【作者】 宫强；

【导师】 孔宪刚；

【作者基本信息】 中国农业科学院 ， 预防兽医学， 2007， 博士

【摘要】 牛结核病是主要由牛分枝杆菌引起的一种慢性消耗性疾病，在许多国家尤其是发展中国家仍广泛流行，该病不仅给畜牧业造成巨大的经济损失，而且严重威胁着人类的身心健康。因此，控制牛结核病对防治人类结核病意义重大。然而，至今仍没有一种疫苗可用于该病的预防。本研究利用PCR技术和重叠延伸剪接技术(splice by overlapping extension，SOE)，以牛分枝杆菌ValleeⅢ菌株的基因组为模板，扩增ag85b、esat-6、hsp65、mpb64基因和ag85b-esat-6、hsp65-esat-6、mpb64-esat-6融合基因，连接到真核表达载体pCDNA3.1(+)中，构建重组质粒pCDNA3.1-Ag85B(简称pCA)、pCDNA3.1．ESAT-6(简称pCE6)、pCDNA3.1-HSP65(简称pCH)、pCDNA3.1-MPB64 (简称pCM)、pCDNA3.1-Ag85B-ESAT-6 (简称pCAE)、pCDNA3.1-HSP65-ESAT-6(简称pCHE)和PCDNA3.1-MPB64-ESAT-6(简称pCME)。转染SP2／0细胞，检测目的基因的表达。以各重组质粒和pCDNA3.1(+)及PBS免疫BALB／c小鼠，免疫3次，每次间隔2周，1免后每周以牛分枝杆菌纯化蛋白衍生物(purified protein derivmive，PPD)为包被抗原，间接ELISA方法检测血清抗体水平；以牛分枝杆菌PPD和刀豆蛋白A(Concanavalin A，ConA)为刺激原，MTT法检测脾淋巴细胞增殖情况。3免2周后以ELISA试剂盒检测脾细胞分泌IFN～γ的情况。结果表明，除pCH组外，其他各重组质粒免疫后小鼠血清抗体水平持续上升，与pCDNA3.1(+)对照组和PBS对照组相比差异显著(p＜0.05)。3免2周后，融合DNA疫苗免疫组的刺激值(SI值)与单价DNA疫苗免疫组相比差异显著(p＜0.05)。PPD刺激后融合基因DNA疫苗免疫组小鼠脾细胞分泌的IFN～γ高于单价DNA疫苗组(p＜0.05)，而ConA刺激后各疫苗组分泌的IFN～γ,水平均高于PPD刺激时(p＜0.05)，其中以pCAE和pCME组最高。以上结果表明在诱导细胞免疫应答方面融合DNA疫苗要优于单价DNA疫苗。同样，利用PCR和SOE技术，获得牛分枝杆菌mpb64-ag85b和mpb64-ag85b-esat-6融合基因，以pCDNA3.1(+)为载体构建了牛分枝杆菌多价组合和多基因融合DNA疫苗：二基因融合(pCDNA3.1-MPB64-Ag85B，简称pCMA)和三基因融合(pCDNA3.1-MPB64-Ag85B-ESAT-6，简称pCMAE)DNA疫苗；二价组合(pCA+pCM)和三价组合(pCA+pCM+pCE6)DNA疫苗，免疫BALB／c小鼠，以牛分枝杆菌BCG免疫组为阳性对照，以pCDNA3.1(+)及PBS免疫组为阴性对照，共免疫3次，每次间隔2周，BCG组仅初免时皮下免疫1次。1免后每周，以原核表达纯化的重组MPB64-Ag85B-ESAT-6蛋白(rMAE)和牛分枝杆菌PPD为包被抗原，以间接ELISA方法检测血清IgG水平及lgG亚类。每次免疫2周后以rMAE蛋白和PPD刺激，MTT法检测脾淋巴细胞增殖情况，试剂盒检测IFN～γ和IL-2的分泌情况。3免3周后以1×10~6CFU的BCG进行攻毒。攻毒3周后，以间接免疫荧光试验、肺脏荷菌数和病理组织学变化评价攻毒后的免疫保护效果。试验结果表明，融合DNA疫苗免疫后产生的血清抗体水平、淋巴细胞增值(SI值)水平、IFN～γ和IL-2水平明显高于多价DNA疫苗(p＜0.05)，以rMAE蛋白为包被抗原和刺激原时BCG免疫组的抗体水平、SI值和分泌的IFN～γ、IL-2的量明显低于融合DNA疫苗组，而与多价DNA疫苗组相当，以牛分枝杆菌PPD为包被抗原和刺激原时BCG免疫组的体液和细胞免疫指标均明显优于各DNA疫苗组。从攻毒保护效果来看，融合DNA疫苗组的保护效果明显优于多价DNA疫苗，达到了BCG疫苗的免疫保护水平，表明本研究制备的融合DNA疫苗具有良好的用前景，从而为牛结核病新型疫苗的研究奠定了基础。更多还原

