乙型脑炎DNA疫苗与ICAM-1编码基因重组子联合免疫BALB/c鼠所致细胞免疫功能的研究
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摘要
目的
分别构建含乙型脑炎病毒(Japanese encephalitis virus, JEV) prME蛋白与BALB/c鼠细胞间粘附分子(Intracellular adhesion molecule-1, ICAM-1)编码基因以及单纯ICAM-1编码基因重组质粒,比较ICAM-1编码基因不同构建方式对乙脑DNA疫苗诱导细胞免疫的免疫佐剂效应并探究ICAM-1编码基因免疫增强效应机制。
材料和方法一、实验材料
1、细胞与动物
中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞与鼠肥大细胞瘤(P815)细胞分别购自中国科学院上海生命科学研究院上海细胞库,雌性、4周龄BALB/c鼠购自中国医学科学院实验动物研究所。
2、质粒与菌株
含JEV prME蛋白基因的重组质粒被命名为pJME,由本实验室构建；原核表达载体pMD19-T simple和真核表达载体pcDNA3.1(+)分别购自Takara宝生物工程(大连)公司和美国Invitrogen公司。大肠杆菌JM109与DH5α购自Takara公司。
3、其他试剂
质粒抽提试剂盒购自QIAGEN公司,Lipofectamine2000购自Invitrogen公司,山羊抗鼠ICAM-1购自Santa公司,异硫氰酸荧光素(FITC)以及辣根酶标记的兔抗山羊IgG均购自北京中杉公司,乳酸脱氢酶(LDH)测定试剂盒购自美国Promega公司。荧光标记的抗体(CD3-PerCP, CD4-APC, CD8-PE, CD54-PE, CD11c-APC, MHCⅡ-FITC, CD80-PE, CD86-FITC)均购自美国BD PharMingen公司。荧光染料羧基荧光素二乙酸盐琥珀酰亚胺脂(CFSE)、异硫氰酸荧光素标记的葡聚糖(FITC-Dextran)均购自美国Molecular Probes公司。Mouse T Cell Lympholyte-Pure购自加拿大Cedarlane公司。丝裂霉素C购自美国Sigma公司。rmIL-2购自美国Peprotech公司。新霉素类似物(G418)购自GiBCO公司,限制性内切酶EcoRI、BamHI以及NotI等均购自Takara公司。IL-4、IL-10、IL-12、IFN-γELISA试剂盒均购自上海森雄科技实业有限公司。ECL发光试剂盒购自美国Pierce公司。
二、实验方法
1、pICAM-1、pJME/ICAM-1的构建和鉴定
从BALB/c鼠脾脏细胞中提取总RNA,参照(Genbank X52264.1),以总RNA为模板,采用套式-RT-PCR法扩增ICAM-1CDS区域序列(1614bp),将后者克隆到pcDNA3.1(+) (EcoRI/NotI),构建重组质粒pICAM-1。限制性内切酶BamHI/EcoRI酶切pJME,获取JEV prME基因(2001bps)并克隆到pICAM-1 BamHI/EcoRI之间,构建融合pJME/ICAM-1.后者通过转化大肠杆菌DH5a、提取、电泳、测序以及酶切等方法进行鉴定。
脂质体法将pJME/ICAM-1转染至CHO细胞,于转染后72h,经G418(800μg/mL)进行细胞筛选7d,再经G418(400μg／mL)持续筛选4周并获得CHO细胞阳性克隆,采用免疫荧光和免疫印迹法检测ICAM-1以及融合蛋白prME/ICAM-1的表达。
2、动物和免疫程序
每组8只BALB/c鼠(购自中国医学科学院实验动物研究所)共5组。分别将溶于100μL灭菌PBS的不同免疫原鼠后肢肌内注射,包括：pJME、pJME/ICAM-1、pJME+pICAM-1、pcDNA3.1(+)(阴性对照)各100μg以及腹腔注射JE灭活疫苗组(阳性对照)。JE灭活疫苗(JEV北京株,辽宁省疾病控制中心惠赠)100μL／次(相当于1／5成人剂量),共免疫3次,间隔2周。
3、小鼠脾脏T淋巴细胞和树突状细胞的分离
脱颈法处死小鼠,无菌打开腹腔,取出脾脏放入冷PBS中,将其捻碎并滤过100目无菌钢网,收集脾细胞悬液,按照产品说明书操作制备脾脏T淋巴细胞；另取部分上述脾细胞悬液,1300 r/min洗涤5 min×3次；用完全培养基20 mL重悬细胞于250 mL培养瓶(Falcon公司)中,37℃,5%CO2培养箱内孵育2 h；用完全培养基20 mL剧烈洗摇培养瓶,并吸弃悬液,共4次；加入完全培养基20 mL,37℃,5%C02培养箱内孵育过夜；收集悬浮细胞。
4、脾脏树突状细胞表型和功能
取制备脾脏DC细胞悬液,流式细胞仪检测DC表面分子CD54、CD11c、MHCⅡ、CD80、CD86、DC摄取FITC-Dextran能力以及DC刺激T淋巴细胞增殖能力。
5、T淋巴细胞亚群与CTL功能检测
取制备脾脏T淋巴细胞悬液,流式细胞仪分析鼠脾细胞中T淋巴细胞亚群特点,乳酸脱氢酶释放法检测CTL活性。
6、ELISA法检测T细胞、DC分泌细胞因子水平
操作步骤按产品说明书进行,显色后即刻用WellscanMK3酶标仪(Lab-systems公司)检测492nm波长的吸光度A值,根据标准曲线计算IL-12、IFN-γ、IL-4、IL-10的含量。
结果
1、pICAM-1、pJME/ICAM-1的构建和鉴定
测序分析显示：JEV prM、E蛋白和ICAM-1编码基因与发表的序列相符。pICAM-1经EcoR I/Not I酶切释出插入子,与ICAM-1基因(1614bp)大小相一致,pJME/ICAM-1经BamHI/EcoRI酶切释出插入子与pJME经相同酶切释出插入子(2001bps)的基因片段大小相一致,pJME/ICAM-1经BamHI/NotI酶切释出的插入子约为3660bp,为鼠ICAM-1编码基因与JEV prME蛋白编码基因之和。
免疫荧光结果显示：pICAM-1、pJME/ICAM-1转染的CHO细胞可见较显著绿色荧光标记,主要分布在胞质,也可见于胞膜。单纯载体转染的CHO细胞及未经质粒转染的正常CHO细胞中未见特异性绿色荧光标记。转染pJME/ICAM-1的CHO细胞的Western blot分析显示,在分子量约为130×103区域处可检测到一蛋白条带,与prME和ICAM-1的分子量之和相一致,转染pICAM-1的CHO细胞的Western blot分析显示,在分子量约为60×103区域处可检测到一蛋白条带,而转染pcDNA3.1(+)的CHO细胞未见相应的条带。
2、pICAM-1、pJME/ICAM-1对小鼠脾脏DC表型和功能的影响
