【作者】 王振华；

【导师】 曾宪垠；

【作者基本信息】 四川农业大学 ， 临床兽医学， 2012， 博士

【摘要】 黏膜免疫系统是人和动物机体整个免疫网络的重要组成部分,又是具有独特结构和功能的独立免疫系统,在抵抗感染方面起着积极重要的作用。首先,黏膜表面与外界抗原(主要指病原微生物)接触,是机体抵抗感染的第一道防线；其次,黏膜免疫系统具有共同黏膜免疫共享机制,这一机制能将全身黏膜免疫状态达成一致；再次,黏膜免疫可同时诱发黏膜免疫和系统免疫应答。因此,黏膜免疫日益受到重视,改变免疫策略,设计黏膜免疫疫苗,对预防传染病的发生具有重要意义,也是传染病预防领域的新技术热点之一猪繁殖与呼吸综合征(Porcine reproductive and respiratory syndrome, PRRS)一直是危害世界养猪业比较严重的疾病,给世界养猪业造成巨大的经济损失。由于其病毒的致病特点,使目前的商品化疫苗存在着缺陷,不能完全阻止经呼吸系统及胎盘传播猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus, PRRSV)。鉴于此,本研究结合PRRSV的致病特点、免疫特性及生物学特性探索性的设计黏膜免疫新型疫苗,对预防该病具有重要意义。试验选取PRRSV的ORF6基因作为目的基因,乳酸乳球菌乳链菌肽控制表达(nisin controlled expression, NICE)系统为抗原递呈载体,构建重组乳酸乳球菌活载体疫苗,并对其免疫反应原性及佐剂效力进行评价。主要研究工作和结果如下：1、试验将PRRSV经Marc-145细胞培养后,进行RT-PCR扩增,获得大小为515bp的抗原基因ORF6,将其克隆到PMD19-T载体后测序,核苷酸序列未发生变异。根据测序结果和乳酸乳球菌表面表达载体PNZ8149特点,设计引物,扩增并纯化出两端含有Sac Ⅰ和NcoⅠ酶切位点的PRRSV的ORF6基因片段,将纯化后的ORF6基因与表达载体pNZ8149进行连接,连接产物采用电转化方法将其转入食品级乳酸乳球菌NZ3900细胞中构建重组菌,命名为PNZ8149/NZ3900-M/PRRS。进一步对PNZ8149/NZ3900-M/PRRS进行诱导,诱导剂乳链杆菌肽(Nisin)添加量为10ng/mL诱导时间分别为2h、4h、6h、8h、10h和12h, SDS-PAGE分析,在19KD处出现了预期的条带,表达了分子量为19KD的蛋白,诱导6h条带比较清晰,是最佳的诱导时间。间接免疫荧光实验表明,重组菌表达蛋白定位于菌体细胞表面且具有反应原性。试验结果表明,成功构建了以乳糖为选择标记的食品级重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS,为开发PRRSV的乳酸乳球菌黏膜免疫疫苗奠定了理论基础。2、将构建的重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS免疫BALB/c小鼠,将小鼠随机分成4组(n=30),组Ⅰ鼻腔免疫PBS缓冲溶液,为空白对照组；组Ⅱ鼻腔免疫空载体菌株培养液,为载体对照组；组Ⅲ鼻腔免疫重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS悬浮菌液,为鼻腔免疫实验组；组Ⅳ口服免疫重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS悬浮菌液,为口服免疫实验组。各组试验动物被连续免疫(即：1d,2d,3d,11d、,12d,13d,21d,22d,23d),免疫剂量为50ul,悬浮菌液细菌含量为1×1011CFU/mL。试验动物在最后一次免疫后0d、7d、14d、21d、28d分别被处死,每次每组处死6只,取血液、肠粘液、呼吸道冲洗液检测抗体,采用间接ELISA方法检测血清中特异性IgG和黏膜中s-IgA抗体含量,评价其动态变化情况,并在最后一次免疫后14d取脾脏进行细胞因子IL-2、IL-4、IL-5、IL-10IFN-γ等活性检测。重组乳酸乳球菌经口服免疫和鼻腔免疫后均能刺激黏膜分泌特异性s-IgA,在整个免疫测定期间内,两试验组在呼吸道黏膜及消化道黏膜产生的特异性s-IgA抗体水平均极显著高于PBS对照组和载体对照组((P<0.01),两试验组间滴鼻免疫试验组高于口服试验组,且呼吸道黏膜s-IgA抗体水平差异极显著((P<0.01),消化道黏膜s-IgA抗体水平差异不显著(P>0.05)。在整个测定期间内粘膜抗体s-IgA保持稳定趋势,可能由于抗体测定时间比较迟或测定周期相对较短；同时在最后一次免疫后14天检测小鼠脾细胞悬液IL-5的活性,重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS免疫组明显高于对照组,差异极显著(p<0.01),口服免疫组和滴鼻组无显著差异(p>0.05),IL-5的活性对黏膜免疫的调节起重要作用,协同其它细胞因子(如IL-2、IL-4、IL-6等)刺激B细胞的增殖与分化,促进活化的B细胞分化成IgA+B细胞,合成分泌型IgA。重组菌PNZ8149/NZ3900-M/PRRS免疫小鼠后,两试验组脾细胞悬液中IL-2和IFN-γ与对照组差异极显著(p<0.01),滴鼻免疫组IL-2和IFN-γ水平高于口服免疫组,滴鼻免疫实验组IL-2和IFN-γ水平分别达到349.848pg/ml和173.276pg/ml,口服免疫实验组IL-2和IFN-γ水平分别达到335.455pg/ml和162.913pg/ml；IL-4水平在各组之间差异不显著(p>0.05),但滴鼻免疫实验组略高于口服实验组,分别为21.466pg/ml和19.799pg/ml。重组菌试验组血清中特异性IgG抗体水显著高于PBS对照组和载体对照组,差异极显著((P<0.01)；鼻腔免疫组抗体水平在最后一次免疫后7天达到最高,在整个测定时间内维持在高水平,口服免疫组抗体水平在最后一次免疫后21天达到最高水平,然后迅速下降。以上结果表明,重组菌PNZ8149/NZ3900-M/PRRS经口服及鼻腔免疫后,能诱导产生粘膜免疫反应,并同时激发Thl型系统免疫应答,即体液免疫和细胞免疫应答,并且滴鼻免疫途径优于口服免疫途径。3、将构建的重组乳酸乳球菌PNZ8149/NZ3900-M/PRRS抗原与黏膜佐剂CpG ODN联合鼻内免疫BALB/c小鼠,评价其佐剂效应。试验选取BALB/c小鼠90只,随机分为3组(n=30),分别为重组菌对照组(组Ⅰ)、佐剂+重组菌实验组(组Ⅱ)和佐剂对照组(组Ⅲ),佐剂免疫剂量每只小鼠为10ug,重组菌免疫剂量、免疫方法及检测指标同第二部分试验。结果得出CpG ODN佐剂协同重组菌抗原组血清特异性抗体IgG高于单独佐剂组和抗原组,呼吸道和肠道黏膜s-IgA水平高于单独佐剂组和抗原组,并且呼吸道黏膜s-IgA抗体水平高于肠道黏膜抗体；最后一次免疫后14天检测脾细胞悬液中IL-2、IFN-γ、IL-4及IL-5的活性。IL-2、IFN-γ和IL-5的活性佐剂CpG ODN协同重组菌抗原组高于单独抗原组(p>0.05),显著高于单独佐剂组(P<0.01)；IL-4水平在各组之间差异不显著(p>0.05)。结果表明CpG ODN能协同重组菌提高小鼠黏膜免疫和系统免疫反应,为研究安全有效的重组菌黏膜疫苗奠定了基础。更多还原

