【作者】 乔薪瑗；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2008， 博士

【摘要】 猪轮状病毒是无囊膜、分节段的双股RNA病毒,隶属于呼肠孤病毒科,是引起仔猪腹泻的重要病原之一。猪轮状病毒感染呈世界范围分布,给养猪业带来了极大的危害。本研究以乳酸乳球菌Lactococcus lactis NZ9000和干酪乳杆菌Lactobacillus casei ATCC 393(L.casei 393)作为递呈抗原的活菌载体,以猪轮状病毒主要免疫保护性抗原VP4蛋白的基因片段为靶基因,并插入大肠杆菌不耐热肠毒素B亚基因(LTB),构建了表达猪轮状病毒VP4基因和共表达VP4-LTB的重组乳酸乳球菌表达系统和干酪乳杆菌表达系统,并对免疫效果进行比较分析。根据目的基因和融合表达载体质粒的特点,应用oligo6.0软件进行设计引物。以VP4-pGEX-6P-1质粒作为模板,通过PCR扩增得到大约756 bp的VP4基因,以pMD-18T-LTB质粒作为模板,通过PCR扩增得到大约375 bp的LTB基因,PCR产物经酶切后,与带有粘性末端的pNZ8112载体连接,得到pNZ8112-VP4和pNZ8112-VP4-LTB的质粒。重组质粒pNZ8112-VP4和pNZ8112-VP4-LTB分别电转化乳酸乳球菌NZ9000 ,得到重组菌pNZ8112-VP4和pNZ8112-VP4-LTB,然后将重组菌进行酶切、PCR鉴定和测序分析。以同样方法得到pPG-1-VP4和pPG-1-VP4-LTB重组菌,然后进行酶切、PCR鉴定和测序分析。构建的重组菌pNZ8112-VP4和pNZ8112-VP4-LTB在GM17培养液中培养,通过Nisin来诱导目的蛋白的表达。重组菌中蛋白的表达和定位通过SDS-PAGE、Western blotting和免疫荧光进行鉴定。考马斯亮蓝染色结果显示:pNZ8112-VP4和pNZ8112-VP4-LTB的菌体裂解物分别表达了27KD和40KD的融合蛋白,而诱导前的菌体没有目的蛋白表达。诱导后表达的VP4和VP4-LTB蛋白经Western blotting分析,可检测到27KD和40KD的特异性反应条带,诱导前的菌体没有相应的条带出现。这些结果表明Nisin能有效诱导乳酸乳球菌NZ9000中异源蛋白的表达。采用抗VP4的鼠血清进行免疫荧光试验的结果显示,pNZ8112-VP4和pNZ8112-VP4-LTB诱导后的菌体表面能看到黄绿色荧光,而诱导前的菌体则没有荧光反应,且细胞被伊文思蓝染成红色。构建的重组菌pPG-1-VP4和pPG-1-VP4-LTB在MRS培养液中进行培养,利用乳糖来诱导目的蛋白的表达。重组菌目的蛋白的表达和定位也是通过SDS-PAGE、Western blotting和免疫荧光来鉴定。考马斯亮蓝染色可看到pPG-1-VP4和pPG-1-VP4-LTB的菌体裂解物分别有大约40KD和53KD的蛋白表达,而未诱导的菌体中没有蛋白表达。通过Western blotting检测诱导后的菌体裂解物,可看到40KD和53KD的特异性反应条带,而未诱导的菌体则没有相应的条带出现,这些结果表明乳糖能有效诱导L.casei 393中异源蛋白的表达。通过间接免疫荧光方法鉴定,pPG-1-VP4和pPG-1-VP4-LTB的菌体表面均能看到黄绿色荧光,而未诱导的菌体则没有荧光反应,且细胞被伊文思蓝染成红色。为检验重组菌是否能诱导黏膜和系统免疫应答,将BALB/c小鼠分为六组,通过口服途径分别免疫pPG-1 , pPG-1-VP4 , pPG-1-VP4-LTB , pNZ8112 , pNZ8112-VP4和pNZ8112-VP4-LTB。每只小鼠分别免疫109 c.f.u.单位的重组菌,每次连续免疫三天,第一次加强免疫在第一次免疫后的17、18、19天,第二次加强免疫在第一次免疫后的33、34、35天。每次免疫后的第7天收集小鼠血清;每次免疫后的1、2、7天收集粪便;眼洗液是在每次免疫后的第7天以50μL的PBS冲洗眼结膜获得;阴道洗液是在每次免疫后的第7天用200μL的PBS冲洗阴道获得;母鼠分娩后的3、5、7天收集乳汁。所有收集的样品保存于–20℃备用。为保持黏膜系统的稳定,许多防御机制都参与进行持久有效地作用。分泌性IgA的参与也是防御机制之一,IgA抗体通过阻止病原微生物的入侵和定植并竞争受体和代谢底物。为评价黏膜免疫反应,通过ELISA方法来检测黏膜样品中特异性IgA抗体的水平。在粪便、眼洗液和阴道洗液中我们检测到了高水平的特异性IgA,与对照组相比差异显著。其中免疫pPG-1-VP4的小鼠中特异性IgA抗体水平高于免疫pNZ8112-VP4的小鼠,免疫pPG-1-VP4-LTB的小鼠中特异性IgA抗体水平也高于免疫pNZ8112-VP4-LTB的小鼠,这是因为L.casei 393同Lactococcus lactis NZ9000相比具有更好的肠道定植能力。免疫pPG-1-VP4-LTB和pNZ8112-VP4-LTB的小鼠粪便、眼洗液和阴道样品中IgA抗体水平均高于免疫pPG-1-VP4和pNZ8112-VP4的小鼠。这是由于添加了黏膜免疫佐剂LTB的缘故,表明LTB能加强黏膜系统反应。具有低免疫原性的疫苗尤其需要强有力的佐剂来加强抗原递呈,本研究证明LTB作为一种安全有效的佐剂具有极大的潜力。免疫pPG-1-VP4和pPG-1-VP4-LTB的小鼠血清中IgG的效价相当,但都高于免疫pPG-1的对照组。免疫pNZ8112-VP4和pNZ8112-VP4-LTB的小鼠血清中IgG抗体的效价相当,但都高于免疫pNZ8112的对照组。在本研究中,口服免疫表达VP4和VP4-LTB的重组乳酸菌不仅能诱导产生黏膜免疫,而且诱导产生了系统免疫。通过口服免疫产生的黏膜免疫反应不只局限于胃肠道,也引起了其他黏膜部位的免疫反应。IgA抗体能和抗原结合并阻止其进入,降低炎性反应的程度,从而阻止了对组织的损伤,通过分泌性IgA抗体在黏膜表面对病原体进行排斥和清除,这对预防轮状病毒感染是至关重要的。将新分离的免疫鼠脾细胞稀释成5×106个/ mL并培养66h,分别用0.5μg/mL,5μg/mL和25μg/mL的VP4抗原进行刺激,收获培养上清用于检测细胞因子。脾淋巴细胞的增殖通过ELISA进行检测,结果表明0.5μg/mL和5μg/mL的VP4与25μg/mL的VP4相比,具有更强的刺激增殖作用。IL-4和IFN-γ的检测采用Biosource检测试剂盒,方法参照说明书。结果表明免疫组中IL-4和IFN-γ的分泌显著高于对照组,组间差异显著,且分泌IL-4的水平显著高于IFN-γ。这些数据表明,通过口服途径免疫重组菌后引起了显著的细胞因子反应,从而加强了抵抗轮状病毒感染的保护作用。免疫母鼠产仔后的3、5、7天收集乳汁,通过ELISA方法检测乳汁中的特异性抗体,结果显示乳汁样品中有较高水平的特异性IgA,表明免疫重组菌产生的抗体对于幼龄动物的被动免疫保护有一定作用。综上所述,本研究所获得的结果表明,乳酸乳球菌和干酪乳杆菌可用做口服免疫传递抗原引起黏膜和系统免疫。乳酸菌作为疫苗传递载体能够定植于不同的黏膜部位,例如胃肠道、生殖道等,可被用于接种在病原体入侵的部位来抵抗感染。乳酸菌安全无毒的特点使它们更适合于用做口服疫苗载体来传递异源抗原,重组乳酸菌具有较好的免疫原性,并能引起较好的黏膜免疫反应和系统免疫反应。当VP4蛋白与LTB共表达时,加强了黏膜免疫反应,LTB具有较好的免疫原性、抗原性和佐剂特性,可被用于基因工程疫苗的研制,很适合于用做黏膜疫苗的免疫佐剂。本研究为进一步研究新型、有效的猪轮状病毒口服疫苗奠定了基础。更多还原

