發(fā)布時間：2018-08-12 11:14

【摘要】： 目的構建單純皰疹病毒2型(HSV-2)糖蛋白gD、gB、gC和早期表達蛋白ICP 27的多表位復合DNA疫苗和免疫佐劑IL2質粒，以及在復合疫苗質粒基礎上修飾的信號肽質粒與泛素化質粒，并探討其誘導小鼠機體免疫應答的能力。 方法國內HSV-2野生株經PCR鑒定后利用PCR技術從其基因組中擴增出HSV-2 ICP27 377-459、gD146-179、gD223-306、gB529-606、gC247-282、gD1-77六個基因片段，PCR鑒定后按先后順序，采用多基因片段一步酶切連接法獲得HV融合基因片段，經pGEMT載體克隆后定向插入真核表達質粒pcDNA3.1載體中，構建出重組真核表達質粒HV-pcDNA3.1，并對其進行酶切分析及測序鑒定。 PCR擴增gD146-179信號肽片段，進一步酶切連接構建重組質粒gDs-HV-his-pcDNA3.1，并對其進行酶切分析及測序鑒定。 PCR擴增人基因組DNA和HSV-2基因組中泛素全長基因和HSV-2 ICP27 377-459，酶切連接獲得融合基因片段，插入真核表達質粒pcDNA3.1載體中，構建出重組質粒Ub-ICP-pcDNA3.1，并對其進行酶切分析及測序鑒定。 PCR擴增人IL-2信號肽基因和人IL-2尾段基因，取上述兩段PCR擴增產物作為融合PCR的模板，擴增IL-2 cDNA全長基因，連入pcDNA3.1質粒中，構建出重組質粒IL-2-pcDNA3.1，并對其進行酶切分析及測序鑒定。 C57／BL6雌性6周齡小鼠40只，隨機分為8組，每組均5只，根據(jù)免疫的質粒類型和免疫方式的不同，各組依次為pcDNA3.1(空質粒對照)、HV-pcDNA3.1、HV-pcDNA3.1+IL2-pcDNA3.1、gDs-HV-his-pcDNA3.1、gDs-HV-his-pcDNA3.1+IL2-pcDNA3.1、Ub-ICP-pcDNA3.1和HV-pcDNA3.1、HV-pcDNA3.1+IL2-pcDNA3.1。 免疫前先用特異性抗原刺激，前6組小鼠每只用5ug的相應質粒(與IL2共表達的組用5+5ug的量)和1ug的脂質體充分混合，，20min后轉染到同批次C57／BL6雌性鼠脾淋巴細胞中，37℃、5％CO_2培養(yǎng)4h后注射入小鼠皮下；后2組小鼠也用5ug的相應質粒(與IL2共表達的組用5+5ug的量)直接注射到雙側脛骨前肌中。14天后正式免疫，前6組小鼠每只用100ug的相應質粒(與IL2共表達的組用100+100ug的量)和10ug的脂質體充分混合，20min后注入小鼠雙側脛骨前肌中；后2組小鼠也用100ug的相應質粒(與IL2共表達的組用100+100ug的量)直接注入小鼠雙側脛骨前肌中。14天后再加強免疫一次，方法和劑量與正式免疫完全一致。 加強免疫后3周小鼠斷尾取血，分離血清。斷頸處死小鼠取脾，分離淋巴細胞。ELISA檢測小鼠血清HSV-2特異性IgG、IL-2和IFN-γ，MTT法檢測小鼠脾T淋巴細胞特異性增殖，乳酸脫氫酶法檢測殺傷性T淋巴細胞(CTL)功能和流式細胞儀檢測CD4+／CD8+T細胞亞群分類。 結果構建的HV-pcDNA3.1質粒、IL2-pcDNA3.1質粒、Ub-ICP-pcDNA3.1質粒、gDs-HV-his-pcDNA3.1質粒經酶切鑒定并進行DNA測序，測序結果正確，表明已成功構建出HSV-2多表位復合DNA疫苗。 免疫小鼠后實驗結果顯示：gDs-HV-his-pcDNA3.1與IL2-pcDNA3.1聯(lián)合免疫后體液免疫最強，特異性IgG效價達到400倍左右；T細胞表面信號分子CD4的陽性率與空載體pcDNA3.1組相比有顯著差異(P＜0.05)。而HV-pcDNA3.1與IL2-pcDNA3.1聯(lián)合免疫后細胞免疫水平最強，特異性T淋巴細胞的刺激指數(shù)SI達到2.75左右，而空質粒對照組為0.7左右；淋巴細胞殺傷實驗CTL反應的殺傷率(效靶比為50：1時)接近50％，而空質粒對照組為8％左右；T細胞表面信號分子CD8的陽性率與空載體pcDNA3.1組相比也有顯著差異(P＜0.05)，但CD4~+／CD8~+的值無顯著差異(P＞0.05)；血清中細胞因子IL2和IFN-γ含量檢測結果顯示：IL2水平(ng／L)為1421.16±220.98，而空質粒對照組為685.21±104.20；IFN-γ水平(ng／L)為1956.19±219.60，而空質粒對照組為547.71±189.33。 另一組有關不同免疫方式比較的實驗總體上看則說明了脂質體包裹的DNA疫苗比單純的裸DNA注射法能顯著提高免疫活性，若用脂質體包裹起來同時轉染IL2質粒和復合疫苗質粒則更強。 結論 1．成功構建了HSV-2多表位復合DNA疫苗，包括質粒HV-pcDNA3.1、信號肽質粒gDs-HV-his-pcDNA3.1、泛素化質粒Ub-ICP-pcDNA3.1和佐劑質粒IL2-pcDNA3.1。 2．小鼠動物實驗表明，我們構建的疫苗質粒HV-pcDNA3.1和信號肽質粒gDs-HV-his-pcDNA3.1能有效誘導小鼠體液免疫和細胞免疫應答，與佐劑質粒IL2-pcDNA3.1共同免疫效果更強。信號肽質粒gDs-HV-his-pcDNA3.1與佐劑質粒IL2-pcDNA3.1共注射誘導的體液免疫反應最強，而質粒HV-pcDNA3.1與佐劑質粒IL2-pcDNA3.1共注射誘導的細胞免疫反應最強。 3．質粒經脂質體包裹并在免疫前通過淋巴細胞體外轉染小鼠皮下預注射方法比單純裸質粒注射方法能明顯提高體液免疫和細胞免疫反應。 4．泛素化質粒Ub-ICP-pcDNA3.1不能誘導有效的體液免疫和細胞免疫應答。