【Abstract】 The mucosal immune system is an important part of the entire immune network in human and animal body, and it is structurally and functionally divided into sites for antigen uptake and processing at inductive sites. The mucosal immune system play an important role in resisting to infection, First of all, mucosal s-IgA can form a barrier to pathogens at the mucosal surface by preventing the initial attachment of the pathogen and its infiltration of the surface layers or by binding to and neutralizing toxins that the pathogen produces, which is the first line of defending infection; Second, the mucosal immune system has a common mucosal immune mechanism which make systemic mucosal immunity status consistent. Against, the mucosal immune system must also be capable of inducing effective cell-mediated and antibody-mediated immune responses towards selected antigens. The information indicates that the mucosal immune is an only management strategies and a one of new technologies in the field of epidemics prevention.Porcine reproductive and respiratory syndrome virus (PRRSV) continues to be a threat, causing economically significant impacts on the swine industry worldwide. However none of the current commercial vaccines is able to completely prevent respiratory infection, trans-placental transmission, pig-to-pig transmission the virus nor maintaining immune protection in sows. We share the information related to PRRSV and review the available options for PRRS control strategies based on pathogenic characteristics, the immune properties and biological characteristics of PRRSV. In this study, the Nisin controlled expression (NICE) system of Lactococcus lactis was selected as vector of recombinant protein expression to express ORF6gene, the aim was to develop the gene engineering L.1acti mucosal vaccines of PRRSV, and evaluate immune activity and adjuvant effect. The results of research are summarized as following.1. In this study, the ORF6structural proteins gene of PRRSV)was amplified by RT-PCR. The ORF6gene was subcloned into the vector PMD19-T and sequenced, the gene consistion of515bp and the nucleotide sequence is not mutate. One pairs of primers was designed according to the nucleotide sequence and the vector PNZ8149characteristics. The DNA of the ORF6gene was amplified by PCR using PMD19-T-M as template, the PCR product with Sac I and Nco I restriction enzyme sites was inserted into vector PNZ8149, and transformed into L.1acti NZ3900by electroporation, result in recombinant strain PNZ8149/NZ3900-M/PRRS. Then, the recombinant protein was detected by Western blot and IFA