重组人IL-12真核表达载体的表达鉴定及其基因佐剂功能研究

发布时间：2018-01-02 10:45

本文关键词：重组人IL-12真核表达载体的表达鉴定及其基因佐剂功能研究　出处：《青岛大学》2006年硕士论文　论文类型：学位论文

【摘要】：目的对重组人IL-12真核载体(pcDNA6-p70)进行表达鉴定并对其表达产物进行体外生物学活性分析;探讨pcDNA6-p70在实验小鼠体内对核酸疫苗的基因佐剂功能。方法 pcDNA6-p70转染HEK293细胞,ELISA检测培养上清液中重组IL-12的表达。重组IL-12作用于献血员外周血单个核细胞(PBMC),分别应用细胞因子流式细胞(CFC)计数、Tetramer染色、NK杀伤实验、非特异性及特异性淋巴细胞增生反应等方法体外分析重组IL-12的生物学活性。pcDNA6-p70与人巨细胞病毒(HCMV)pp65基因重组腺病毒(adeno-pp65)共免疫小鼠(昆明小鼠及Balb/C)1次/2周,共8周。分别应用CFC,CTL杀伤实验,特异性淋巴细胞增生反应及受试小鼠血清及脾淋巴细胞的IFN-γ产生等评价pcDNA6-p70对核酸疫苗的基因佐剂功能。并对pcDNA6-p70在小鼠体内应用的安全性进行了初步的探讨。结果 pcDNA6-p70转染HEK293细胞后,最高表达量达到517gp/ml(5×10~4细胞)。献血员PBMC经重组IL-12作用后,其对PHA及HCMV刺激的CD4/IFN-γ及CD8/IFN-γ双标记阳性细胞的百分率(PHA:5.0、4.55;HCMV:7.78、5.82)高于对照组(PHA:0.3、1.05;HCMV:3.76、2.46);N9V特异性CD8+细胞百分率(1.19%)高于对照组(0.98%);NK杀伤活性及非特异性(PHA)、特异性(HCMV)淋巴细胞增生反应也显著增高,表明重组IL-12具有良好的生物学活性。pcDNA6-p70与adeno-pp65dx鼠体内共免疫结果显示,共免疫组NK杀伤活性(52.2%;单独免疫组46.4%;对照组44.9%)、CTL伤活性(74.9%;单独免疫组52.2%;对照组53.0%)、特异性淋巴细胞增生反应(1.204;单独免疫组1.113;对照组1.110)、特异性CD4/IFN-γ(5.17%;单独免疫组2.92%;对照组2.05%)、CD8/IFN-γ(4.24%;单独免疫组2.02%;对照组1.95%)双标记阳性细胞的百分率及特异性(150.5pg/ml;单独免疫组143.5pg/ml;对照组139.0pg/ml)、非特异性IFN-γ(146.7pg/ml;单独免疫组138.2pg/ml;对照组140.8pe/ml)释放均较adeno-pp65单独免疫组及生理盐水对照组增高。安全性评价结果表明,pcDNA6-p70实验组体温、体重及一般情况与对照组无显著差异。结论 pcDNA6-p70在HEHEK293细胞中可表达较高水平的重组IL-12;表达的重组IL-12在体外表现预期的生物学活性;pcDNA6-p70与核酸疫苗共免疫实验小鼠可发挥较好的基因佐剂功能。初步安全性评价显示,其在实验小鼠体内应用具有较高的
[Abstract]:Objective to identify recombinant human IL-12 eukaryotic expression vector (pcDNA6-p70) and analyze its biological activity in vitro, and to explore the function of pcDNA6-p70 in gene adjuvant of nucleic acid vaccine in mice.
Methods pcDNA6-p70 was transfected into HEK293 cells, ELISA culture medium was detected in the supernatant of recombinant IL-12. The recombinant IL-12 on blood peripheral blood mononuclear cells (PBMC), respectively by cytokine flow cytometry (CFC) count, Tetramer staining, NK cytotoxicity assay of recombinant IL-12 in vitro non-specific and specific lymphocyte proliferation reaction method the biological activity of.PcDNA6-p70 and human cytomegalovirus (HCMV) pp65 Gene Recombinant Adenovirus (adeno-pp65) were immunized mice (Kunming mice and Balb/C) 1 /2 weeks, 8 weeks respectively. The application of CFC CTL, killing experiment, specific lymphocyte proliferative response and produced by IFN- gamma test mice serum and spleen lymphocytes the evaluation of pcDNA6-p70 nucleic acid vaccine gene adjuvant function. And the pcDNA6-p70 is discussed in the safety of mice.
Results pcDNA6-p70 after transfection into HEK293 cells, the highest expression level reached 517gp/ml (5 x 10~4 cells). Blood donors by recombinant PBMC after IL-12, CD4/IFN- and CD8/IFN- of the gamma gamma PHA and HCMV stimulated double labeled positive cells percentage (PHA:5.0,4.55; HCMV:7.78,5.82) is higher than that of the control group (PHA:0.3,1.05; HCMV:3.76,2.46); specific N9V the percentage of CD8+ cells (1.19%) was higher than the control group (0.98%); and nonspecific cytotoxic activity of NK (PHA), specificity (HCMV) lymphocyte proliferative response was significantly increased, indicating that the recombinant IL-12 has good biological activity of.PcDNA6-p70 and adeno-pp65dx mice were immunized results showed that CO immunization group the killing activity of NK alone (52.2%; immune group 46.4%; control group 44.9%), CTL (74.9%; immune injury activity alone group 52.2%; control group 53%), specific lymphocyte proliferation responses (1.204; immunized 1.113; control group 1.110), specific CD4/IF N- gamma (5.17%; immunized 2.92%; control group 2.05%), CD8/IFN- (4.24%; gamma immunized 2.02%; control group 1.95%) double labeled positive cells percentage and specificity (150.5pg/ml; immunized group 143.5pg/ml; control group 139.0pg/ml), nonspecific IFN- gamma (146.7pg/ml; immunized 138.2pg/ml; control group 140.8pe/ml) release were lower than adeno-pp65 immunized group and saline control group increased. Safety evaluation results show that the pcDNA6-p70 experimental group, body temperature, body weight and general condition had no significant difference with control group.
Conclusion pcDNA6-p70 high level expression of recombinant IL-12 in HEHEK293 cells; recombinant IL-12 expression expected biological activity in vitro; pcDNA6-p70 mice were immunized with DNA vaccine can play genetic adjuvant better. Preliminary safety evaluation, has higher application in the in vivo

【学位授予单位】：青岛大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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