hCGβ-C3d3融合蛋白疫苗的体液免疫效应及其抗生育潜能

【作者】 王秀丽；

【导师】 李大金；

【作者基本信息】 复旦大学 ， 妇产科学， 2004， 博士

【摘要】 目的:构建带有 6 个组氨酸(6His)纯化标签的 hCGβ-C3d3 融合蛋白和 hCGβ蛋白的分泌型真核表达质粒,体外稳定表达与纯化具有生物学活性的 hCGβ-C3d3融合蛋白和 hCGβ 蛋白。通过融合分子佐剂 C3d3,增强 hCGβ 蛋白抗原在不同品系小鼠的免疫原性,实现 hCGβ 蛋白疫苗的 Th2 型体液免疫优势效应,并增强hCGβ-C3d3 融合蛋白疫苗抗血清中和 hCG 生物学活性的能力。 方法:以带有信号肽的 hCGβ 为模板,经 PCR 反应在 hCGβ 的氨基端加上 6个组氨酸(6His)纯化标签,将其构建入高效真核表达载体 phCMV1 即得phCMV1-6His-hCGβ。将 C3d3 与 hCGβ 进行基因融合,并构建入 phCMV1 即得phCMV1-6His-hCGβ-C3d3 。 脂 质 体 法 介 导 phCMV1-6His-hCGβ-C3d3 、phCMV1-6His-hCGβ、pcDNA3-hCGβ-C3d3 和 pcDNA3-hCGβ 转染中国仓鼠卵巢(CHO)细胞,G418 (800μg/ml)筛选抗药性克隆。放射免疫法检测抗性克隆无血清培液中 hCGβ 表达量,Western blotting 鉴定所表达的重组蛋白,Raji 细胞免疫化学染色法鉴定 hCGβ 是否与 C3d 分子成功融合。随后,采用针对于 6His 的镍柱结合凝胶过滤层析纯化表达产物。分别用 hCGβ-C3d3 融合蛋白、单用 hCGβ蛋白和 hCGβ 蛋白加用弗氏佐剂免疫生育期雌性 BALB/c 和 C57BL/6 小鼠,共免疫两次,间隔 4 周。ELISA 测定血清中抗 hCGβ 抗体滴度,比较各组的免疫效果。BALB/c 小鼠于末次免疫后 3 周处死,制备脾淋巴细胞悬液,hCG 体外脉冲培养48 h,ELISA 检测培养上清中 Th1 型(IFNγ、IL-2)和 Th2 型(IL-4、IL-10)细胞因子分泌水平。为检测 hCGβ-C3d3 融合蛋白疫苗的抗生育潜能,把 hCG 分别与正常鼠血清、hCGβ-C3d3 免疫鼠血清、hCGβ 免疫鼠血清和 hCGβ+ CFA/IFA免疫鼠血清共培养 MLTC-1 细胞,化学发光法检测培养前及共培养 7 小时后培养上清中孕酮的含量。此外,选用 3 周龄雌性 BALB/c 小鼠,皮下注射 hCG 的同时分别腹腔内注射系列稀释的正常鼠血清、hCGβ-C3d3 免疫鼠血清、hCGβ 免疫鼠血清和 hCGβ+ CFA/IFA 免疫鼠血清,每天 2 次,共 3 天。3 天后取 子宫称 湿重, 计 算每 克 体重的 子宫湿重(mg/g)。 1<WP=7>中 文 摘 要 结 果 : 酶 切 鉴 定 及 测 序 结 果 显 示 : phCMV1-6His-hCGβ-C3d3 和phCMV1-6His-hCGβ 构建正确,hCGβ 与 C3d3 连接子为稳定、高度柔性的 Gly.Ser接头。放免检测结果显示,在 CHO 细胞抗性克隆无血清培液中可检测到 hCGβ的表达,其中 phCMV1 表达 hCGβ 的效率是 pcDNA3 载体的 1.6 倍,表达hCGβ-C3d3 融合蛋白的效率是 pcDNA3 的 1.4 倍。 Western blotting 分析显示,phCMV1-6His-hCGβ 的表达产物为 24KD,phCMV1-6His-hCGβ-C3d3 的表达产物有三种,大小分别为 128、96 和 62KD。镍柱结合凝胶过滤层析纯化可获得较高纯度的 hCGβ-C3d3 融合蛋白和 hCGβ 蛋白。无论是 BALB/c 小鼠还是 C57BL/6小鼠,hCGβ-C3d3 融合蛋白诱导产生抗 hCGβ 抗体的出现时间都明显早于单用hCGβ 蛋白和 hCGβ 蛋白加用弗氏佐剂免疫组。BALB/c 小鼠的免疫结果显示,C3d3使hCGβ的免疫原性增强了1995倍,C3d3的佐剂能力是弗氏佐剂的10倍(初次免疫)～32 倍(再次免疫)。C57BL/6 小鼠所显示出的体液免疫反应强度虽不如 BALB/c 小鼠,但 C3d3 也使 hCGβ 的免疫原性增强了 1259 倍, C3d3 的佐剂能力是弗氏佐剂的 10 倍(初次免疫)～20 倍(再次免疫)。细胞因子分析结果显示,hCGβ-C3d3 融合蛋白和 hCGβ+CFA/IFA 免疫鼠 IL-4 和 IL-10 分泌水平均显著高于 hCGβ 免疫组,以 hCGβ-C3d3 融合蛋白免疫组最明显。从 IL-4/ IFNγ比值分析,hCGβ-C3d3 融合蛋白免疫组为 8.0,显示 Th2 型细胞因子明显优势表达,而 hCGβ 免疫组为 1.77,Th2 型优势效应不如 hCGβ-C3d3 融合蛋白。抗血清特性研究结果显示,hCGβ-C3d3 融合蛋白和 hCGβ+CFA/IFA 抗血清均能明显抑制hCG 刺激 MLTC-1 细胞分泌孕酮的作用,两组间无显著性差异。hCGβ 抗血清对hCG 的生物学活性几乎没有影响。从抗血清原液至 1:400 的 hCGβ-C3d3 抗血清稀释液和 1:200 的 hCGβ+CFA/IFA 抗血清稀释液均可显著抑制 hCG 诱导的小鼠子宫增重,hCGβ 抗血清只能在 1:50 的稀释度显示抑制作用。 结论:成功构建了 hCGβ-C3d3 融合蛋白和 hCGβ 蛋白的真核表达质粒phCMV1-6His-hCGβ-C3d3 和 phCMV1-6His-hCGβ。在 CHO 细胞中可成功获得hCGβ-C3d3 融合蛋白和 hCGβ 蛋白的分泌性、稳定表达。phCMV1 真核载体的表达效率高于 pcDNA3。镍柱结合凝胶过滤层析可纯化获得较高纯度的重组蛋白。在个体差异很大的不同品系小鼠,分子佐剂 C3d3 均可大幅增强 hCGβ 避孕疫苗 2<WP=8>中 文 摘 要的免疫原性,使 hCGβ-C3d3 融合蛋白呈现一个 Th2 型体液免疫优势效应。对于hCGβ 抗原而言,C3d3 的佐剂能力强于弗氏佐剂。hCGβ-C3d3 融合蛋白抗血清具有很强的中和 hCG 生物学活性的作用。更多还原

【Abstract】 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<WP=10>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更多还原