【Abstract】 Bovine tuberculosis (BT) is a chronic wasting disease primary caused by Mycobacterium bovis thatis prevalent throughout the world, particularly in developing countries. This disease not only causestremendous economic hardship to the livestock industry, but it is also a serious human health threat.Hence, the development of strategies to control the disease is tremendously important to preventinghuman tuberculosis. Currently, no clinical vaccine against BT is available.In the present study, the DNA fragments of ag85b, esat-6, hsp65, mpb64 and ag85b-esat-6,hsp65-esat-6, mpb64-esat-6 were amplified by PCR and spliced by overlapping extension (SOE) fromthe genome DNA of Mycobacterium bovis Vallee HI.These seven fragments were inserted intopCDNA3.1(+) vector to construct recombinant plasmids pCA, pCE6, pCH, pCM, pCAE, pCHE andpCME. The seven plasmids were transfected into SP2/0 cell in vitro to detect the expression of targetgenes. BALB/c mice were intramuscularly vaccinated with the seven plasmids and the control vectorpCDNA3.1(+) and PBS respectively. The animals in each group were immunized 3 times with 2-wkintervals.The serum antibody titers were determined using indirect enzyme-linked immunosorbent assay(iELISA) coated with with M. bovis PPD every week. Two weeks after each immunization, spleniclymphocytes proliferation (SLP) assay were detected by MTT method using M. bovis PPD and ConA asstimulator. Two weeks after the third immunization, secreted IFN～γ, of spleen were tested. The results ofindirect ELISA showed the levels of antibodies in all recombinant plasmids groups were significantlyhigher than the two control groups(p＜0.05). SLP experiments indicated the SI value in the groups offusion gene were higher than the single gene groups (p＜0.05). The IFN～γexperiments showed thelevels of IFN～γ, induced with PPD in the groups of fusion gene were significantly higher than the singlegene (p＜0.05). The levels of IFN～γinduced with ConA were higher than with PPD, and the levelswere higher in groups pCAE and pCME than in other groups. So the results showed fusion gene DNAvaccines surpass single gene DNA vaccines in the induction of cellullar immunologic response.The fusion DNA fragments of ag85b-mpb64 and ag85b-mpb64-esat-6 were obtained by PCR andSOE technique. Various DNA vaccines were constructed with the pcDNA3.1(+): fusion of two genes(pCMA), and of three genes (pCMAE), bivalent combinations (pCA+pCM) and trivalent combinations(pCA+pCM+pCE6). BALB/c mice were vaccinated with this DNA vaccines.The mice injected withBCG were positive control and the mice injected with pCDNA3.1(+) and PBS were negative control.The mice were immunized 3 times with 2-wk intervals. The animals in group BCG were only inoculatedsubcutaneously with 1×10~6 CFU BCG at initial vaccination. The serum IgG titers and IgG isotype weredetermined using iELISA coated with M. bovis PPD and rMAE protein expressed and depurated inprokaryotic expression system every week. Two weeks after each immunization, SLP and IFN～γ, IL-2assay were detected using rMAE protein and M. bovis PPD as stimulator. Three weeks after the 3rdDNA immunization, mice were challenged with 1×10~6 CFU of BCG. Three weeks after the challengethe protective efficacy were evaluated based on immunofluorescence assay, lung tissue bacterial loadsand pathohistologic changes. The data demonstrate that immunization with fusion (of two or three genes) DNA vaccines results in significantly higher serum antibody levels, lymphocyte proliferation (SI value), IFN-γ, and IL-2 levels than immunization with polyvalent DNA vaccines (p＜0.05).These index in group BCG were significantly lower than the groups of fusion DNA vaccines and equivalent to groups of polyvalent DNA vaccines when rMAE protein was custodite antigen and stimulator. However, these index in group BCG were significantly higher than other groups when M. bovis PPD was custodite antigen and stimulator. The protective efficacy of the fusion DNA vaccines was surpass the polyvalent DNA vaccines and equivalent to that of the BCG vaccine, suggesting that fusion DNA vaccines represent a promising approach for the prevention of BT. So our experiment may be useful for the development of Bovis tuberculosis vaccine.更多还原