摘要
布鲁氏菌病，简称布病（Brucellosis），是由布鲁氏菌（Brucella spp.）引起的一种的人畜共患传染病。目前，该病已在世界范围内广泛流行，并呈一定上升态势，严重危害畜牧业发展和人类健康。建立方便快捷的布鲁氏菌检测技术，研发安全有效的新一代布病疫苗已成为控制和消灭动物布病的必然趋势。本研究主要内容如下：
1.选择布鲁氏菌OMP25诊断基因和P39、18KD、L7/L12和BCSP31优势抗原基因，扩增后分别与pMD18-T载体连接，制备了克隆质粒。分析比对GenBank中OMP25基因序列，设计2套特异性引物，通过反应物浓度和反应条件的优化，建立了布鲁氏菌快速检测LAMP方法。该方法能从奶样和血样中检出6个种的布鲁氏菌病原，所能检测出的最低限度为9fg/μl，较PCR方法高出10倍，具有很高的特异性、敏感性和稳定性。对田间样品进行检测发现，LAMP方法与巢式PCR方法符合率达98.95%，可为临床布鲁氏菌的快速检测提供新的技术手段。
2.借助pQCXIX和pcDNA3.1真核载体，成功构建了P39和18KD双基因共表达核酸质粒pcDNA-P39/18KD。体外转染293AD细胞，经RT-PCR鉴定、间接免疫荧光和Western blot检测，证实P39和18KD目的蛋白可在293AD细胞中有效共表达，反应原性良好。
3.经测序鉴定成功构建了L7/L12和BCSP31双基因穿梭载体pSh-LL/BP。然后与复制缺陷型Ad5腺病毒于BJ5183工程菌内同源重组，成功筛选到腺病毒重组子质粒pAd-LL/BP。将其转染293AD细胞，成功包装并纯化出共表达重组腺病毒Ad-LL/BP，滴度达10~(9.68)TCID50/mL。连续传代后经PCR检测、电镜观察、IFA、Western blot等实验证实该重组病毒没有发生外源基因缺失，两个目的蛋白获得了稳定共表达，且反应性良好。
4.以上述构建的共表达DNA疫苗和腺病毒载体疫苗单独和联合免疫BALB/c小鼠，共免疫三次。ELISA检测血清特异性抗体发现，各免疫组均能产生IgG特异性抗体，联合免疫组显著高于单独免疫组，抗体水平最高的为pcDNA-P39/18KD（prime）和Ad-LL/BP（boost）组，最低的为pcDNA-P39/18KD组；除IgA外，IgG、IgG1、IgG2a水平随免疫次数和免疫时间增加逐渐升高，IgG2a较IgG1升高更快（p<0.05）。MTT法检测脾淋巴细胞增殖指数（SI）显示，抗原和ConA刺激均能使各组SI升高，但以ConA略高（p>0.05），组间对比Cp>V3>V4>V1>V2>Cn。FCM脾脏T淋巴细胞亚群分析显示，除CD~(8+)及CD~(4+)/CD~(8+)外，各疫苗免疫组CD3+、CD4+脾脏T淋巴细胞数均显著升高（P<0.05），联合免疫组显著高于其单独免疫组。ELISA检测脾淋巴细胞诱导上清IL-12和IL-10显示，联合免疫组IL-12水平，DNA/Ad和Ad/DNA分别为70.7±8.1pg/mL和64.2±7.5pg/mL，明显高于单独DNA免疫组36.4±4.4pg/mL或重组腺病毒免疫组38.7±5.15pg/mL，但都低于A19弱毒苗免疫组85.3±8.2pg/mL（p<0.05）；而各组IL-10水平与阴性对照组相比，均无显著差异（p>0.05）。由此可见，本研究所构建的双基因表达DNA疫苗和腺病毒活载体疫苗均能产生高水平的IgG抗体，能有效诱导以细胞免疫为主的免疫应答反应。首次证实布鲁氏菌DNA和重组腺病毒疫苗联合免疫效果优于单独免疫，以pcDNA-P39/18KD（prime）和Ad-LL/BP（boost）免疫效果最好。
Brucellosis, caused by Brucella spp., is a highly infectious zoonotic disease occurring in humans andvarious species of domesticated and wild animals worldwide. At present, the incidence of human andanimal brucellosis trends to be increasing. It causes a great economic impact on livestock industry andthreats to human health. It is urgent to prevent and control the disease with new diagnostic methods andnew generation vaccines with high efficacy and safety. The main contents of this study are as thefollowing:
1. Complete coding sequences of Brucella genes OMP25, P39,18KD, L7/L12and BCSP31wereamplified and cloned into pMD18-T vector, and a series of cloning plasmids were verified by sequencesanalysis. Then based on reported OMP25sequence, two sets of four LAMP primers were designed withonline softwares. Through the optimization of the reaction system and conditions, a novel LAMP assaywas established to detect Brucella DNA from six classical species. It was confirmed that the assay wasable to detect a limit9fg/ml Brucella DNA with good sensitivity, which was10times higher than thatof a reference nested PCR. The results in detection of field samples showed that the LAMP assaydeveloped in this study yielded a99.0%concordance rate in comparison with nested PCR. These resultsindicate that the LAMP assay is a potential for the diagnosis of Brucellosis with high convenience,rapidity, sensitivity and specificity.
