【作者】 王晓凌；

【导师】 王永祥； 金玉怀；

【作者基本信息】 河北医科大学 ， 病原生物学， 2005， 硕士

【摘要】 目的:柯萨奇病毒B 组3 型(Coxsackievirus group B type3, CVB3)是引起人类病毒性心肌炎的主要病原体。虽然病毒的分子结构已经研究得很清楚,然而目前还没有特异性的防治措施,基因疫苗则为预防CVB3 感染提供了可能性。VP1是CVB3 主要的衣壳蛋白,可诱导机体产生免疫应答。编码VP1 的DNA 疫苗免疫小鼠后可诱导机体产生中和抗体,但效价较低,不能有效阻止致死量病毒感染。因此如何增强VP1 的免疫原性,尤其是抗体应答,成为目前的研究热点。抗原提呈细胞(Ag-presenting cells,APCs)在诱发免疫应答过程中处于核心地位。将抗原定向提呈给APCs 来增强DNA疫苗的免疫原性的靶向策略已经发展起来,抗原和诸如CTLA-4 and L-selectin 之类的分子相连,把抗原靶向APCs增强免疫应答,在不同物种取得了一定的效果。巨噬细胞源趋化因子(macrophage-derived chemokine,MDC) 首先是从人单核细胞来源的巨噬细胞cDNA 克隆随机片段分离得到的,具有趋化单核细胞、巨噬细胞、树突细胞、IL-2 活化的NK 细胞的活性。随后相似的趋化因子分别从人活化的巨噬细胞,鼠B 细胞和树突细胞分离,被称为STCP-1 和ABCD-1。MDC 属CC 趋化因子家族,现在已经证实的MDC 受体是CCR4,主要表达于Th2 细胞,B 细胞、巨噬细胞等。树突细胞和单核细胞虽对MDC 应答,但不表更多还原

