【作者】 李华萍；

【导师】 李大金； 姚晓英；

【作者基本信息】 复旦大学 ， 妇产科学， 2006， 博士

【摘要】 目的从人肝cDNA文库克隆C3基因片段hC3d，将三个拷贝的hC3d与hCGβ基因融合，构建携分子佐剂的hCGβ-hC3d3融合蛋白和hCGβ蛋白的分泌型真核表达质粒，体外获得稳定高效表达细胞株，并获得较高纯度的目的蛋白hCGβ-hC3d3和hCGβ。目的蛋白体外作用于人外周血免疫细胞，以了解融合蛋白hCGβ-hC3d3的免疫原性及免疫效应，探讨分子佐剂hC3d的作用机制，为重组hCGβ-hC3d3融合蛋白疫苗最终用于人类奠定基础。方法以人肝cDNA文库为模板，PCR扩增hC3d基因片段，插入pMD18-T simple载体，构建含三个拷贝hC3d的pMD18-T-hC3d3质粒；以phCMV1-6his-hCGβ为模板扩增带有相应酶切位点的hCGβ基因片段，将其构建入高效真核表达载体pCI-gs得到pCI-gs-signal-6His-hCGβ真核表达质粒。将hCGβ基因片段插入pMD18-T-hC3d3质粒，使hCGB与hC3d3基因融合，再构建入pCI-gs获得pCI-gs-signal-6His-hCGβ-hC3d3真核表达质粒。脂质体法介导重组质粒pCI-gs-signal-6His-hCGβ、pCI-gs-signal-6His-hCGβ-hC3d3转染中国仓鼠卵巢(CHO)细胞，序列浓度MSX筛选抗性细胞克隆；化学发光法检测抗性克隆培养上清hCGβ含量，以选择高效表达克隆。Western blotting鉴定目的蛋白；Raji细胞免疫化学染色法鉴定hCGβ-hC3d3融合蛋白。采用针对6His的镍柱结合凝胶过滤层析纯化表达产物。用L-亮氨酸甲基酯(L-LME)或B细胞磁珠阴性分离试剂盒对人外周血单个核细胞(PBMC)进行细胞分离纯化；实验分四组：B细胞、B+T细胞组、PBMC和Raji细胞组。分别应用1nM、10nM、100nM不同浓度的hCGβ、hCGβ-hC3d3及美洲商陆(PWM)等抗原体外作用于以上细胞10-12d，用ELISA检测培养上清中总免疫球蛋白(Ig)及抗hCGβ抗体的分泌水平；用氚标记的胸腺嘧啶核苷(~3H-TdR)掺入法了解不同抗原刺激后各组细胞增殖情况，而ELISPOT可测定各种抗原刺激后Ig或特异抗体的分泌细胞数量。另外，为探讨分子佐剂hC3d3的作用机制，用100nM的hCGβ、hCGβ-hC3d3或PWM等抗原与B细胞、B+T细胞、PBMC细胞体外作用48h，用ELISA检测培养上清中IL-2浓度；用荧光抗体标记培养细胞，流式细胞仪检测B细胞、T细胞表面协同刺激分子CD80、CD86、CD154的表达情况及Th细胞活化后IL-2Rα链(CD25)的表达水平。结果分子克隆的hC3d基因片段的测序结果与目的基因一致。酶切鉴定及测序结果显示，pMD18-T-hC3d3、pCI-gs-signal-6His-hCGβ、pCI-gs-signal-6His-hCGβ-hC3d3重组质粒构建正确。转染CHO细胞后，经抗性克隆筛选，各挑选出一个高表达细胞株，化学发光法检测其培养上清中hCGβ的含量结果显示，pCI-gs表达效率明显高于pcDNA和phCMV1。Western blotting分析显示，pCI-gs-signal-6His-hCGβ的表达产物为24KDa；而pCI-gs-signal-6His-hCGβ-hC3d3的表达产物有三种，大小分别为136 KDa、100 KDa、64 KDa。镍柱结合分子筛纯化可获得较高纯度的融合蛋白hCGβ-hC3d3和hCGβ。用不同浓度hCGβ、hCGβ-hC3d3及PWM作用于B细胞、B+T细胞、PBMC、Raji细胞10～12d后，~3H-TdR掺入法分析显示：与hCGβ处理组相比，融合蛋白hCGβ-hC3d3能使各组细胞增殖明显升高，且呈浓度依赖性。总Ig水平检测结果表明，100nM hCGβ-hC3d3与前三组细胞共培养上清中总Ig的含量分别为hCG B刺激组的4倍、10倍和10.85倍；且呈浓度依赖性。ELISPOT结果与上清检测结果类似，经与hCGβ-hC3d3共培养后Ig生成细胞较hCGβ组明显增加。用100nM的不同抗原共培养12d后，取培养上清进行ELISA检测，结果表明，经hCGβ及PWM刺激后，上清中均未检测到抗hCGβ特异性抗体；与hCGβ-hC3d3培养后则在B+T细胞、PBMC组可见低水平抗hCGβ抗体。用ELISPOT法分析抗hCGβ特异性抗体生成细胞，结果发现，hCGβ刺激的细胞仅有数个散在的阳性细胞；而经hCGβ-hC3d3刺激后，阳性细胞明显增多(P＜0.05)。流式细胞术分析结果提示：与hCGβ比较，hCGβ-hC3d3蛋白能显著上调B细胞表面CD80、CD86分子的表达，尤其是CD86分子的表达；能明显增强T细胞表面CD154、CD25分子的表达(P＜0.05)。hCGβ-hC3d3蛋白能提高B细胞抗原提呈的能力及T细胞活化后分泌IL-2的能力(P＜0.05)。结论成功克隆了人hC3d基因片段，实现了hC3d3与hCGβ的基因融合；构建了pCI-gs-signal-6His-hCGβ，pCI-signal-6His-hCGβ-hC3d3真核表达质粒；并在CHO细胞中获得hCGβ蛋白、hCGβ-hC3d3融合蛋白的高效分泌性稳定表达。镍柱结合分子筛可获得较高纯度的目的蛋白。hCGβ-hC3d3可通过提高人外周血B细胞及T细胞表面协同刺激分子的表达，促进B细胞活化，改善其抗原提呈能力，加强B细胞T细胞间相互作用，有效活化T辅助细胞；分子佐剂hC3d能增强人外周免疫活性细胞对hCGβ抗原的反应性，显著提高B细胞合成Ig及特异性抗体的能力。更多还原

【Abstract】 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.更多还原