ABSTRACT
Objective:To establish an experimental model for intracellular antibacteria and endotoxin neutralization in vitro to detect the antibacterial and endotoxin neutralization activity of the muBPI_(25) protein.Methods: RAW264.7 cells were transfected with pcDNA3.1(+)muBPI_(36-259), and then were infected with intracellular bacterial of either G ~+/G~-to establish the experimental model of intracellalar antibacteria.The RAW264.7 cells were co-transfected with the pSecTag2B-muBPI_(36-259) and dual-luciferase reporter gene plasmids for establishment of the experimental model of endotoxin neutralization.Results:The experimental model of intracellular antibacteria confirmed that the muBPI_(25) protein could inhibit/kill Salmonella typhi.The experimental model of endotoxin neutralization indicated that the muBPI_(25) protein could neutralize endotoxin.Conelusion: We firstly demonstrate that murine BPI N-terminal functional fragment(muBPI_(25) protein)can inhibit/kill Salmonella typhi,and can neutralize, its lysating product, endotoxin.
ABSTRACT
<p><b>OBJECTIVE</b>To construct pBV-BPI600-Fcgamma1(700) recombinant expression vector, to transform it into Escherichia coli DH5alpha, and to induce the expression of BPI23-Fcgamma1 anti-bacterial recombinant protein.</p><p><b>METHODS</b>Genes coding for BPI23 and Fcgamma1 were amplified by RT-PCR from mRNA extracted from HL-60 cell and normal human leukocytes; recombinant cloning vector and recombinant expression vector were then constructed. pBV-BPI600-Fcgamma1(700) recombinant expression vector was transformed into the competent Escherichia coli DH5alpha and BPI23-Fcgamma1 recombinant protein was expressed by a temperature-induced method.</p><p><b>RESULTS</b>(1) Expected amplified products BPI600hp and Fcgamma1(700bp) were obtained by RT-PCR method. (2) pUC18-BPI180, pUC18-BPI420 and pUC18-Fcgamma1(700) recombinant cloning vector were successfully constructed, and sequences were identical with the reported ones. 3) pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and the enzyme digestion analysis showed an expected result. (4) The expression level of BPI23-Fcgamma1 recombinant protein accounted for 20% of total bacterial proteins. (5) The renatured BPI23-Fcgamma1 recombinant protein showed bacteriocidal activity and biological function of complement fixation, and opsonization.</p><p><b>CONCLUSION</b>pBV-BPI600-Fcgamma1(700) recombinant expression vector was successfully constructed, and BPI23-Fcgamma1 recombinant protein with double biological activity of BPI and IgGFc was expresed in Escherichia coli.</p>
Subject(s)
Humans , Antimicrobial Cationic Peptides , Blood Bactericidal Activity , Blood Proteins , Genetics , Pharmacology , Carrier Proteins , Genetics , Pharmacology , Cell Adhesion Molecules , Escherichia coli , Genetics , Metabolism , Genetic Vectors , HL-60 Cells , Membrane Proteins , Recombinant Fusion Proteins , Genetics , PharmacologyABSTRACT
Objective:To clone Fab genes of anti-p185 monoclonal antibody 5E12 and express it in E.coli.Methods:Fd and ? genes were cloned by RT-PCR, inserted into Fab expression vector and expressed in E.coli. The N-terminal sequences of V regions was resumed by PCR mediated mutagenesis. The antigen-binding activity of the Fab were tested by ELISA and immunohistochemistry.Results:Fd and ? genes were cloned and expressed in E.coli. But the bacterially expressed Fab fragments showed no antigen binding activity. After the N-terminal sequences of V regions was corrected to original sequences, the Fab expressed in bacterial was able to target HER2/neu-expressing cells(NIH3T3/erbB-2 cells). Correction of Fd N-terminal sequences could partially resume the antigen binding activity. But correction of ? chain N terminal sequences was shown no expected result.Conclusion:Successful in constructing and expressing anti-p185 Fab, which will benefit the construction of other engineering antibody and humanization of murine anti-p185 McAb. We also found that the V region N terminal changes introduced with PCR primers may affect antigen binding activity seriously, to which more attention should be paid during antibody engineering.
ABSTRACT
Objective:To express and secrete rBPI m23 bactericidal protein in Pichia pastoris expression system. Methods:pPICZ? synBPI m600 recombinant expression vector was constructed with prepared vectors (pUC18 synBPI 414 and PBV220 BPI m420 ) according to Molecular Cloning Laboratory Manual. The Pichia pastoris GS115 were electroporated with linearized pPICZ? synBPI m600 vectors , positive clones were screened on low salt LB medium with Zeocin , then the positive clones were identified by PCR and Mut phenotype analysis. rBPI m23 was induced to express in BMMY ,purified by SP Sepharose beads , identified by SDS PAGE and Western blot and measured with BCA method. Results:①pPICZ? synBPI m600 expression vector was successfully constructed.②Pichia pastoris GS115 strains integrated with synBPI m600 stably were obtained.③SDS PAGE showed the molecular weight of the recombinant protein was approximate 23 kD,and Western blot confirmed that the protein could bind to the anti BPI antibody specially.④The supernate protein concentration was about 2 9 mg/L.Conclusion:rBPI m23 was expressed and secreted effectively in Pichia pastoris GS115.