各组DC表面表达高水平MHCⅡ, pJME/ICAM-1组、pJME+pICAM-1组、pJME组高于JE灭活疫苗组及空载体免疫组(P<0.05),三组间比较差异无统计学意义(P>0.05)；pJME/ICAM-1组DC表面CD80表达水平最高,高于pJME组、JE灭活疫苗组及空载体免疫组(均P<0.05), pJME/ICAM-1组与pJME+pICAM-1组比较,差异无统计学意义(P>0.05); pJME组与JE灭活疫苗组DC表面CD86比较差异无统计学意义(P>0.05),其余各组间比较差异均有统计学意义(均P<0.05); pJME/ICAM-1组、pJME+pICAM-1组DC表面表达高水平ICAM-1,高于pJME组,JE灭活疫苗组及空载体免疫组(均P<0.05),两组间比较差异有统计学意义(P<0.05), pJME组,JE灭活疫苗组DC表面ICAM-1表达水平高于空载体免疫组(P<0.05),两组间比较差异无统计学意义(P>0.05)。
pJME/ICAM-1组与pJME+pICAM-1组内吞能力较强,显著高于其它各组(P<0.05)。pJME/ICAM-1组与pJME+pICAM-1组比较差异无统计学意义(P>0.05)。
比较不同免疫原对细胞分裂的作用,以pJME/ICAM-1组最强,(73.69±7.32)%CD4+T细胞发生分裂,(45.40±2.57)%CD8+T细胞发生分裂,显著高于对照组(P<0.05); pJME+pICAM-1组次之,(53.48±5.23)%CD4+T细胞发生分裂,(35.85±4.46)%CD8+T细胞发生分裂,显著高于pJME组、JE灭活疫苗组及空载体免疫组(均P<0.05)。
3、细胞免疫反应的检测
pJME/ICAM-1组CD3+CD4+T淋巴细胞比例为(36.1±6.45)%,明显高于其它组(P<0.05); pcDNA3.1(+)组CD3+CD4+T淋巴细胞比例为(20.87±4.45)%,低于其它组(P<0.05); pJME+pICAM-1组CD3+CD4+T淋巴细胞比例为(31.23±6.55)%,高于pJME组(25.26±9.72)%及JE灭活疫苗组(25.35±3.82)%(P<0.05)。各免疫组间CD3+CD8+T淋巴细胞比例无显著性差异(P>0.05)。
各组免疫鼠CTL活性由高至低依次为：pJME/ICAM-1组(56.38±5.69)%、pJME+pICAM-1组(50.37±6.33)%、pJME组(35.25±4.78)%、JE灭活疫苗组(26.72±3.78)%、pcDNA3.1(+)组(12.34±7.73)%,各组间比较,差异有统计学意义(均P<0.05)。
4、小鼠脾T淋巴细胞和DC分泌细胞因子水平的测定
pJME/ICAM-1组与pJME+pICAM-1组T淋巴细胞和DC分泌IL-12、IFN-γ水平较pJME组、JE灭活疫苗组及空载体免疫组明显升高(均P<0.05),两组间比较,前者T淋巴细胞和DC分泌IL-12、IFN-γ水平显著升高(P<0.05)。各组间T淋巴细胞IL-4分泌水平比较差异无统计学意义(均P>0.05),JE灭活疫苗组T淋巴细胞分泌IL-10的水平高于其它实验组(均P<0.05)；JE灭活疫苗组DC分泌IL-10、IL-4的水平高于其它实验组(均P<0.05),其余各组间比较差异无统计学意义(均P>0.05)。
结论
1、成功构建了融合表达重组质粒pJME/ICAM-1以及单质粒pICAM-1,并能在真核细胞内有效表达目的蛋白；
2、ICAM-1编码基因能够促进T细胞分泌细胞因子,促使CD4+T细胞向Thl细胞分化；
3、ICAM-1编码基因能够增强T细胞对靶细胞的细胞毒效应；
4、树突状细胞能够通过ICAM-1编码基因调节Th1/Th2免疫平衡；
5、ICAM-1编码基因能够促进JE DNA疫苗脾脏DC的成熟；
6、ICAM-1编码基因能够提供独立或放大B7分子的协同刺激信号；
7、相对于联合免疫组,融合质粒免疫组诱导更强的细胞免疫反应。
Objective
We constructed fusion plasmid named pJME/ICAM-1 with Japanese encephalitis virus (Japanese encephalitis virus, JEV) prME protein and BALB/c mouse intercellular adhesion molecule(intracellular adhesion molecule-1, ICAM-1) encoding gene, and pure plasmid named pICAM-1 encoding ICAM-1. To compare the adjuvant effect of ICAM-1 encoding gene in different ways on cell-mediated immunity induced by JE DNA vaccine and explore ICAM-1 coding gene immune-enhancing effect mechanism.
Materials and methods
Materials
1、Cells and animals
Chinese hamster ovary (CHO) cells, P815 cells were purchased from Shanghai Institutes for Biological Sciences, China. CHO cells were used for the transfection experiment and P815 cells were used as target cells; Female,4-week-old BALB/c mice were obtained from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, and maintained in sterile cages under specific pathogen-free conditions. The mice were later used directly for the experiment on DNA immunization.
2、Plasmids and strains
The recombinant plsmid named pJME encoding JEV prME was provided in our laboratory; Prokaryotic expression vector pMD19-T simple and Eukaryotic expression vector pcDNA3.1(+) were purchased separately from Takara Biotechnology (Dalian) Company and the U.S. Invitrogen corporation for plasmid sequencing and construction; Escherichia coli JM109 and DH5a were purchased from Takara company and used for expansion of recombinant plasmids.
