Fusion Of Molecular Adjuvant HC3d3 To HCGβ Enhances Primary Responsiveness Of Human Peripheral Immuno-competent Cells Upon HCGβ Antigen

Objective To clone the fragment of human C3 gene (hC3d) from human hepatic cDNA library, and construct the eukaryotic expression plasmids of pCI-gs-signal-6His-hCGp, pCI-gs-signal-6His-hCGp-hC3d3. To establish the CHO lines with a high-efficient secreting expression of hCGp and hCGp-hC3d3 fusion protein. To testify the immunogenicity of hCGp-hC3d3 fusion protein to human peripheral immuno-competent cells, and to probe into the effective mechanisms of the molecular adjuvant hC3d.Methods Human C3d gene fragment was cloned from human hepatic cDNA library by PCR techniques, and the eukaryotic expression plasmids of pCI-gs-signal-6His-hCGp and pCI-gs-signal-6His-hCGp-hC3d3 were constructed respectively by way of molecular cloning. The CHO cells were transfected by the recombinant expression plasmids with aid of Lipofectaine 2000 reagent. The resistant clones secreting the protein of interest were screened on gradient concentrations of MSX. The clones were selected with the highest resistance to MSX, and screened for expression following the concentration of the secreted hCGP in the cultural supernatants assayed by chemiluminescent assay. Western blotting and Raji cell immunocytochemistry were used to identify the hCGP and hCGP-hC3d3 fusion protein, respectively. The recombinant proteins of interest were purified using the immobilized metal affinity chromatography under native condition and Sephadex G150 column gel filtration chromatography. The isolated B cells, the combined B cells and T cells, PBMC and Raji cells were treated in vitro respectively with lnM, 10nM,100 nM hCGp, hCGp-hC3d3 or PWM for 2-12 days. The expressions of CD80, CD86, CD154 and CD25 on the cells above were analyzed by flow cytometry. The IL-2 production in the supernatant was assayed by the enzyme-linked immunosorbent assay (ELISA). The cell proliferation was determined by incorporation of [³H]thymidine. Immunoglobulin(Ig) and anti- hCGp antibody levels in the 12-day culture supernatants were measured by an indirect ELISA. TheIg/Ab-secreting cells in the 10-day cultured lymphocytes were detected by the enzyme-linked immunospot (ELISpot) assay.Results The human C3d gene fragment has been successfully cloned from human hepatic cDNA library, and the eukaryotic expression plasmids of pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3 have been successfully constructed. The sequence analysis revealed that PCR products and colonies appeared to successful construction of signal-6His-hCGβ and signal-6His-hCGβ-hC3d3 in the correct reading frame. The correspondent recombinant proteins were secreted into the culture supernatant by CHO cells. The pCI-gs was able to produce more hCGβ and hCGβ-hC3d3 fusion protein than pcDNA3 and phCMV1 vector in CHO cells, respectively. The recombinant protein can be successfully purified with Ni²⁺-chelating chromatography and Sephadex G150 column gel filtration chromatography.It was found that the proliferation of B cell, the combined B and T cell, PBMC and Raji following exposure to hCGβ-C3d3 fusion protein was significantly higher than that of hCGβ alone. The Ig levels the in 12-day culture supernatants of B cell, the combined B and T cells, and PBMC treated with 100nM hCGβ-C3d3 fusion protein were 4-fold, 10-fold and 10.85-fold more than that of hCGβ alone. The low level of anti-hCGβ antibody could be produced in the combined B cells and T cells, and PBMC with 100nM hCGβ-C3d3, but no anti-hCGβ antibody was observed in all co-cultured cells with hCGβ alone. Both the Ig-secreting cells and anti-hCGβ antibody-secreting cells were significantly increased after the B cell, B and T cell, and PBMC were treated with hCGp-C3d3 fusion protein compared to the hCGβ alone.The hCGp-hC3d3 enhanced significantly the expressions of CD80 and CD86 molecules in B cell, especially CD86 (P<0.05), and that of CD154 and CD25 molecules in T cell compared to the hCGβ alone (P<0.05). The hCGβ-hC3d3 promoted human PBMC producing more IL-2 than the hCGβ.Conclusions We have successfully cloned the human C3d gene fragment and constructed the eukaryotic expression plasmids of pCI-gs-signal-6His-hCGβ and pCI-gs-signal-6His-hCGβ-hC3d3, and gained a more efficient secretive expression, and quick purification procedure of hCGβ and hCGβ-hC3d3 fusion protein in CHO cells. The molecular adjuvant hC3d3 enhanced greatly proliferative responses andinduction of more Ig and/or anti-hCGβ antibody synthesis of cultured lymphocytes compared to hCGβ alone. The human molecular adjuvant hC3d3 improves expressions of co-stimulatory molecules in human immuno-competent cells and their responsiveness upon the hCGβ antigen if fusing to the antigen.