【Abstract】 Porcine rotaviruses are nonenveloped, segmented, doublestranded RNA viruses classified in the family Reoviridae. Porcine rotaviruses are the major cause of diarrhea in the piglets .Porcine rotavirus has been widely distributed in the world and cost pig producers serious loss.In this study, Lactococcus lactis NZ9000 and Lactobacillus casei ATCC 393 (L.casei 393) were selected as an antigen delivery vehicle for the development of live mucosal vaccine. The main protective antigen VP4 was selected as the target gene and E.coli heat-labile toxin B subunit protein was inserted. We constructed recombinant Lactococcus lactis NZ9000 and recombinant L.casei 393 systems expressing VP4 and co-expressing VP4-LTB.The immunogenicity responses induced by oral mucosal immunizations with recombinant strains were compared.We designed primers with oligo6.0 software according with target gene and considering the characters of fusion expression vector plasmid. About 756bp gene fragment (VP4) and 375bp gene fragment were amplified by PCR using the plasmid VP4-pGEX-6P-1 and the plasmid pMD-18T-LTB. The PCR products were digested by restriction enzyme, linked with expression vector pNZ8112 digested by restriction enzyme , giving rise to pNZ8112-VP4 and pNZ8112-VP4-LTB.The recombinant plasmids pNZ8112-VP4 and pNZ8112-VP4-LTB were electroporated into Lactococcus lactis NZ9000 respectively, generating pNZ8112-VP4/ NZ9000 and pNZ8112-VP4-LTB/ NZ9000 followed enzyme digestion, PCR identification and sequence analysis.At the same time, we obtained pPG-1-VP4/ L.casei 393 and pPG-1-VP4-LTB/ L.casei 393 by the same way.They also followed enzyme digestion, PCR identification and sequence analysis.The recombinant strains pNZ8112-VP4 and pNZ8112-VP4-LTB constructed were induced by Nisin in GM17 medium to express interest protein. The expression and localization of the protein from recombinant strains were detected via SDS-PAGE , Western blotting , and immunofluorescence. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 27KD and 40KD fusion protein were expressed in lysates of pNZ8112-VP4 and pNZ8112-VP4-LTB, but not expressed when the same cells were grown in GM17 medium without Nisin . The localization of the VP4 and VP4-LTB protein nisin-induced were analyzed via Western blotting .Immunoreactive bands (27KD,40KD) were detected , and corresponding immunoreactive bands did not display when they were not induced. These results show that Nisin promoter from Lactococcus lactis NZ9000 could efficiently induce the expression of heterologous protein. The immunofluorescence was developed with the mouse anti-VP4 serum. The results indicated that there was green-yellow fluorescence on the cell surface of pNZ8112-VP4 and pNZ8112-VP4-LTB when induced. The cells uninduced did not display an immunofluorescence reaction and were dyed red by Evans blue.The recombinant strains pPG-1-VP4 and pPG-1-VP4-LTB constructed in this study were induced by lactose in MRS medium to express interest protein. The expression and localization of the protein from pPG-1-VP4 and pPG-1-VP4-LTB were also detected via SDS-PAGE, Western blotting, and immunofluorescence. The lysates of the cells were analyzed by SDS-PAGE. Coomassie blue gel staining