3、Main reagents
Plasmid Abstraction Kits were purchased from QIAGEN company; Lipofectamine2000 was purchased from Invitrogen company and was used for transfection;Goat-Anti-Mouse ICAM-1 were purchased from Santa company, and Fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP) labeled rabbit anti-goat IgG were purchased from Beijing Zhong Shan company, and they were used for immunofluorescence and western-blot analysis; The CytoTox96 Non-Radioactive Cytotoxicity assay was purchased from Promega company and used to detect mouse splenocyte CTL activites; Anti-CD3-PerCP, CD4-APC, CD8-PE, CD54-PE, CDllc-APC, MHCⅡ-FITC, CD80-PE, CD86-FITC monoclonal antibody were purchased from BD company and used to detect phenotype of spleen dendritic cells(DC); Anti-CD4-APC, CD8-PE monoclonal antibody were purchased from BD company and used to detect spleen T-lymphocytes subsets; Carboxyfluorescein diacetate, succinimidyl ester(CFSE) and FITC-dextran were purchased from Molecular Probes company and respectively used to detect mixed lymphocyte reaction(MLR) and phagocytic function of DC; Lympholyte-M was purchased from Canada Cedarlane company. Mitomycin C was purchased from Sigma company. IL-2 was purchased from Peprotech company. G418 was purchased from GiBCO company and used for screening of resistant clone; Restriction endonuclease EcoRI、BamHI and NotI were all purchased from Takara company, which were used construction and identification of recombinant plasmid. IL-4, IL-10, IL-12, IFN-y ELISA kit were purchased from the Shanghai SenXiong Technology company. ECL luminescence kit purchased from the
United States Pierce company.
Methods
1、Construction and identification of pICAM-1、pJME/ICAM-1
The coding sequence of murine ICAM-1 (1614 bp) was amplified by nested RT-PCR from total RNA isolated from stimulated BALB/c murine splenocytes.A 1659-bp fragment of murine ICAM-1and Gly-linker was digested with restriction enzymes Eco R I and Not I from pMD19-T simple-ICAM-1 and subcloned into the pcDNA3.1(+) vector at the EcoRI/Not I site. The construct containing the murine ICAM-1 gene was designated pICAM-1. To construct a DNA vaccine encoding fusion protein of prME and ICAM-1 (pJME/ICAM-1), a fragment of 2,001 bp of JEV prME isolated from pJME by BamH I and EcoR I digestion was inserted into the BamH I/EcoR I site of p ICAM-1.
pICAM-1 and pJME/ICAM-1 transforming competent Escherichia coli DH5αwere amplified and extracted, and then verfied by restriction enzyme digestion and DNA sequencing.
Plasmid pICAM-1 and pJME/ICAM-1 was transfected into CHO cells by Lipofectamine TM2000 according to the manufacturer's instruction. After 72h of transfection, the cells were selected with G418 (800μg/mL) for one week and then cultured with G418 (400μg/mL) for four weeks for establishment of stable transfectants. CHO cells transfected with an empty pcDNA3.1(+) vector and normal CHO were used as a control, stable transfectants were detected for expression of ICAM-1 and fusion protein prME/ICAM-1 by immunofluorescence and Western blot analysis.
2、Animals and immunization procedure
We used 5 groups of 4-week-old female BALB/c mice (n=8 per group) which were as follows:pJME, pJME/ICAM-1, pICAM-1. For the i.m. immunization, mice were injected with 100μg pJME/GM-CSF,100μg pJME with 100μg of plasmid pICAM-1 into the quadriceps muscle mass of the left hind leg. All the mice received 2 booster doses in the same muscle (containing the same amount of plasmid as in the primary dose) 3 and 5 weeks after the primary injection. For the positive control group, each mouse was immunized with inactivated vaccine, a formalin-inactivated mouse brain-derived JEV vaccine (Beijing-1 strain) obtained from Liaoning Province Center of Disease Control and Prevention, and each mouse of the inactivated vaccine group was given an injection of 100μl (1/5 of a recommended adult dose) of inactivated vaccine and boosted with the same dose 3 and 5 weeks after the first immunization. The pcDNA3.1(+) immunized group was used as the negative control. The volume of vaccine solution injected into each thigh was adjusted to 100μl per mouse with PBS.
3、Mouse spleen T lymphocytes and dendritic cells Separation
Mouse spleen cell suspension were enriched according to the published methods. Briefly, mouse spleens were disrupted and cut up into small pieces, and passed through a fine screen mesh. A clean suspension was adjusted the cell concentration to a maximum of 2 x 107 nucleated cells per mL. Preparation of splenic T lymphocytes were performed in strict accordance with the instructions within the reagent kit. Another part of the spleen cell suspension were centrifuged at 1300 rpm for 5 min, resuspended in RPMI 1640 medium supplemented with 10% heat-activated fetal calf serum,2 mmol/1 Lglutamine,1 mmol/1 pyruvate,50 mmol/1 mercaptoethanol,100 U/mL penicillin, and 100 mg/mL streptomycin, and then incubated for 2 h at 37℃in a 5% CO2 atmosphere in plastic cell cultures plates. Culture plates were then washed vigorously four times with supplemented RPMI 1640 medium, and the nonadherent cells were discarded. The residual adherent cells were maintained in the culture medium and incubated overnight at 37℃in a 5% CO2 atmosphere. After incubation, DCs(which exhibit adherence capacity in the first hours of culture) become nonadherent and float in the medium. The DCs were collected and immediately used in our assays.
4、Measurement of phenotype and function of spleen DC
Spleen DC were isolated and Flow cytometry detected DC surface molecules CD54, CD11c, MHCⅡ, CD80, CD86, DC uptake of FITC-Dextran capability and capacity of DC to stimulate T lymphocyte proliferation.
5、Spleen DC T-lymphocyte subsets and CTL function detection
Single-cell spleen suspensions were prepared, from which erythrocytes were removed. T-lymphocyte subsets of mouse spleen cells were assayed with flow cytometry technique, and CTL activity of mouse spleen cells was detected with lactic dehydrogenase release test method.
6、Dectection of cytokines secreted by T cells and DC
Steps were carried out by product specification. WellscanMK3 microplate reader (Lab-syste ms Dragon Company) detected immediately 492nm wavelength absorbance A value after color and IL-12, IFN-γ, IL-4, IL-10 levels were calculated according to the standard curve.
