ABSTRACT
The establishment of in vitro model will provide optimal conditions for the study of human papillomavirus (HPV)-associated cervical cancer. In this study, E6 and E7 gens of HPV31 were cloned and expressed in E. coli. The recombinant proteins were purified and used as antigens to immunize mice for the production of polyclonal antibody. Mammalian expression plasmid pBudCE4. 1-HPV31-E6/E7 was also constructed and transfected into C33A cells. The transfected cells were then selected by Zeocin. The expressions of the E6 and E7 mRNAs and proteins were detected by RT-PCR and Western blot respectively. A stable cervical cancer cell line was established as an in vitro model for the study of human papillomavirus type 31(HPV31) associated cervical cancer.
Subject(s)
Animals , Female , Humans , Mice , Cell Line , Virology , Human papillomavirus 31 , Genetics , Metabolism , Oncogene Proteins, Viral , Genetics , Metabolism , Papillomavirus E7 Proteins , Genetics , Metabolism , Papillomavirus Infections , Virology , Recombinant Proteins , Genetics , Metabolism , TransfectionABSTRACT
PBD-1 is an antibacterial peptide that plays an important role in defence system of porcine. To produce PBD-1 with bioactivity in Pichia pastoris, according to published amino acid sequence of porcine beta-defensin 1(PBD-1) and the partiality codon of yeast, the PBD-1 gene was synthesized by PCR and cloned into pPIC9K to construct the recombinant expression vector pPIC9K-PBD-1, the obtained recombinant plasmid was linearized by Sal I, and then transformed into SMD1168 by electroporation. Under the control of the promoter AOX1, an approximately 4.5 kD PBD-1 peptide was expressed. Antibacterial activity assay shows that the PBD-1 has the antibacterial activity on Staphylococcus aureus. This is the first secreted expression of porcine beta-defensin 1 gene in Pichia pastoris.
Subject(s)
Animals , Anti-Bacterial Agents , Pharmacology , Cloning, Molecular , Gene Expression , Genetic Vectors , Genetics , Pichia , Genetics , Protein Engineering , Staphylococcus aureus , Swine , Genetics , beta-Defensins , Genetics , PharmacologyABSTRACT
Objective:To obtain the high expression of Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D gene. Methods:The Herpes Simplex Virus Type 1(HSV-1)Glycoprotein D(gD1) gene fragment containing dominant antigen epitopes confirmed by computer analysis was cloned by PCR technical and inserted into plasmid vector pTrxA. Then the recombinant plasmid was transformed into Rosetta. The expressed product was analyze by SDS-PAGE. Results:930 bp gene fragment was amplified by PCR as anticipated. Nucleotide sequencing showed a 100 % homology with that of the published sequence in GenBank. The molecular weight of the expressed protein was about 48kDa, Western blotting indicated that the antigenicity of the protein was good. Conclusion:The plasmid pTrxA-gd1 was constructed and a high efficiency expression of the gd1 gene from Herpes Simplex Virus Type 1(HSV-1)strain was made. The expressed product shows a good antigenicity.
ABSTRACT
In present study,bovine Lactoferricin was first secretly expressed in Pichia pastors yeast expression system.The synthesized LfcinB gene fragment was cloned into expression vector pPIC9K,and then obtained recombinant plasmid,designated as pPIC9K-LfcinB,was linearized and transformed into Pichia pastors strains SMD1168 by electroporation.The transformants were screened with Geneticin and multiply-copy colonies were harvested,in which LfcinB gene was verified to inserted into yeast chromosome stably.The positive recombinant Pichia strains were induced with methanol to express LfcinB in culture supernatant.It's expressive products has high activity of killing bacteria.We concluded that LfcinB gene was cloned and integrated into yeast chromosomes,and obtained expression peptide was tested to have high antibacterial activity.