[Abstract]:Objective To construct a multi-epitope compound DNA vaccine and immune adjuvant IL-2 plasmid of herpes simplex virus-2 (HSV-2) glycoprotein gD, gB, gC and early expression protein ICP-27, as well as a signal peptide plasmid and a ubiquitination plasmid modified on the basis of the composite vaccine plasmid, and to investigate the ability of the plasmid to induce immune response in mice.
Methods Six gene fragments of HSV-2 ICP27 377-459, gD146-179, gD223-306, gB529-606, gC247-282 and gD1-77 were amplified from the genome of HSV-2 wild strain in China by PCR. The fragments of HV fusion gene were cloned by pGEMT vector and inserted directionally. The recombinant eukaryotic expression plasmid HV-pcDNA3.1 was constructed and identified by enzyme digestion and sequencing.
The gD146-179 signal peptide fragment was amplified by PCR, and the recombinant plasmid gDs-HV-his-pcDNA3.1 was constructed by enzyme digestion and ligation.
Full-length ubiquitin gene and HSV-2 ICP27 377-459 in human genome DNA and HSV-2 genome were amplified by PCR. Fusion gene fragments were obtained by enzyme digestion and ligation. The recombinant plasmid Ub-ICP-pcDNA3.1 was constructed by inserting into eukaryotic expression plasmid pcDNA3.1 vector. The recombinant plasmid Ub-ICP-pcDNA3.1 was analyzed by enzyme digestion and sequenced.
The full-length gene of IL-2 cDNA was amplified by PCR. The recombinant plasmid IL-2-pcDNA3.1 was constructed and identified by restriction enzyme digestion and sequencing.
C57/BL6 female mice aged 6 weeks were randomly divided into 8 groups. Each group consistof 5 mice randomly divided into 8 groups. According to the different immunplasmid types and immunimmunmodes, each group was pcDNA3.1 (blankplasmid control), HV-pcDNA3.1, HV-pcDNA3.1, HV-pcDNA3.1+IL2-pcDNA3.1, HV-pcDNA3.1+IL2-pcDNA3.1, HV-pcDNA3.1+IL2-pcDNA3.1, gDs-HV-HV-his-pcDNA3.1, gDs-HV-HV-pcDNA3.1+his-pcDNA3.1, gDDs-HDs-HV-HV-pcDNA3.1+IL2-ppcDNA 3.1.