Results
1、Construction and identification of pICAM-1 and pJME/ICAM-1
Upon restriction enzyme digestion, pICAM-1 was digested with EcoR I/Not I into 1 fragment, as predicted, at 1,614bp in size, and confirmed that the DNA fragment released from the recombinant plasmid corresponded to the ICAM-1 coding sequence from the published murine ICAM-1 gene sequence. Meanwhile, the pJME/ICAM-1 was cut with BamH I/ EcoRI and Bam HI/Not I into 2 fragments as predicted at 2,001 and 3,660 bp in size, and confirmed that 2,001 bp was the same as that of the JEV prME gene and 3,660 bp was the sum of the murine ICAM-1 gene and the JEV prME gene. With sequence analysis using the T7 promoter primer located in pcDNA3.1(+), it was indicated that there was a normal junction of BamH I site between vectors and the prME gene of JEV, and EcoR I site between vectors and the murine ICAM-1 gene.
Immunofluorescence result showed that the green fluorescence mark was mainly distributed obviously in endochylema of CHO cells transfected with pICAM-1 and pJME/ICAM-1, and not much in membrane. There was no specific green fluorescence in CHO cells untransfected and transfected with pcDNA3.1(+).
In Western blots, the specific ICAM-1 protein and fusion protein prME/ICAM-1 were clearly visible in CHO cells transfected with both pICAM-1 and pJME/ICAM-1, but almost undetectable specificity protein in cells transfected with pcDNA3.1(+). Anti-murine ICAM-1 reactive protein bands of about 60 and 130 kDa were respectively in correspondence with ICAM-1 protein expressed by pICAM-1 and prME/ICAM-1 fusion protein expressed by pJME/ICAM-1, and there was no a corresponding protein band by Western blot analysis of CHO transfected with pcDNA3.1(+).
2、The impact of pICAM-1 and pJME/ICAM-1 on mouse spleen DC phenotype and function
DC of each group express high levels MHCⅡ, pJME/ICAM-1, pJME+ pICAM-1 and pJME group was higher than JE inactivated vaccine and empty vector vaccinated groups (P<0.05),there were no statistically significant difference among the three groups (P>0.05). DC surface CD80 expression in spleen from the pJME/ICAM-1 vaccinated group was the highest in all groups, although there was no significant difference between pJME/ICAM-land pJME+ pICAM-1 group (P>0.05). There was significant difference in the expression of DC surface CD86 between 2 random groups except that there was no significant difference between JE inactivated vaccine and pJME vaccinated groups. DC surface ICAM-1 expression in spleen from the pJME/ICAM-1 and pJME+pICAM-1 vaccinated group was higher than that in the other groups, and there was significant difference between the two groups (P<0.05); DC surface ICAM-1 expression in spleen from the pJME and JE inactivated vaccine vaccinated group was higher than that in the pcDNA3.1(+) vaccinated group, and there was no significant difference between the two groups(P>0.05).
Phagocytic capacity of dendritic cells in the pJME/ICAM-1 and pJME+pICAM-1 vaccinated groups was obviously higher than those in other groups(P<0.05), and there was no significant difference between the two groups (P>0.05).
In order to further analyze the antigen-presenting function of mouse spleen dendritic cell, we determined the stimulating ability of dendritic cells to T lymphocytes in spleens of DNA-immunized mice by flow cytometry. The percentage of CD4+T and CD8+T cells division in spleen cells was respectively (73.69±7.32)% and (45.40±2.57)% in the pJME/ICAM-1 vaccinated group, which significantly increased the division capacity of CD4+T cell compared with other groups (P<0.05). The percentage of CD4+T and CD8+T cells division in spleen cells was respectively (53.48 +5.23)% and (35.85±4.46)% in the pJME+pICAM-1 vaccinated group, obviously higher than those in the pJME, JE inactivated vaccine and pcDNA3.1(+) vaccinated groups (P< 0.05).
3、Dectection of cellular immune responses
The adjuvant effects of the ICAM-1 DNA on the generation of CD4+T cells or CD8+T cells from spleen were compared. The percentage of CD4+T cells in spleen cells was (36.1±6.45)% in the pJME/ICAM-1 vaccinated group, which significantly increased the relative frequency of CD4+T subpopulations compared with other groups (P<0.05). The percentage of CD4+T cells in the pcDNA3.1(+) vaccinated group was (20.87±4.45)%, and lower than in all other groups(P<0.05). The percentage of CD4+T cells in the pJME+pICAM-1 vaccinated group was (31.23±6.55)%, and higher than pJME and JE inactivated vaccine group(P<0.05). There was no significant difference in the levels of CD8+T cells between 2 random groups(P>0.05).
Mice immunized with 100μg pJME/ICAM-1 had higher CTL activity than those immunized with the same amount of other naked DNA(P<0.05). The CTL activities of the spleens of BALB/c mice immunized 3 times with indicated immunogens were as follows:pJME/ICAM-1 (56.38±5.69)%; pJME+pICAM-1 (50.37±6.33)%; pJME (35.25±4.78)%; JE inactivated vaccine (26.72±3.78)%, and pcDNA3.1(+) (12.34±7.73)%. There was significant difference in CTL activity between 2 random groups(P<0.05).
4、Dectection of cytokines secreted by T cells and DC
Secretion of T cells and DC in the pJME/ICAM-1 and pJME+pICAM-1 vaccinated group was obviously higher than those in the pJME, JE inactivated vaccine and pcDNA3.1(+) vaccinated groups(P<0.05), pJME/ICAM-1 group acquired higher levels of IL-12, IFN-γthan pJME+pICAM-1 vaccinated group did(P<0.05). There was no significant difference in the levels of IL-4 secreted by T lymphocytes between 2 random groups(P>0.05). JE. inactivated vaccine vaccinated group obtained higher levels of IL-10 secreted by T lymphocytes compare with other groups(P<0.05). JE inactivated vaccine vaccinated group showed a great increase in levels of IL-10 and IL-4 secreted by DC compared with other groups(P<0.05), and there was no significant difference between any other groups(P>0.05).
Conclusions
1、The constructed recombinant pJME/ICAM-1 and pICAM-1 were confirmed by restrict enzyme digestion and DNA sequencing, and could express effectively the target protein in eukaryotic cells;
2、ICAM-1 coding gene can promote T-cell cytokine secretion, and induced CD4+T cells to Thl cell differentiation;
3、ICAM-1 coding gene can enhance the cytotoxic effects of T cells to target cells;
4、Dendritic cells can regulate Thl/Th2 immune balance through ICAM-1 coding gene;
5、ICAM-1 coding gene can promote the maturation of spleen DC induced by JE DNA vaccine;
6、ICAM-1 coding gene can amplify costimulatory signals that be independent of B7 molecules;
7、The fusion plasmid immunization group induced a stronger cellular immune response relative to the combined immune group.