experiments after the bacteria was respectively induced2h,4h,6h,8h,10h,12h by10ng/mL Nisin. The result indicated that the molecular weight of the expressed recombinant protein was about19kDa, protein bands is relatively clear at6h, the protein maintain the antigenicity of PRRSV as expected, moreover the protein was secreted and located on the surface of the bacteria. The result showed that we successfully constructed the food-grade recombinant L.1acti PNZ8149/NZ3900-M/PRRS in which the lactose operon, Furthermore, the recombinant protein was located on the surface of the bacteria and had reactogenicity with antibody against PRRSV. The study is expected to lay a theoretical foundation on developing of the gene engineering L. lacti s mucosal vaccines.2. To evaluate the changes of immune response of recombinant L.1acti PNZ8149/NZ3900-M/PRRS strain that expresses ORF6gene of PRRSV, BALB/c mice were immunized with the bacterial supernatant. Six to eight week old BAI B/C mice were randomly divided into four groups (group I, Ⅱ,Ⅲand Ⅳ, respectively), with each group consists of30animals. The control group I were immunized intranasally (i.n.) with50ul PBS, the control group Ⅱ were immunized i.n.with50ul L.lactis cell suspension (1011CFU) carrying the control vector, the experimental groupⅢ were vaccinated i.n. administration of50ul recombinant L.1actis cell suspension (1011CFU), the experimental groupIV were vaccinated orally (o.r) administration of50ul recombinant L.1acti s cell suspension (10"CFU). The mice were were vaccinated on days1,2,3,11,12, and13and on days21, 22, and23.)-The mice were respectively sacrificed on day23,30,37,44, and51, respectively. Blood samples, lung lavage fluid and intestinal washes were obtained on different days after immunization, IgG in serum and s-IgA in lung lavage fluid and intestinal washes were analyzed by using a similar enzyme-linked immunosorbent assay (ELISA). For the purpose of evaluating of changes about cytokines IL-2、IL-4、IL-5、 IL-10and IFN-y, the spleen collected on day37were analyzed.The results clearly shows that mice immunized with recombinant strain PNZ8149/NZ3900-M/PRRS could induce remarkable special s-IgA in intestinal washes and lung lavage fluid, at the whole experimental session, compared with the control group Ⅰ and Ⅱ, the level of special s-IgA showed significantly increase ((P<0.01), the production of the s-IgA antibody in lung lavage fluid from the i.n immunized mice was higher significantly than that from the o.r immunized mice ((P<0.01), the production of the s-IgA antibody in intestinal washes from the i.n immunized mice was higher than the o.r immunized mice ((P<0.05). The s-IgA antibody from the immunized mice get it’s maximum number and maintain a stable trend at the whole experimental session, it is possible that the measurement time of s-IgA is late or the detection cycle