showed that 40KD and 53KD fusion protein were expressed in lysates of pPG-1-VP4 and pPG-1-VP4-LTB, but not expressed when the same cells were grown in MRS medium without lactose . The localization of the VP4 and VP4-LTB protein lactose-induced were analyzed via Western blotting .Immunoreactive bands (40KD,53KD) were detected , and corresponding immunoreactive bands did not display when they were not induced. These results show that lactose promoter from L.casei 393 could efficiently induce the expression of heterologous protein. The immunofluorescence was developed with the mouse anti-VP4 serum. The results indicated that there was green-yellow fluorescence on the cell surface of pPG-1-VP4 and pPG-1-VP4-LTB when induced. The cells uninduced did not display an immunofluorescence reaction and were dyed red by Evans blue.To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses, six groups of ten female mice were immunized via oral route with pPG-1, pPG-1-VP4,pPG-1-VP4-LTB,pNZ8112,pNZ8112-VP4 and pNZ8112-VP4-LTB respectively. Every mouse received dose of 109 colony-forming units (c.f.u.)/mL of recombinant strains, respectively. The immune protocol was administered on three consecutive days at days 1,2 and 3. A booster immunization was given at days 17,18 and 19 and a second booster was given at days 33,34 and 35. Serum of mice were collected 7 days after every immunization. Fecal pellets were collected 1, 2, and 7 days after every immunization. Ophthalmic wash were obtained by washing the eyes with 50μL phosphate-buffered saline (PBS) 7 days after every immunization. Vaginal samples were collected by washing the vagina with 200μL PBS 7 days after every immunization . Milk were collected 3、5、7 days after delivery. All samples were stored at–20℃until required.To maintain homeostasis in the mucosae,different defense mechanisms are involved in permanent and effective surveillance. One of these mechanisms is the secretory immune system through IgA antibodies. IgA prevents the invasion and colonization of pathogenic microorganisms and competes for receptors and metabolic substrates. To assess mucosal immune responses, specific IgA levels in mucosal samples were determined by ELISA. Specific IgA reached a high level in the fecal pellets,ophthalmic and vaginal wash. In contrast,only background levels of antibodies were detected in control animals.The IgA levels of mice administered with pPG-1-VP4 are higher than those administered with pNZ8112-VP4. The IgA levels of mice administered with pPG-1-VP4-LTB are higher than those administered with pNZ8112-VP4-LTB. This is because L.casei 393 has better colonization ability in intestinal tracts than Lactococcus lactis NZ9000. The IgA levels of mice administered with pPG-1-VP4-LTB or pNZ8112-VP4-LTB are higher than those administered with pPG-1-VP4 or pNZ8112-VP4 in fecal, ophthalmic and vaginal samples. This is due to the addition of mucosal immunoadjuvant LTB. This indicates that LTB can enhance the mucosal system response. Vaccines with low immunogenicity in particular require the use of a strong adjuvant to enhance the antigen-presenting .This study has highlighted the potential of LTB as a safe and effective adjuvant.IgG titers of serum in mice given