分别构建含乙型脑炎病毒(Japanese encephalitis virus, JEV) prME蛋白与BALB/c鼠细胞间粘附分子(Intracellular adhesion molecule-1, ICAM-1)编码基因以及单纯ICAM-1编码基因重组质粒,比较ICAM-1编码基因不同构建方式对乙脑DNA疫苗诱导细胞免疫的免疫佐剂效应并探究ICAM-1编码基因免疫增强效应机制。
材料和方法一、实验材料
1、细胞与动物
中国仓鼠卵巢(Chinese hamster ovary, CHO)细胞与鼠肥大细胞瘤(P815)细胞分别购自中国科学院上海生命科学研究院上海细胞库,雌性、4周龄BALB/c鼠购自中国医学科学院实验动物研究所。
2、质粒与菌株
含JEV prME蛋白基因的重组质粒被命名为pJME,由本实验室构建；原核表达载体pMD19-T simple和真核表达载体pcDNA3.1(+)分别购自Takara宝生物工程(大连)公司和美国Invitrogen公司。大肠杆菌JM109与DH5α购自Takara公司。
3、其他试剂
质粒抽提试剂盒购自QIAGEN公司,Lipofectamine2000购自Invitrogen公司,山羊抗鼠ICAM-1购自Santa公司,异硫氰酸荧光素(FITC)以及辣根酶标记的兔抗山羊IgG均购自北京中杉公司,乳酸脱氢酶(LDH)测定试剂盒购自美国Promega公司。荧光标记的抗体(CD3-PerCP, CD4-APC, CD8-PE, CD54-PE, CD11c-APC, MHCⅡ-FITC, CD80-PE, CD86-FITC)均购自美国BD PharMingen公司。荧光染料羧基荧光素二乙酸盐琥珀酰亚胺脂(CFSE)、异硫氰酸荧光素标记的葡聚糖(FITC-Dextran)均购自美国Molecular Probes公司。Mouse T Cell Lympholyte-Pure购自加拿大Cedarlane公司。丝裂霉素C购自美国Sigma公司。rmIL-2购自美国Peprotech公司。新霉素类似物(G418)购自GiBCO公司,限制性内切酶EcoRI、BamHI以及NotI等均购自Takara公司。IL-4、IL-10、IL-12、IFN-γELISA试剂盒均购自上海森雄科技实业有限公司。ECL发光试剂盒购自美国Pierce公司。
二、实验方法
1、pICAM-1、pJME/ICAM-1的构建和鉴定
从BALB/c鼠脾脏细胞中提取总RNA,参照(Genbank X52264.1),以总RNA为模板,采用套式-RT-PCR法扩增ICAM-1CDS区域序列(1614bp),将后者克隆到pcDNA3.1(+) (EcoRI/NotI),构建重组质粒pICAM-1。限制性内切酶BamHI/EcoRI酶切pJME,获取JEV prME基因(2001bps)并克隆到pICAM-1 BamHI/EcoRI之间,构建融合pJME/ICAM-1.后者通过转化大肠杆菌DH5a、提取、电泳、测序以及酶切等方法进行鉴定。
脂质体法将pJME/ICAM-1转染至CHO细胞,于转染后72h,经G418(800μg/mL)进行细胞筛选7d,再经G418(400μg／mL)持续筛选4周并获得CHO细胞阳性克隆,采用免疫荧光和免疫印迹法检测ICAM-1以及融合蛋白prME/ICAM-1的表达。
2、动物和免疫程序
每组8只BALB/c鼠(购自中国医学科学院实验动物研究所)共5组。分别将溶于100μL灭菌PBS的不同免疫原鼠后肢肌内注射,包括：pJME、pJME/ICAM-1、pJME+pICAM-1、pcDNA3.1(+)(阴性对照)各100μg以及腹腔注射JE灭活疫苗组(阳性对照)。JE灭活疫苗(JEV北京株,辽宁省疾病控制中心惠赠)100μL／次(相当于1／5成人剂量),共免疫3次,间隔2周。
3、小鼠脾脏T淋巴细胞和树突状细胞的分离
脱颈法处死小鼠,无菌打开腹腔,取出脾脏放入冷PBS中,将其捻碎并滤过100目无菌钢网,收集脾细胞悬液,按照产品说明书操作制备脾脏T淋巴细胞；另取部分上述脾细胞悬液,1300 r/min洗涤5 min×3次；用完全培养基20 mL重悬细胞于250 mL培养瓶(Falcon公司)中,37℃,5%CO2培养箱内孵育2 h；用完全培养基20 mL剧烈洗摇培养瓶,并吸弃悬液,共4次；加入完全培养基20 mL,37℃,5%C02培养箱内孵育过夜；收集悬浮细胞。
4、脾脏树突状细胞表型和功能
取制备脾脏DC细胞悬液,流式细胞仪检测DC表面分子CD54、CD11c、MHCⅡ、CD80、CD86、DC摄取FITC-Dextran能力以及DC刺激T淋巴细胞增殖能力。
5、T淋巴细胞亚群与CTL功能检测
取制备脾脏T淋巴细胞悬液,流式细胞仪分析鼠脾细胞中T淋巴细胞亚群特点,乳酸脱氢酶释放法检测CTL活性。
6、ELISA法检测T细胞、DC分泌细胞因子水平
操作步骤按产品说明书进行,显色后即刻用WellscanMK3酶标仪(Lab-systems公司)检测492nm波长的吸光度A值,根据标准曲线计算IL-12、IFN-γ、IL-4、IL-10的含量。
结果
1、pICAM-1、pJME/ICAM-1的构建和鉴定
测序分析显示：JEV prM、E蛋白和ICAM-1编码基因与发表的序列相符。pICAM-1经EcoR I/Not I酶切释出插入子,与ICAM-1基因(1614bp)大小相一致,pJME/ICAM-1经BamHI/EcoRI酶切释出插入子与pJME经相同酶切释出插入子(2001bps)的基因片段大小相一致,pJME/ICAM-1经BamHI/NotI酶切释出的插入子约为3660bp,为鼠ICAM-1编码基因与JEV prME蛋白编码基因之和。
免疫荧光结果显示：pICAM-1、pJME/ICAM-1转染的CHO细胞可见较显著绿色荧光标记,主要分布在胞质,也可见于胞膜。单纯载体转染的CHO细胞及未经质粒转染的正常CHO细胞中未见特异性绿色荧光标记。转染pJME/ICAM-1的CHO细胞的Western blot分析显示,在分子量约为130×103区域处可检测到一蛋白条带,与prME和ICAM-1的分子量之和相一致,转染pICAM-1的CHO细胞的Western blot分析显示,在分子量约为60×103区域处可检测到一蛋白条带,而转染pcDNA3.1(+)的CHO细胞未见相应的条带。
2、pICAM-1、pJME/ICAM-1对小鼠脾脏DC表型和功能的影响
各组DC表面表达高水平MHCⅡ, pJME/ICAM-1组、pJME+pICAM-1组、pJME组高于JE灭活疫苗组及空载体免疫组(P<0.05),三组间比较差异无统计学意义(P>0.05)；pJME/ICAM-1组DC表面CD80表达水平最高,高于pJME组、JE灭活疫苗组及空载体免疫组(均P<0.05), pJME/ICAM-1组与pJME+pICAM-1组比较,差异无统计学意义(P>0.05); pJME组与JE灭活疫苗组DC表面CD86比较差异无统计学意义(P>0.05),其余各组间比较差异均有统计学意义(均P<0.05); pJME/ICAM-1组、pJME+pICAM-1组DC表面表达高水平ICAM-1,高于pJME组,JE灭活疫苗组及空载体免疫组(均P<0.05),两组间比较差异有统计学意义(P<0.05), pJME组,JE灭活疫苗组DC表面ICAM-1表达水平高于空载体免疫组(P<0.05),两组间比较差异无统计学意义(P>0.05)。
