【作者】 王楠；

【导师】 刘松财；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2016， 博士

【摘要】 布鲁氏菌病(Brucellosis,以下简称布病),是一种严重威胁人类健康的动物源性人畜共患病。动物感染后导致繁殖能力和生产性能下降,经济损失巨大。我国预防牛、羊布病主要使用的是布病活疫苗Brucella suis Stain 2株(B.suis S2株),该疫苗在使用过程中无法区分疫苗免疫与自然感染抗体,给该病的流行病学调查和临床血清学诊断等造成了困难。口蹄疫(Foot and mouth disease,FMD)是由口蹄疫病毒(Foot and mouth disease virus,FMDV)引起的牛、羊等的一种急性、热性、高度接触性传染病。VP1蛋白是FMDV的主要结构蛋白,也是病毒感染细胞的关键,具有与细胞受体结合的位点,所以VP1基因是研制基因工程疫苗的首选靶抗原。本研究以我国广泛使用的布病活疫苗B.suis S2株为亲本株,利用蔗糖自杀质粒载体,将FMDV的VP1基因表达框部分替代了布鲁氏菌wbo A基因,成功构建了重组口蹄疫病毒VP1基因的布鲁氏菌病活疫苗。首先,以布病活疫苗B.suis S2株基因组为模板,利用含氯霉素抗性载体分段缺失了wbo A基因,体外活力实验筛选了6个分段缺失重组菌的活力。然后,构建了包含wbo A基因的上、下游同源臂、VP1基因表达框和蔗糖负筛选基因的重组穿梭质粒p UC-wbo A-/VP1+,电转化进布病活疫苗B.suis S2株感受态细胞中,利用氨苄抗性基因(Amp+)和蔗糖负筛选基因(Sac B-)筛选得到阳性重组菌,经PCR和测序鉴定,命名为S2?wbo A/VP1株,进一步开展重组疫苗的生物学特性研究,结果如下:(1)表型鉴定:热凝集试验、吖啶黄试验和菌落结晶紫染色试验结果证明,重组疫苗S2?wbo A/VP1株的表型为粗糙型,表明亲本株部分缺失了wbo A基因后布鲁氏菌表型从光滑型改变为粗糙型。(2)安全性评价:以不同剂量的重组疫苗分别注射小鼠和牛,通过直肠测温、采食情况和病理组织学观察等结果均证明重组疫苗具有良好的安全性。(3)口蹄疫VP1基因的鉴定和体液免疫研究:将VP1基因进行原核表达,制备抗血清与重组疫苗进行Western Blot分析,结果表明VP1在重组疫苗中成功表达,具有免疫反应原性。用地高辛探针标记VP1基因进行Southern杂交结果显示,重组疫苗与亲本株相比VP1基因已经成功插入布鲁氏菌基因组。间接ELISA实验证实,与亲本株不同,重组疫苗免疫组小鼠14d检测到VP1基因抗体产生,第二次免疫后抗体水平呈快速增加趋势。(4)布鲁氏菌病免疫原性研究:重组疫苗的抗血清与不同表型布鲁氏菌菌株的凝集实验结果表明,重组疫苗与粗糙型菌株B.melitensis M111株和B.canine RM6/66株呈阳性反应,与其他光滑型菌株呈阴性反应。以小鼠为动物模型,通过脾脏称重和细菌分离实验研究重组疫苗在小鼠体内的存活时间。结果表明,免疫后14d重组疫苗小鼠脾脏重量高于亲本株,与对照组差异显著(P<0.05)。免疫后42d,疫苗株几乎不能引起小鼠脾脏肿大,与对照组差异不显著(P>0.05),脾脏中分离不到细菌。用微量试管凝集实验方法研究抗体消长规律,结果表明免疫后7d即可检测到布鲁氏菌抗体,从第7d~14d抗体显著增加,第14d~28d抗体继续增长,但趋势缓慢,到第42d抗体呈降低趋势。细胞因子检测试验结果表明,重组疫苗与亲本株免疫小鼠后产生的细胞免疫反应增长趋势基本一致,免疫后第7d开始产生IFN-γ,第7d~28d IFN-γ值持续增加,28d后IFN-γ水平呈下降趋势。小鼠攻毒保护实验结果表明,重组疫苗1×109CFU/只免疫组可以对羊种B.meltensis M28强毒株攻击提供100%的保护。以羊为动物模型进行攻毒保护试验,羊的脾脏和腹股沟淋巴结细菌分离结果表明,1×1011 CFU/只免疫组感染强毒后有2/10只分离到强毒菌,免疫保护率为80%,与1×1010 CFU/只亲本株提供相似的免疫保护率(80%)。由于布病和口蹄疫是两种感染牛、羊的重要家畜传染病,布鲁氏菌疫苗作为载体能有效提供对目的基因编码蛋白的细胞免疫作用。因此,本研究构建并鉴定的重组口蹄疫VP1基因的布鲁氏菌活疫苗为实现对布病和口蹄疫的双重免疫提供候选疫苗株。更多还原