pPG-1-VP4 or pPG-1-VP4-LTB were similar but higher than the mice given pPG-1. IgG titers of serum in mice given pNZ8112-VP4 or pNZ8112-VP4-LTB were similar but higher than the mice given pNZ8112. In this study, oral administration of recombinant strains displaying VP4 or VP4-LTB protein antigens induced both systemic and mucosal immune responses. Oral immunization elicited specific mucosal responses at the site of gastrointestinal tract, as well as the remote mucosal sites. The IgA antibodies can bind the antigen and minimize its entry with a consequent reduction in inflammatory reactions, which prevents a potentially harmful effect on the tissue . The immune exclusion and elimination of the pathogen at the mucosal surfaces by secretory IgA is crucial in preventing porcine rotavirus .Freshly isolated mouse splenocytes were seeded at 5×106 cells/mL and incubated for 66 h. 0.5μg/mL,5μg/mL and 25μg/mL VP4 were added to challenge.Cell culture supernatants were harvested and analyzed for the presence of cytokines. Splenic lymphocyte proliferation assay was measured by ELISA. The result indicated that 0.5μg/mL and 5μg/mL VP4 have stronger challenge than 25μg/mL VP4. Interleukin-4 (IL-4) and gamma interferon (IFN-γ) cytokine assays were performed using Biosource ELISA kits according to the manufacturer’s instructions. The result indicated that all immunized groups secreted significantly more IL-4 and IFN-γthan the control group.Significant differences were seen among the groups. Immunized mice secreted increased levels of IL-4 that were significantly higher than IFN-γ. Overall, these data show that immunization of recombinant strains via oral route produced significant innate-type cytokine responses. It suggested that enhanced protection against infection of rotavirus .Furthermore, we collected the milk of mice 3、5、7 days after delivery.Specific IgA in the milk were detected by ELISA.The result indicated that there were high levels of specific IgA in the samples.It shows that antibodies produced by administration with recombinant strains can serve passive immunoprotection to the immature animals.In conclusion , the results obtained so far demonstrate that Lactococcus lactis and lactobacillus are capable of delivering antigen to the mucosal and systemic immune systems following oral immunization. Lactic acid bacteria as vaccine delivery vehicles are able to colonize different mucosal sites such as gastrointestinal tracts, genital tracts and could thus be used specifically to vaccinate against pathogens at their site of entry. Their harmLess nature make them more appropriate as an oral vaccine carrier to deliver heterologous antigens. The recombinant strains have better immunogenicity and can elicit stronger specific mucosal immune responses and systematic immune responses. When VP4 was co-expressed with LTB, it showed much stronger mucosal immune responses. It indicated that LTB with qualified immunoreactivity, antigenicity and adjuvanticity could be used to develop genetically engineered vaccine. It should be suitably applicable as an immunoadjuvant for mucosal vaccines.All theses work established a good foundation for further study on the new and effective recombinant oral vaccine of porcine rotavirus.更多还原