Expression Of Hcg��-c3d3 To Fusion Protein Vaccine Humoral Immune Anti-fertility Potential

Objective: To construct the eukaryotic expression vectors of the secreted hCGβand hCGβ-C3d3 with a hexahistidyl (6His) purified tag and gained an effectivesecreting expression in CHO cells, and high-effective and quick purificatioin of hCGβand hCGβ-C3d3 fusion protein. To enhance the immunogenity and Th2-biasedhumoral response of hCGβ protein vaccine by being fused with the molecularadjuvant C3d3 and immunizing mice of different strains. To testify the hCG biologicalneutralization capacity of the anti-serum of the hCGβ-C3d3 fusion proteinimmunization. Methods: phCMV1-6his-hCGβ and phCMV1-6his-hCGβ-C3d3 expressionvectors, based on the phCMV1, were constructed by way of molecular cloning. PCRtechniques were used to successfully add 6His residues to the C-terminal of signalpeptide and the N-terminal of hCGβ. Potential proteolytic cleavage sites between thejunctions of hCGβand C3d3 were mutated by ligating BamHⅠ and Bgl Ⅱ restrictionendonuclease site to mutate an Arg codon to a Gly codon. The CHO cells weretransfected by the recombinant expression vectors with aid of Lipofectamine reagent.The resistant clones secreting the recombinant proteins were established under theselection of G418 (800 μg/ml). The concentration of the secreted hCGβ in the culturesupernatants was assayed by the radioimmunoassay. Western blotting and Raji cellimmunocytochemistry were used to identify the hCGβ and hCGβ-C3d3 fusion protein.The recombinant proteins of interest were purified using the immobilized metal-ionaffinity chromatography (IMAC) under native conditions and Sephadex G-150column gel filtration chromatography. BAL B/c and C57BL/6 mice were inoculatedwith hCGβ-C3d3 fusion protein, hCGβ and hCGβ plus CFA/IFA respectively,followed by a boost at a 28-day interval. The antibody titers of serum weredetermined by enzyme-linked immunosorbent assay (ELISA). BAL B/c mice havingbeen immunized with hCGβ-C3d3 fusion protein, hCGβ and hCGβ plus CFA/IFArespectively were sacrificed in 3 weeks of the booster, and the spleen lymphocyteswere restimulated with hCG in vitro for 24 h. Then the cytokines of Th1, IFNγ and 4英 文 摘 要IL-2, and Th2, IL-4 and IL-10 in the supernatant were determined by ELISA. In orderto analyze the contraceptive potential of hCGβ-C3d3 fusion protein, MLTC-1 cells,which can produce progesterone under the action of hCG, were cocultured with hCGand antiserum of immunization of hCGβ-C3d3 fusion protein, hCGβ alone andhCGβ plus CFA/IFA, respectively. The concentration of progesterone in thesupernatant was determined at 7 h later. Moreover, the inhibiting test of hCG-inducedincrease of murine uterus weight was used to investigate the hCG-neutralizingcapacity of anti-hCGβ antiserum. Results: We have successfully constructed the eukaryotic expression vectors ofphCMV1-6his-hCGβ and phCMV1-6his-hCGβ-C3d3. The sequence revealed that allPCR products and clones appeared to the correct insert cDNA of interest, and thesequences of 6his-hCGβ and 6his-hCGβ-C3d3 were in the correct reading frame. Wehave successfully gained adequately the correspondent recombinant proteins from thetransfected CHO cell culture supernatnats. The phCMV1 vector can produce 1.6-foldand 1.4-fold higher level of hCGβ and hCGβ-C3d3 fusion protein than pcDNA3vector in CHO cells. With Ni2+-chelating chromatography and gel filteringchromatography, the recombinant protein can be successfully purified. ThehCGβ-C3d3 fusion protein was 1995–fold and 1259-fold more immunogenicrespectively in BAL B/c and C57BL/6 mice than hCGβ alone. In BAL B/c mice, theC3d3, as an adjuvant, fused to hCGβ was 10-fold (primer) and 32-fold (booster) moreimmunogenic than CFA/IFA. In C57BL/6 mice, the adjuvant effectiveness of C3d3was 10-fold (primer) and 20-fold (booster) more than that of CFA/IFA. Production ofIL-4 and IL-10 were significantly increased (p<0.05) i...