本文關(guān)鍵詞：重組人IL-12真核表達(dá)載體的表達(dá)鑒定及其基因佐劑功能研究　出處：《青島大學(xué)》2006年碩士論文　論文類型：學(xué)位論文

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【摘要】：目的 對(duì)重組人IL-12真核載體(pcDNA6-p70)進(jìn)行表達(dá)鑒定并對(duì)其表達(dá)產(chǎn)物進(jìn)行體外生物學(xué)活性分析;探討pcDNA6-p70在實(shí)驗(yàn)小鼠體內(nèi)對(duì)核酸疫苗的基因佐劑功能。 方法 pcDNA6-p70轉(zhuǎn)染HEK293細(xì)胞,ELISA檢測(cè)培養(yǎng)上清液中重組IL-12的表達(dá)。重組IL-12作用于獻(xiàn)血員外周血單個(gè)核細(xì)胞(PBMC),分別應(yīng)用細(xì)胞因子流式細(xì)胞(CFC)計(jì)數(shù)、Tetramer染色、NK殺傷實(shí)驗(yàn)、非特異性及特異性淋巴細(xì)胞增生反應(yīng)等方法體外分析重組IL-12的生物學(xué)活性。pcDNA6-p70與人巨細(xì)胞病毒(HCMV)pp65基因重組腺病毒(adeno-pp65)共免疫小鼠(昆明小鼠及Balb/C)1次/2周,共8周。分別應(yīng)用CFC,CTL殺傷實(shí)驗(yàn),特異性淋巴細(xì)胞增生反應(yīng)及受試小鼠血清及脾淋巴細(xì)胞的IFN-γ產(chǎn)生等評(píng)價(jià)pcDNA6-p70對(duì)核酸疫苗的基因佐劑功能。并對(duì)pcDNA6-p70在小鼠體內(nèi)應(yīng)用的安全性進(jìn)行了初步的探討。 結(jié)果 pcDNA6-p70轉(zhuǎn)染HEK293細(xì)胞后,最高表達(dá)量達(dá)到517gp/ml(5×10~4細(xì)胞)。獻(xiàn)血員PBMC經(jīng)重組IL-12作用后,其對(duì)PHA及HCMV刺激的CD4/IFN-γ及CD8/IFN-γ雙標(biāo)記陽性細(xì)胞的百分率(PHA:5.0、4.55;HCMV:7.78、5.82)高于對(duì)照組(PHA:0.3、1.05;HCMV:3.76、2.46);N9V特異性CD8+細(xì)胞百分率(1.19%)高于對(duì)照組(0.98%);NK殺傷活性及非特異性(PHA)、特異性(HCMV)淋巴細(xì)胞增生反應(yīng)也顯著增高,表明重組IL-12具有良好的生物學(xué)活性。pcDNA6-p70與adeno-pp65dx鼠體內(nèi)共免疫結(jié)果顯示,共免疫組NK殺傷活性(52.2%;單獨(dú)免疫組46.4%;對(duì)照組44.9%)、CTL傷活性(74.9%;單獨(dú)免疫組52.2%;對(duì)照組53.0%)、特異性淋巴細(xì)胞增生反應(yīng)(1.204;單獨(dú)免疫組1.113;對(duì)照組1.110)、特異性CD4/IFN-γ(5.17%;單獨(dú)免疫組2.92%;對(duì)照組2.05%)、CD8/IFN-γ(4.24%;單獨(dú)免疫組2.02%;對(duì)照組1.95%)雙標(biāo)記陽性細(xì)胞的百分率及特異性(150.5pg/ml;單獨(dú)免疫組143.5pg/ml;對(duì)照組139.0pg/ml)、非特異性IFN-γ(146.7pg/ml;單獨(dú)免疫組138.2pg/ml;對(duì)照組140.8pe/ml)釋放均較adeno-pp65單獨(dú)免疫組及生理鹽水對(duì)照組增高。安全性評(píng)價(jià)結(jié)果表明,pcDNA6-p70實(shí)驗(yàn)組體溫、體重及一般情況與對(duì)照組無顯著差異。 結(jié)論 pcDNA6-p70在HEHEK293細(xì)胞中可表達(dá)較高水平的重組IL-12;表達(dá)的重組IL-12在體外表現(xiàn)預(yù)期的生物學(xué)活性;pcDNA6-p70與核酸疫苗共免疫實(shí)驗(yàn)小鼠可發(fā)揮較好的基因佐劑功能。初步安全性評(píng)價(jià)顯示,其在實(shí)驗(yàn)小鼠體內(nèi)應(yīng)用具有較高的
[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

【學(xué)位授予單位】：青島大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2006
【分類號(hào)】：R392

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