide with cosmic site were synthesised using E.coli biased codons according to the amino acid sequence of β-defensin 3, connected and amplified by PCR. The PCR product encoding human β-defensin 3 was cloned into pET30a vector.The recombinant vector was transformed into E.coli BL21(DE3)PlysS and the expression was induced by IPTG. The recombinant fusion protein was analyzed by SDS-PAGE and purified by affinity column. The mass of the fusion protein consisted of 30.9% in total bacteria proteins. The recombinant fusion protein was digested by enterokinase, resulting in the recombinant hBD-3. Antimicrobial activity analysis showed that both recombinant hBD-3 fusion protein and recombinant hBD-3 had similar potency as the native protein in suppressing growth of both gram positive bacteria S.aureus and gram negative one E.coli in a dose dependent manner.
文摘The present study is aimed at studying the gene for TIMP-3,a mammalian tissue inhibitor,by constructing a recombinant eukaryotic cell vector for gene therapy in human breast cancer.We obtained the TIMP-3 gene from the human placent by RT-PCR.TIMP-3 gene was subcloned into pcDNA3.1 vetor from pMD18T vector by means of gene cloning to construct pcDNA3.1 recombinant vector.Human breast cancer cell line MDA-MB-453 was transfected with pcDNA3.1-TIMP3 recombinant vector using lipofectamine reagent.Then the expression of TIMP-3 and the effect on the metastasis of MDA-MB-453 were examined.The correct construction of pcDNA-TIMP3 was identified by means of restriction enzyme analysis,PCR amplication and nucleotide sequencing.Western blotting showed that the transfected cells were able to express TIMP-3, indicating that our construction of the pcDNA-TIMP3 eukaryotic expression vector was constructed successfully.Our experiments further indicated that the potential of metastasis was significantly reduced for the transfected cell line MDA-MB-453.Cellular & Molecular Immunology.2004;1(4):308-310.
