ABSTRACT
AIM: To analyze the capability of cytomegalovirus (CMV)-infected human embryonic lung fibroblasts (HELFs) to induce immune response. METHODS: HELFs were infected with cytomegalovirus and stained with antibody against HLA-A2 molecular, the expression of HLA-A2 was detected by FCM. The infected HELFs were incubated with individual pp65 peptide NLVPMVATV. While the uninfected and unloaded infected HELFs served as control respectively. After PBMC was added to the differently treated HELFs and incubated, the immune response was measured with IFN-gamma release as readout. RESULTS: The expression of HLA-A molecular on infected fibroblasts diminished markedly compared with that on the uninfected. The peptides expressed on the infected HELFs together with those pulsed externally induced a stronger response than the infected HELFs alone. CONCLUSION: Although CMV can down-regulate the expression of MHC I on the infected cells, it can not decrease the capacity of cells to present peptides loaded externally, and therefore still induce immune response to some extent.
Subject(s)
Cytomegalovirus/immunology , Fibroblasts/immunology , Fibroblasts/virology , Amino Acid Sequence , Animals , Cell Line , Fibroblasts/metabolism , Flow Cytometry , Gene Expression Regulation/immunology , HLA-A Antigens/metabolism , Humans , Lung/cytology , Peptide Fragments/chemistry , Peptide Fragments/immunology , Phosphoproteins/chemistry , Viral Matrix Proteins/chemistryABSTRACT
Human placental ribonuclease inhibitor is an acidic protein of Mr approximately 50 kDa with unusually high contents of leucine and cysteine. It is a cytosolic protein that protects cells from the adventitious invasion of pancreatic-type ribonuclease. HRI has 32 cysteine residues, and the oxidative formation of disulfide bonds from those cysteine residues is a rapid cooperative process that inactivates HRI. The most proximal cysteine residues in native HRI are two pairs that are adjacent in sequence. In the present paper, two molecules of alanine to substitute for cys328/cys329 were performed by site-directed mutagenesis. The site-mutated RI cDNA was constructed into plasmid pPIC9K, and then transformed Pichia pastoris GS115 by electroporation. After colony screening , the bacterium was cultured and the product was purified with affinity chromatography. The affinity of the recombinant human RI with double site mutation was examined for RNase A and its anti-oxidative effect. The results indicated that there was no much change in the affinity for RNase A detected when compared with the wild type of RI. But the capacity of anti-oxidative effect was increased by 7-9 times. The enhance in anti-oxidative effect might be the reason for preventing the formation of disulfide bond between cys328 and cys329 and the three dimensional structure of RI was thereby maintained.