2. Using pMD18-T, pQCXIX and pcDNA3.1, we successfully constructed a recombinant DNAvaccine pcDNA-P39/18KD with IRES sequence. The pcDNA-P39/18KD recombinant plasmids weretransfected into293AD cells using lipofectamine. The insertion of two genes was validated by RT-PCR,IFA and Western blot. The co-expression of P39and18KD genes cloned into pcDNA3.1was confirmedand its specific reactivity with antisera was observed.
3. L7/L12and BCSP31genes were excised from pQCXIX vector with IRES sequence and clonedinto a transfer vector pShuttle-CMV. The recombinant plasmid pSh-LL/BP was constructed and verifiedby sequence analysis. Through homologous recombination between the plasmid pSh-LL/BP andskeleton vector pAdEasy-1in E. coli BJ5183, a recombinant plasmid pAd-LL/BP was obtained. Then293AD cells were successfully transfected and a recombinant adenovirus Ad-LL/BP was packed. Thetiter of the recombinant adenovirus containing L7/L12and BCSP31genes of Brucella spp. was109.68TCID50/mL in293AD monolayer cells. After continuously passaging, maintenance and stableco-expression of the two target genes in the recombinant adenovirus Ad-LL/BP were confirmed by PCR,IFA, Western blot and transmission electron microscope, and the two target proteins showed specificreactivity.
4. The pcDNA-P39/18KD DNA vaccine and the pAd-LL/BP adenoviral vector vaccine wereevaluated in BALB/c mice by intramuscular injection alone or in combination after proliferated andpurified. Fifty BALB/c mice were divided into ten groups (5mice in each group), and inoculated threetimes at a two-week interval. Blood samples were taken before each injection, and15days after final administration the mice were sacrificed for blood and aplenocytes collection.
ELISA results showed that the IgG was elicited in serum of all the mice immunized with DNA andrecombinant adenovirus vaccines alone or in combination from the15th-day post first inoculation. Inprime-boost, combination immunization groups showed significantly higher antibody levels than eithervaccine alone. The highest level of specific IgG was observed in the group with pcDNA-P39/18KD(prime) and Ad-LL/BP (boost) regimen， and the lowest in the group with pcDNA-P39/18KDimmunization. Except for IgA, the levels of all IgG, IgG1and IgG2a all rose with the increase ofvaccination times, in which IgG2a levels were great higher than IgG1(p<0.05). MTT results showedthat lympho-spleencytes stimulation index (SI) of the mice in each group immunized with DNA vaccineand recombinant adenovirus vaccine alone or in combination increased after A19antigen or ConAstimulation, but a little higher with ConA stimulation than A19antigen (p>0.05). A SI order in differentgroups was Cp>V3>V4>V1>V2>Cn. Analysis of spleen T lymphocyte subsets by FCM showed that thepercentage of CD~(3+)and CD~(4+)from mice was significantly higher in immunized groups than in negativecontrol groups, and also higher in prime-boost combination immunization groups than each vaccinealone. But there were no remarkable differences in the percentage of CD~(8+)and CD~(4+)/CD~(8+)from mice indifferent immunized groups. ELISA results of the induced IL-12levels from spleen lymphocytesshowed that the expression levels of IL-12were70.7±8.1pg/mL and64.2±7.5pg/mL in DNA/Ad andAd/DNA combination immunization group, respectively, which were significantly higher than36.4±4.4pg/mL and38.7±5.15pg/mL in respective DNA and recombinant adenovirus immunization alone(p<0.05). Conversely, there were no obvious differences in IL-10levels between the immunized groupsand the negative control groups.
In summary, the DNA and recombinant adenovirus vaccines co-expressing two genes of Brucella spp.constructed in this study were able not only to induce high level IgG specific antibody, but also toinduce both specific humoral immunity and predominant Th1-type cellular immune responses inBALB/c mice. The present study is the first demonstration that Brucella-specific immune responses canbe enhanced more significantly using prime-boost combination immunization than either the DNAvaccine or the recombinant adenovirus vaccine. The immune response induced by thepcDNA-P39/18KD priming and the Ad-LL/BP boosting appeared to be efficient against Brucellosis inmouse.
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