pJME/ICAM-1组与pJME+pICAM-1组内吞能力较强,显著高于其它各组(P<0.05)。pJME/ICAM-1组与pJME+pICAM-1组比较差异无统计学意义(P>0.05)。
比较不同免疫原对细胞分裂的作用,以pJME/ICAM-1组最强,(73.69±7.32)%CD4+T细胞发生分裂,(45.40±2.57)%CD8+T细胞发生分裂,显著高于对照组(P<0.05); pJME+pICAM-1组次之,(53.48±5.23)%CD4+T细胞发生分裂,(35.85±4.46)%CD8+T细胞发生分裂,显著高于pJME组、JE灭活疫苗组及空载体免疫组(均P<0.05)。
3、细胞免疫反应的检测
pJME/ICAM-1组CD3+CD4+T淋巴细胞比例为(36.1±6.45)%,明显高于其它组(P<0.05); pcDNA3.1(+)组CD3+CD4+T淋巴细胞比例为(20.87±4.45)%,低于其它组(P<0.05); pJME+pICAM-1组CD3+CD4+T淋巴细胞比例为(31.23±6.55)%,高于pJME组(25.26±9.72)%及JE灭活疫苗组(25.35±3.82)%(P<0.05)。各免疫组间CD3+CD8+T淋巴细胞比例无显著性差异(P>0.05)。
各组免疫鼠CTL活性由高至低依次为：pJME/ICAM-1组(56.38±5.69)%、pJME+pICAM-1组(50.37±6.33)%、pJME组(35.25±4.78)%、JE灭活疫苗组(26.72±3.78)%、pcDNA3.1(+)组(12.34±7.73)%,各组间比较,差异有统计学意义(均P<0.05)。
4、小鼠脾T淋巴细胞和DC分泌细胞因子水平的测定
pJME/ICAM-1组与pJME+pICAM-1组T淋巴细胞和DC分泌IL-12、IFN-γ水平较pJME组、JE灭活疫苗组及空载体免疫组明显升高(均P<0.05),两组间比较,前者T淋巴细胞和DC分泌IL-12、IFN-γ水平显著升高(P<0.05)。各组间T淋巴细胞IL-4分泌水平比较差异无统计学意义(均P>0.05),JE灭活疫苗组T淋巴细胞分泌IL-10的水平高于其它实验组(均P<0.05)；JE灭活疫苗组DC分泌IL-10、IL-4的水平高于其它实验组(均P<0.05),其余各组间比较差异无统计学意义(均P>0.05)。
结论
1、成功构建了融合表达重组质粒pJME/ICAM-1以及单质粒pICAM-1,并能在真核细胞内有效表达目的蛋白；
2、ICAM-1编码基因能够促进T细胞分泌细胞因子,促使CD4+T细胞向Thl细胞分化；
3、ICAM-1编码基因能够增强T细胞对靶细胞的细胞毒效应；
4、树突状细胞能够通过ICAM-1编码基因调节Th1/Th2免疫平衡；
5、ICAM-1编码基因能够促进JE DNA疫苗脾脏DC的成熟；
6、ICAM-1编码基因能够提供独立或放大B7分子的协同刺激信号；
7、相对于联合免疫组,融合质粒免疫组诱导更强的细胞免疫反应。
Objective
We constructed fusion plasmid named pJME/ICAM-1 with Japanese encephalitis virus (Japanese encephalitis virus, JEV) prME protein and BALB/c mouse intercellular adhesion molecule(intracellular adhesion molecule-1, ICAM-1) encoding gene, and pure plasmid named pICAM-1 encoding ICAM-1. To compare the adjuvant effect of ICAM-1 encoding gene in different ways on cell-mediated immunity induced by JE DNA vaccine and explore ICAM-1 coding gene immune-enhancing effect mechanism.
Materials and methods
Materials
1、Cells and animals
Chinese hamster ovary (CHO) cells, P815 cells were purchased from Shanghai Institutes for Biological Sciences, China. CHO cells were used for the transfection experiment and P815 cells were used as target cells; Female,4-week-old BALB/c mice were obtained from the Institute of Laboratory Animal Sciences, Chinese Academy of Medical Sciences, and maintained in sterile cages under specific pathogen-free conditions. The mice were later used directly for the experiment on DNA immunization.
2、Plasmids and strains
The recombinant plsmid named pJME encoding JEV prME was provided in our laboratory; Prokaryotic expression vector pMD19-T simple and Eukaryotic expression vector pcDNA3.1(+) were purchased separately from Takara Biotechnology (Dalian) Company and the U.S. Invitrogen corporation for plasmid sequencing and construction; Escherichia coli JM109 and DH5a were purchased from Takara company and used for expansion of recombinant plasmids.