【Abstract】 Objective: Coxsackievirus B3(CVB3) infections arecommon causes of acute and chronic myocarditis in human.Despite the well charactered molecular structure of Coxsackieviruses, no virus-specific preventive procedures against CVB3are clinically available today. Immunization with DNA vaccinemakes it possible to establish a new preventive procedureagainst CVB infections. VP1 is a major capsid protein of CVB3,which can induce the production of effective neutralizingantibody. Although the vaccine constructed with VP1 alonecould induce the production of antibody, the titers of antibodyproduced were usually too low to protect the host from lethalCVB3 challenge. Therefore, improving the immunologicalpotency ,particularly the Ab response, has been a seriouse hurdleto enhancing the protective efficacy of DNA vaccine encondingvp1. Antigen-presenting cells (APCs) play a central role inpriming immune responses . Targeting strategies,which targetAg to APCs, are being developed to enhance theimmunogenicity of DNA vaccines. Ags linked to molecules suchas CTLA-4 and L-selectin have been used to targete Ags toAPCs , and have been proved that the immune responses wreenhanced in different species with some degrees of success.Macrophage-derived chemokine (MDC, CCL22) was firstisolated by random sequencing of cDNA clones from humanmonocyte-derieved macrophages and showed chemotacticactivity of monocytes, macrophages, dendritic cells, and IL-2activated natural killer cells. Similar chemokines were alsoisolated from human activated microphages, and murine B cellsand dendritic cells,and were termed stimulated T-cellchemotactic protein (STCP)-1 and ABCD-1, respectively. MDCis a member of the CC chemokine family , and has been shownto bind to CC receptor 4 (CCR4), which are mainly expressedon Th2 cells, B cells, Macrophages, et al. Although dendriticcells and monocytes, are responsive to MDC, they do notexpress CCR4. It han been suggested that MDC may recognizeanother, yet unidentified receptor, such as CCR4 variant (GarciaGE, 2003). MDC ia a strong chemotactic agent to T cells,especially Th2 cells , plays an important role for the traffickingof Th2 cells and the induction of Th2-related immune responses.Therefore , we postulated that fusing MDC to VP1 couldenhance the effect of CVB3 vaccines. In this study, MDC genewas linked to CVB3 VP1 gene to construct an eukaryoticexpressing plasmid pcDNA3/MDC-VP1, based on an idea thatthe fusion protein MDC-VP1 would be expressed in the animalcells after inoculation and targeted VP1 to APC by the MDCprotein recognizing MDC receptors on the cell surface of APCs.To maintain the natural spatial stuctions of MDC protein andVP1 protein, a DNA sequence which encode a flexiblepolypeptide (Linker) was used.Methods: (1) The total RNA of spleen cells from scaldmice, which have been stimulated with LPS for 24 hours, wasextracted. The DNA fragments of MDC and MDC-Linker(MDC-L) was amplified by RT-PCR from the total RNA ofspleen cells. Similarly, the DNA fragments of VP1 andLinker-VP1(L-VP1) was amplified by RT-PCR from the RNAof CVB3. (2) The PCR products of the four DNA fragments waslinked to plasmid pGEM-T , E.coli DH5αwas transformed andcultured in medium containing Ampicilin (100 ug/ml) 2YT.Then plasmids were extracted, recombinant clones wereselected and identified by endonuclease cutting and sequencing.(3) The constructed plasmid were cut by proper endonucleasesand the target gene fragments were linked to pcDNA3 whichwas cut by the same endonucleases to construct the eukaryoticexpressing plasmids of pcDNA3/MDC , pcDNA3/VP1, andpcDNA3/ MDC-VP1. (4)Large preparation were performed toobtain the plasmids of pcDNA3/MDC , pcDNA3/VP1, andpcDNA3/ MDC-VP1 and pcDNA3 from transformed E.coliDH5α, and purified by PEG. (5) BALB/c mice aged 6-8 weekswere divided into 6 groups at random : Saline control group,pcDNA3 control group, pcDNA3/MDC group, pcDNA3/VP1group, pcDNA3/VP1 and pcDNA3 /MDC group, andpcDNA3/MDC-VP1 group. Every group consisted of 26 mice.The plasmid were inoculated to mice in a dose of 100ug ofDNA/mouse, intramuscularly (i.m.) in quadriceps muscle at3-week intervals. Twenty days after every injection , sera werecollected and CVB3-specific neutralizing antibodies weretitrated. Three weeks after the third immunization, 20 mice fromeach group were subjected to intraperitoneal (i.p.) challengewith 10 LD50 of CVB3 and the number of surviving animalswas monitored up to 3 weeks post infection. The rest mice ofeach group challenge with 3 LD50 CVB3 and were sacrificed at7th day, the titers of blood viruses and the ratio of heatweight versus body weight (HW/BW) were evaluated. Then thehearts fixed with 10% formalin ,dehydrated with gradingethanol, embedded in paraffin, and cut into 5 μm sections.Some sections were stained with hemotoxylin and eosin toobserve the changes of the myocardium.Results: (1) The gene fragments of MDC, MDC-L, VP1and L-VP1 was amplified by RT-PCR. The length of thefragments was same as expected.(2)The prokaryotic clonevectors pGEM-T/MDC, pGEM-T/MDC-L, pGEM-T/VP1 andpGEM-T/L-VP1 were successfully constructed , DNAsequencing showed that the sequences is identical to thesequence recorded in Genbank.(3) After these gene fragmentswere inserted to the pcDNA3, the recombined plasmid wasidentified by enzyme cutting and the results were identical asexpected and suggested that the eukaryotic expressing plasmidof pcDNA3/MDC , pcDNA3/ VP1 and pcDNA3/ MDC-VP1had been constructed successfully. (4) The mean titers ofneutralizing antibody after every immunization in pcDNA3/VP1group were 1:5.95, 1: 9.01, 1: 16.82, respectively, and that inpcDNA3/VP1+ pcDNA3/MDC group were 1: 6.37, 1: 10.72, 1:26.39, respectively, and that in pcDNA3/MDC-VP1 group were1: 6.83, 1: 18.03, 1: 33.64 ,respectively. The mean neutralizingantibody titers in pcDNA3 group , N.S. and pcDNA3/MDCgroup were all lower than 1:5. The statistics analysis showedthat the diffrences of neutralizing antibody titers of every groupproduced after three times of inoculation were significant; Afterthe third inoculation, the mean neutralizing antibody titers inpcDNA3/MDC-VP1 group and pcDNA3/VP1+pcDNA3/MDCgroup were higher than pcDNA3/VP1. The difference weresignificant when analysed by the multiple comparison (P<0.05).But the difference between pcDNA3/MDC-VP1 group andpcDNA3/VP1+ pcDNA3/MDC group was not significant. (5)Up to the 21th day after CVB3 challenge, the survival rates ofpcDNA3/MDC-VP1 group, pcDNA3/VP1+ pcDNA3/MDCgroup, pcDNA3/VP1 group, pcDNA3/MDC group, pcDNA3group and N.S. group were 50%, 35%, 25%, 20%, 10% and15%, respectively. Chi-square test indicated that there weredifferences among groups(P=0.048), but differences beteen anytwo groups wre not significant by the multiple comparison.Kaplan-Meier test was used to compare differences of survivalcurves, the result showed that the difference was significant(P<0.05). The result of multiple comparison indicated that thesurvive state of pcDNA3/MDC-VP1 group is better than that ofpcDNA3/VP1 group (P<0.05), but difference beteenpcDNA3/VP1 + pcDNA3/MDC group and pcDNA3/VP1 wasnot significant (P>0.05). (6)The virus titers of blood inpcDNA3/MDC-VP1 group and pcDNA3/vp1 + pcDNA3/MDCgroup were lower than that of any other group, the differencewas significant (P ≤0.05), but difference betweenpcDNA3/MDC-VP1 group and pcDNA3/VP1 + pcDNA3/MDCgroup was not significant. (7) HW/BW in all group, exceptpcDNA3 group, are smaller than N.S. group, the multiplecomparison indicate the differences were significant (P<0.05).HW/BW in pcDNA3/MDC-VP1 group and pcDNA3/VP1+pcDNA3/MDC group were smaller than other groups (P<0.05).(8) The histopathology changes in pcDNA3/MDC-VP1 groupand pcDNA3/vp1 + pcDNA3/MDC group were less seriouscompared with that of the other groups, but no significantdifference were found between them. (9) Differences in micebody weight between 0th day (just before inoculation) and 63thday (just before challeng with CVB3) were analysed. Thedifferences were not significant.Conclusion: (1) The gene fragments of MDC and CVB3VP1 was succesfully cloned by RT-PCR. (2) The eukaryoticexpressing plasmid pcDNA3/MDC, pcDNA3/MDC-VP1 andpcDNA3/VP1 was constructed successfully; (3) The survivalrates of pcDNA3/MDC-VP1 group, pcDNA3/VP1+pcDNA3/MDC group, pcDNA3/VP1 group were 50%, 35%,25%, respectively, the differences were not significant. The更多还原