【Abstract】 Brucellosis is an animal sourced zoonosis severely threatening public health. After infection, animal’s reproduction and growth performance decrease greatly, result huge economic losses. In China, Brucella suis Stain 2(B.suis S2) is mainly used to prevent brucellosis in cow and sheep; however, this vaccine cann’t differentiate antibody of vaccine immunization from spontaneous infection, bringing difficulties to epidemiological investigation and clinical serological diagnosis of brucellosis.Foot and mouth disease(FMD) is an acute, febrile and highly contagious infectious disease in cow and sheep, etc. caused by foot and mouth disease virus(FMDV). VP1, the major structural protein of FMDV, is the critical factor for virus infection of cells. With binding site for cell receptors, VP1 gene is the first target antigen choice for the research and development of recombinant vaccine.In this study, live B.suis S2 extensively used in China was used as a parent strain, which utilizes suicide plasmid vector to substitute gene cassette of FMDV VP1 to wbo A gene of B.suis S2, and successfully constructed recombinant B.suis vaccine carrying FMDV VP1. First, genome of B.suis S2 was used as the template, considering that wbo A gene was missed in carrier segment with chloramphenicol resistance, vitalities of 6 nonrecombined segments were screened by in vitro viability test. Then the recombinant shuttle plasmid was constructed, which included upstream and downstream homologous sequences of wbo A gene, and sucrose negative screening gene. The plasmid was electrically transformed into competent cell of B.suis S2 strain, and ampicillin resistance gene(Amp+) and sucrose negative screening gene(Sac B-) were utilized to screen the recombinant. The positive recombinant was identified by PCR and sequencing, named S2?wbo A/VP1 strain, and then biologically characterized.(1) Phenotype identification: Heat agglutination test, acriflavin test and colony crystal violet stain test demonstrated that, phenotype of recombinant S2?wbo A /VP1 was a rough phenotype, suggesting that the phenotype of parent strain without wbo A gene changed from smooth type to rough type.(2) Safety evaluation: Different doses of recombinant were injected into mice, cow and sheep, and rectal temperature, food intake and pathological findings, etc were observed. All results suggested that recombinant is safe for mice.(3) Identification of VP1 gene and study on humoral immunity of FMD: prokaryotic expression was conducted for VP1 gene to prepare antiserum and recombinant for Western Blot analysis, which suggested VP1 gene was successfully expressed in recombinant, and it had immunoreactivity. VP1 gene was marked with digoxin probe to perform Southern hybridization, and the result suggested that compared to parent strain, VP1 gene successfully inserted into a genome copy of B.suis S2. Indirect ELISA confirmed that, compared with parent strain, VP1 antibody generated 14 days later in mice of recombinant inoculation group, and the antibody level tended to quickly increase after the 3rd inoculation.(4) Study on immunogenicity of Brucelosis: Agglutination test on recombinant with different phenotypes demonstrated that, recombinant showed positive response with rough phenotype strains of B.melitensis M111 and B.canine RM6/66, and negative response to other strains of smooth type.Survival time of recombinant in mice was investigated by weighing spleen and separating bacteria. The results suggested that, day 14 of inoculation, spleen weight of mice was higher in recombinant group than that in parent strain group, with significant difference compared to control group(P<0.05). On day 42, vaccine strain even did not result in splenomegaly in mice(P>0.05, compared with the control), and no bacterium was separated from spleen. ELISA method was used to investigate the change of antibody. The results suggested that recombinant antibody was detected 7 days after inoculation, significantly increased from day 7 to 14, continued to increase from day 14 to 28 at a slow rate, and tended to decrease till day 42. Cytokine test indicated that, mice inoculated with recombinant strain S2?wbo A/VP1 and parent strain B.suis S2 showed basically consistent cellular immune response, started to generate IFN-γ since day 7 of inoculation. IFN-γ value continued to increase from day 7 to 28, and tended to decrease after day 7. Mice challenge test suggested that, 1×109CFU/mouse could provide 100% protection to challenge by B.meltensis M28.Challenge test was performed with sheep, Bacterial separation results from spleen and inguinal lymph nodes suggested that the inoculation group at 1×1011 CFU/sheep held 80% of protection percentage, similar to that provided by 1×1010 CFU/sheep parent strain of B.suis S2(80%).Brucellosis and FMD are important infectious diseases of domestic animals including cow and sheep. As the carrier with characteristics of intracellular parasitism, Brucellosis vaccine can sufficiently mediate T cell immunity in the host. Therefore, live recombinant B.suis vaccine carrying VP1 gene constructed and identified in this study provides an alternative vaccine strain to realize double immunization against brucellosis and FMD.更多还原