3、Main reagents
Plasmid Abstraction Kits were purchased from QIAGEN company; Lipofectamine2000 was purchased from Invitrogen company and was used for transfection;Goat-Anti-Mouse ICAM-1 were purchased from Santa company, and Fluorescein isothiocyanate (FITC) and horseradish peroxidase (HRP) labeled rabbit anti-goat IgG were purchased from Beijing Zhong Shan company, and they were used for immunofluorescence and western-blot analysis; The CytoTox96 Non-Radioactive Cytotoxicity assay was purchased from Promega company and used to detect mouse splenocyte CTL activites; Anti-CD3-PerCP, CD4-APC, CD8-PE, CD54-PE, CDllc-APC, MHCⅡ-FITC, CD80-PE, CD86-FITC monoclonal antibody were purchased from BD company and used to detect phenotype of spleen dendritic cells(DC); Anti-CD4-APC, CD8-PE monoclonal antibody were purchased from BD company and used to detect spleen T-lymphocytes subsets; Carboxyfluorescein diacetate, succinimidyl ester(CFSE) and FITC-dextran were purchased from Molecular Probes company and respectively used to detect mixed lymphocyte reaction(MLR) and phagocytic function of DC; Lympholyte-M was purchased from Canada Cedarlane company. Mitomycin C was purchased from Sigma company. IL-2 was purchased from Peprotech company. G418 was purchased from GiBCO company and used for screening of resistant clone; Restriction endonuclease EcoRI、BamHI and NotI were all purchased from Takara company, which were used construction and identification of recombinant plasmid. IL-4, IL-10, IL-12, IFN-y ELISA kit were purchased from the Shanghai SenXiong Technology company. ECL luminescence kit purchased from the
United States Pierce company.
Methods
1、Construction and identification of pICAM-1、pJME/ICAM-1
The coding sequence of murine ICAM-1 (1614 bp) was amplified by nested RT-PCR from total RNA isolated from stimulated BALB/c murine splenocytes.A 1659-bp fragment of murine ICAM-1and Gly-linker was digested with restriction enzymes Eco R I and Not I from pMD19-T simple-ICAM-1 and subcloned into the pcDNA3.1(+) vector at the EcoRI/Not I site. The construct containing the murine ICAM-1 gene was designated pICAM-1. To construct a DNA vaccine encoding fusion protein of prME and ICAM-1 (pJME/ICAM-1), a fragment of 2,001 bp of JEV prME isolated from pJME by BamH I and EcoR I digestion was inserted into the BamH I/EcoR I site of p ICAM-1.
pICAM-1 and pJME/ICAM-1 transforming competent Escherichia coli DH5αwere amplified and extracted, and then verfied by restriction enzyme digestion and DNA sequencing.
Plasmid pICAM-1 and pJME/ICAM-1 was transfected into CHO cells by Lipofectamine TM2000 according to the manufacturer's instruction. After 72h of transfection, the cells were selected with G418 (800μg/mL) for one week and then cultured with G418 (400μg/mL) for four weeks for establishment of stable transfectants. CHO cells transfected with an empty pcDNA3.1(+) vector and normal CHO were used as a control, stable transfectants were detected for expression of ICAM-1 and fusion protein prME/ICAM-1 by immunofluorescence and Western blot analysis.
2、Animals and immunization procedure
We used 5 groups of 4-week-old female BALB/c mice (n=8 per group) which were as follows:pJME, pJME/ICAM-1, pICAM-1. For the i.m. immunization, mice were injected with 100μg pJME/GM-CSF,100μg pJME with 100μg of plasmid pICAM-1 into the quadriceps muscle mass of the left hind leg. All the mice received 2 booster doses in the same muscle (containing the same amount of plasmid as in the primary dose) 3 and 5 weeks after the primary injection. For the positive control group, each mouse was immunized with inactivated vaccine, a formalin-inactivated mouse brain-derived JEV vaccine (Beijing-1 strain) obtained from Liaoning Province Center of Disease Control and Prevention, and each mouse of the inactivated vaccine group was given an injection of 100μl (1/5 of a recommended adult dose) of inactivated vaccine and boosted with the same dose 3 and 5 weeks after the first immunization. The pcDNA3.1(+) immunized group was used as the negative control. The volume of vaccine solution injected into each thigh was adjusted to 100μl per mouse with PBS.
3、Mouse spleen T lymphocytes and dendritic cells Separation
Mouse spleen cell suspension were enriched according to the published methods. Briefly, mouse spleens were disrupted and cut up into small pieces, and passed through a fine screen mesh. A clean suspension was adjusted the cell concentration to a maximum of 2 x 107 nucleated cells per mL. Preparation of splenic T lymphocytes were performed in strict accordance with the instructions within the reagent kit. Another part of the spleen cell suspension were centrifuged at 1300 rpm for 5 min, resuspended in RPMI 1640 medium supplemented with 10% heat-activated fetal calf serum,2 mmol/1 Lglutamine,1 mmol/1 pyruvate,50 mmol/1 mercaptoethanol,100 U/mL penicillin, and 100 mg/mL streptomycin, and then incubated for 2 h at 37℃in a 5% CO2 atmosphere in plastic cell cultures plates. Culture plates were then washed vigorously four times with supplemented RPMI 1640 medium, and the nonadherent cells were discarded. The residual adherent cells were maintained in the culture medium and incubated overnight at 37℃in a 5% CO2 atmosphere. After incubation, DCs(which exhibit adherence capacity in the first hours of culture) become nonadherent and float in the medium. The DCs were collected and immediately used in our assays.
4、Measurement of phenotype and function of spleen DC
Spleen DC were isolated and Flow cytometry detected DC surface molecules CD54, CD11c, MHCⅡ, CD80, CD86, DC uptake of FITC-Dextran capability and capacity of DC to stimulate T lymphocyte proliferation.
5、Spleen DC T-lymphocyte subsets and CTL function detection
Single-cell spleen suspensions were prepared, from which erythrocytes were removed. T-lymphocyte subsets of mouse spleen cells were assayed with flow cytometry technique, and CTL activity of mouse spleen cells was detected with lactic dehydrogenase release test method.
6、Dectection of cytokines secreted by T cells and DC
Steps were carried out by product specification. WellscanMK3 microplate reader (Lab-syste ms Dragon Company) detected immediately 492nm wavelength absorbance A value after color and IL-12, IFN-γ, IL-4, IL-10 levels were calculated according to the standard curve.
