ABSTRACT
ObjectiveTo investigate the adjuvant effect of heatshock protein 110(HSP110) on the immune responses induced by an altered peptide ligand of human papilloma virus type 16 E711-20 peptide (HPV16E711-20).Methods The complex of HSP110 and an altered peptide ligand of HPV16E711-20 was constructed in vitro.Fifteen 6-week-old C57BL/6 female mice were randonly and equally divided into 3 groups,including complex group,ligand group,and phosphate buffered solution (PBS) group,to receive intraperitoneal immunization with the complex (100 μg),peptide (10 μg),and PBS (100 μl) respectively.Immunization was carried out with an interval of 2 weeks for 2 times.Two weeks after the last immunization,the mice were sacrificed followed by the isolation of splenocytes.MTT assay was performed to evaluate the proliferation activity of splenocytes,intracellular staining for interferon(INF)-γ to detect cytotoxic T lymphocytes (CTLs),standard chromium-51 (51Cr) release assay to estimate the lethal effect of specific CTLs on target cells.Statistical analysis was performed by using t test with SPSS 10.0 software.Differences were considered as statistically significant when the P value was less than 0.05.ResultsA significant increase was observed in the proliferation index ( 1.87 ± 0.122 vs.0.32 ± 0.071,t =4.01,P ＜ 0.01 ) of,and percentage of CD8+IFN-γ+ T lymphocytes(3.9％ vs.0.4％,t =3.88,P ＜ 0.01 ) among splenocytes from the complex group compared with the ligand group.At the effector-to-target ratio of 100 ∶ 1,50 ∶ 1,25∶ 1 and 12.5 ∶ 1,the death rate of target cells was 54.7％,72.2％,61.5％ and 39.8％ respectively after incubation with CTLs from the compleximmunized mice,higher than that from the ligand-immunized mice (35.2％,49.3％,28.1％,17.4％,respectively).ConclusionHSP110 could enhance the immunological effect of the altered peptide ligand of HPV16E711-20,and can serve as an immunological adjuvant.
ABSTRACT
Objective To investigate the immunogenicity of immunodominant cytotoxic T lymphocyte epitope E749-57 of human papilloma virus (HPV) 16 oncoprotein E7 chaperoned by heat shock protein (HSP)110. Methods Mouse HSP110 gene was cloned into prokaryotic expression vector pQE-80L for the expression of HSP110 protein, which was purified using Ni-NTA column. SDS-PAGE and Western-blot were conducted to confirm the purified mHSP110 protein, which was subsequently incubated with E749-57 peptide under heat shock condition, and high-performance liquid chromatography (HPLC) was used to evaluate the binding efficiency of the recombinant protein and E749-57 peptide. Twenty mice were divided into 4 groups to be immunized with mHSP110 protein, E749-57 peptide, mHSP110-E749-57 complex and phosphate buffered saline (PBS),respectively. Two weeks after the last immunization, spleen cells were collected from the immunized mice and divided into 2 parts: one were stimulated by E749-57 peptide followed by the detection of CD8+ INF-γ+ T cells with flow cytometry; the other one were subjected to MTT analysis for the estimation of cell proliferation. The mHSP110-E749-57 complex was also used to immunize TC-1 tumor bearing mice to observe its anti-tumor effect.Results The full-length 2577 bp-sized mHSP110 gene was amplified from mouse liver cDNA and cloned into pQE-80L vector. Direct sequencing confirmed the correctness of the cloning. SDS-PAGE and Western-blot demonstrated the successful purification of mHSP110. HPLC assay showed that the purified mHSP110 protein could bind with E749-57 to form a relatively stable protein complex. The percentage of IFN-γ+ CD8+ T cells in and proliferation index of spleen cells from the complex-immunized mice were statistically higher than those from the other 3 groups of mice. Moreover, the complex could obviously inhibit the growth of TC-1 tumor in mice. Conclusion The mHSP110-E749-57 complex could enhance the generation of specific cytotoxic T lymphocytes and exert anti-tumor effects in mice.