The mice in the first six groups were stimulated with specific antigens before immunization. Each mouse in the first six groups was adequately mixed with the corresponding plasmids of 5+5 UG (the amount of 5+5 UG in the group co-expressed with IL2) and the liposome of 1 ug. The spleen lymphocytes of the same batch of C57/BL6 female mice were transfected 20 minutes later. The spleen lymphocytes were cultured at 37 C for 4 hours with 5% CO_2 and injected subcutaneously into the mice in the second two groups with the corresponding plasmids of 5 UG (the L2 co-expression group was injected directly into bilateral anterior tibial muscles with the dose of 5+5ug. 14 days later, the mice in the first six groups were immunized with 100 UG of corresponding plasmids (100+100ug of the group co-expressed with IL2) and 10 UG of liposome. After 20 minutes, the mice in the latter two groups were injected into bilateral anterior tibial muscles with 100 UG of corresponding plasmids (with IL2). The expression group was injected directly into the bilateral anterior tibial muscles of mice with the dose of 100 + 100 ug. The method and dose of the immunotherapy were completely consistent with the formal immunization.
Three weeks after the immunization, the mice were killed by neck-cutting and the spleens were taken out for lymphocyte isolation. The specific IgG, IL-2 and IFN-gamma of HSV-2 in serum were detected by ELISA, the specific proliferation of splenic T lymphocyte was detected by MTT, the killer T lymphocyte (CTL) function was detected by lactate dehydrogenase assay and the CD4 + / CD8 + T cell was detected by flow cytometry. Classification of subgroups.
Results The constructed HV-pcDNA3.1 plasmid, IL2-pcDNA3.1 plasmid, Ub-ICP-pcDNA3.1 plasmid and gDs-HV-his-pcDNA3.1 plasmid were identified by enzyme digestion and DNA sequencing. The results showed that the HSV-2 multi-epitope compound DNA vaccine was successfully constructed.
The results showed that the combined immunization of gDs-HV-his-pcDNA3.1 and IL2-pcDNA3.1 had the strongest humoral immunity, and the specific IgG titer was about 400 times. The positive rate of CD4 on T cell surface was significantly different from that of pcDNA3.1 group (P < 0.05). The stimulus index SI of specific T lymphocyte reached about 2.75, while that of blank plasmid control group was about 0.7; the killing rate of CTL reaction in lymphocyte killing experiment (the effective target ratio was 50:1) was close to 50%, while that of blank plasmid control group was about 8%; the positive rate of CD 8 on T cell surface signal molecule was also obvious compared with blank vector pcDNA3.1 group. There was significant difference (P < 0.05), but there was no significant difference (P > 0.05) in CD4 + / CD8 +; the levels of IL 2 and IFN - gamma in serum were 1421.16 + 220.98, 685.21 + 104.20 in blank plasmid control group, 1956.19 + 219.60 in ng / L, 547.71 + 189.33 in empty plasmid control group.
Another group of experiments comparing different immune modes showed that liposome-encapsulated DNA vaccines could significantly improve the immune activity compared with bare DNA vaccines, and the liposome-encapsulated DNA vaccines could transfect IL2 plasmids and composite vaccine plasmids simultaneously.
conclusion
1. HSV-2 multi-epitope DNA vaccine was successfully constructed, including plasmid HV-pcDNA3.1, signal peptide plasmid gDs-HV-his-pcDNA3.1, ubiquitinated plasmid Ub-ICP-pcDNA3.1 and adjuvant plasmid IL2-pcDNA3.1.
2. Animal experiments in mice showed that the Vaccine Plasmid HV-pcDNA3.1 and the signal peptide plasmid gDs-HV-his-pcDNA3.1 could effectively induce humoral and cellular immune responses in mice, and the co-immunization effect was stronger with the adjuvant plasmid IL2-pcDNA3.1. The signal peptide plasmid gDs-HV-his-pcDNA3.1 and the adjuvant plasmid IL2-pcDNA3.1 co-injected to induce humoral immunity. The strongest immune response was induced by co-injection of plasmid HV-pcDNA3.1 and adjuvant plasmid IL2-pcDNA3.1.
3. Subcutaneous pre-injection of plasmid encapsulated in liposome and transfected with lymphocytes in vitro before immunization can significantly improve humoral and cellular immune responses compared with bare plasmid injection.
4. ubiquitin plasmid Ub-ICP-pcDNA3.1 can not induce effective humoral and cellular immune responses.
【學位授予單位】：復旦大學
【學位級別】：博士
【學位授予年份】：2006
【分類號】：R392;R752.1

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