Results
1、Construction and identification of pICAM-1 and pJME/ICAM-1
Upon restriction enzyme digestion, pICAM-1 was digested with EcoR I/Not I into 1 fragment, as predicted, at 1,614bp in size, and confirmed that the DNA fragment released from the recombinant plasmid corresponded to the ICAM-1 coding sequence from the published murine ICAM-1 gene sequence. Meanwhile, the pJME/ICAM-1 was cut with BamH I/ EcoRI and Bam HI/Not I into 2 fragments as predicted at 2,001 and 3,660 bp in size, and confirmed that 2,001 bp was the same as that of the JEV prME gene and 3,660 bp was the sum of the murine ICAM-1 gene and the JEV prME gene. With sequence analysis using the T7 promoter primer located in pcDNA3.1(+), it was indicated that there was a normal junction of BamH I site between vectors and the prME gene of JEV, and EcoR I site between vectors and the murine ICAM-1 gene.
Immunofluorescence result showed that the green fluorescence mark was mainly distributed obviously in endochylema of CHO cells transfected with pICAM-1 and pJME/ICAM-1, and not much in membrane. There was no specific green fluorescence in CHO cells untransfected and transfected with pcDNA3.1(+).
In Western blots, the specific ICAM-1 protein and fusion protein prME/ICAM-1 were clearly visible in CHO cells transfected with both pICAM-1 and pJME/ICAM-1, but almost undetectable specificity protein in cells transfected with pcDNA3.1(+). Anti-murine ICAM-1 reactive protein bands of about 60 and 130 kDa were respectively in correspondence with ICAM-1 protein expressed by pICAM-1 and prME/ICAM-1 fusion protein expressed by pJME/ICAM-1, and there was no a corresponding protein band by Western blot analysis of CHO transfected with pcDNA3.1(+).
2、The impact of pICAM-1 and pJME/ICAM-1 on mouse spleen DC phenotype and function
DC of each group express high levels MHCⅡ, pJME/ICAM-1, pJME+ pICAM-1 and pJME group was higher than JE inactivated vaccine and empty vector vaccinated groups (P<0.05),there were no statistically significant difference among the three groups (P>0.05). DC surface CD80 expression in spleen from the pJME/ICAM-1 vaccinated group was the highest in all groups, although there was no significant difference between pJME/ICAM-land pJME+ pICAM-1 group (P>0.05). There was significant difference in the expression of DC surface CD86 between 2 random groups except that there was no significant difference between JE inactivated vaccine and pJME vaccinated groups. DC surface ICAM-1 expression in spleen from the pJME/ICAM-1 and pJME+pICAM-1 vaccinated group was higher than that in the other groups, and there was significant difference between the two groups (P<0.05); DC surface ICAM-1 expression in spleen from the pJME and JE inactivated vaccine vaccinated group was higher than that in the pcDNA3.1(+) vaccinated group, and there was no significant difference between the two groups(P>0.05).
Phagocytic capacity of dendritic cells in the pJME/ICAM-1 and pJME+pICAM-1 vaccinated groups was obviously higher than those in other groups(P<0.05), and there was no significant difference between the two groups (P>0.05).
In order to further analyze the antigen-presenting function of mouse spleen dendritic cell, we determined the stimulating ability of dendritic cells to T lymphocytes in spleens of DNA-immunized mice by flow cytometry. The percentage of CD4+T and CD8+T cells division in spleen cells was respectively (73.69±7.32)% and (45.40±2.57)% in the pJME/ICAM-1 vaccinated group, which significantly increased the division capacity of CD4+T cell compared with other groups (P<0.05). The percentage of CD4+T and CD8+T cells division in spleen cells was respectively (53.48 +5.23)% and (35.85±4.46)% in the pJME+pICAM-1 vaccinated group, obviously higher than those in the pJME, JE inactivated vaccine and pcDNA3.1(+) vaccinated groups (P< 0.05).
3、Dectection of cellular immune responses
The adjuvant effects of the ICAM-1 DNA on the generation of CD4+T cells or CD8+T cells from spleen were compared. The percentage of CD4+T cells in spleen cells was (36.1±6.45)% in the pJME/ICAM-1 vaccinated group, which significantly increased the relative frequency of CD4+T subpopulations compared with other groups (P<0.05). The percentage of CD4+T cells in the pcDNA3.1(+) vaccinated group was (20.87±4.45)%, and lower than in all other groups(P<0.05). The percentage of CD4+T cells in the pJME+pICAM-1 vaccinated group was (31.23±6.55)%, and higher than pJME and JE inactivated vaccine group(P<0.05). There was no significant difference in the levels of CD8+T cells between 2 random groups(P>0.05).
Mice immunized with 100μg pJME/ICAM-1 had higher CTL activity than those immunized with the same amount of other naked DNA(P<0.05). The CTL activities of the spleens of BALB/c mice immunized 3 times with indicated immunogens were as follows:pJME/ICAM-1 (56.38±5.69)%; pJME+pICAM-1 (50.37±6.33)%; pJME (35.25±4.78)%; JE inactivated vaccine (26.72±3.78)%, and pcDNA3.1(+) (12.34±7.73)%. There was significant difference in CTL activity between 2 random groups(P<0.05).
4、Dectection of cytokines secreted by T cells and DC
Secretion of T cells and DC in the pJME/ICAM-1 and pJME+pICAM-1 vaccinated group was obviously higher than those in the pJME, JE inactivated vaccine and pcDNA3.1(+) vaccinated groups(P<0.05), pJME/ICAM-1 group acquired higher levels of IL-12, IFN-γthan pJME+pICAM-1 vaccinated group did(P<0.05). There was no significant difference in the levels of IL-4 secreted by T lymphocytes between 2 random groups(P>0.05). JE. inactivated vaccine vaccinated group obtained higher levels of IL-10 secreted by T lymphocytes compare with other groups(P<0.05). JE inactivated vaccine vaccinated group showed a great increase in levels of IL-10 and IL-4 secreted by DC compared with other groups(P<0.05), and there was no significant difference between any other groups(P>0.05).
Conclusions
1、The constructed recombinant pJME/ICAM-1 and pICAM-1 were confirmed by restrict enzyme digestion and DNA sequencing, and could express effectively the target protein in eukaryotic cells;
2、ICAM-1 coding gene can promote T-cell cytokine secretion, and induced CD4+T cells to Thl cell differentiation;
3、ICAM-1 coding gene can enhance the cytotoxic effects of T cells to target cells;
4、Dendritic cells can regulate Thl/Th2 immune balance through ICAM-1 coding gene;
5、ICAM-1 coding gene can promote the maturation of spleen DC induced by JE DNA vaccine;
6、ICAM-1 coding gene can amplify costimulatory signals that be independent of B7 molecules;
7、The fusion plasmid immunization group induced a stronger cellular immune response relative to the combined immune group.
引文
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