is short. Cytokines IL-5responses play an important role in inducing s-IgA antibody responses, therefore, we examined the activity of IL-5in splenocytes on day37, there was significant production of IL-5in mice administered with recombinant strain by the way of oral and intranasal, but the production of IL-5in intranasal immunized mice was no significant compared to that from the oral immunized mice ((P>0.05).To assess cell responses induced by recombinant L.1actis expressing ORF6gene of PRRSV, we investigated the activity of IL-2and IFN-y in splenocytes on day37. IL-2and IFN-y contents of the experimental groupIIIandIV were significant higher than those of the control group I and II (P<0.01), furthermore the i.n immunized mice was higher significantly than that from the o.r immunized mice (P<0.01), IL-2and IFN-y contents in splenocytes from mice immunized intranasally was respectively349.848pg/ml and173.276pg/ml. as well as, IL-2and IFN-y contents in splenocytes from mice immunized orally was respectively335.455pg/ml and162.913pg/ml. Notably, it is worthy to note that cytokines of IL-4contents was not different to the all groups. IgG anti-PRRSV was detected in serum, in contrast, the level of special IgG was higher significantly following experimental groupⅢ and Ⅳ compared to the control group Ⅰ and Ⅱ((P<0.01) at all time points the i.n immunized mice get it’s maximum number on day30, which lasted at least4weeks post-immunization; as well as the o.r immunized mice get it’s maximum number on day44, following decreased rapidly. Regretful, the titer of IgG was not investigated, for the reason that whether is able to resistant to the virus.All the results indicated that mice immunized by the recombinant L.1actis expressing ORF6gene of PRRSV can significantly induce mucosal immune responses, humoral immunity responses and enhanced Thl immunity response against the PRRSV, in contrast, Intranasal immunization is superior to oral immunization.3. Oligonucleotides containing CpG motifs (CpG ODN) are strong adjuvant for immune responses, particularly in mice. Recently, it has been showed that CpG ODN is a promising mucosal adjuvant, but data on mucosal immune responses induced by CpG ODN plus the recombinant L.1actis are scarce. To investigate the immune responses induced by intranasal immunization, with some doses of CpG ODN plus the recombinant L.lactis expressing ORF6gene of PRRSV antigen. Six to eight week old BAI B/C mice were randomly divided into three groups (group Ⅰ, Ⅱ, andⅢ, respectively), with each group consists of30animals. The control group Ⅰ were immunized i.n. administration of50ul recombinant L.1actis cell suspension (1011CFU), the experimental groupⅡ were immunized i.n.10ul CpG ODN plus50ul administration of recombinant L.1actis cell suspension, the control groupⅢ were vaccinated i.n.10ul CpG ODN. Immunization dose, immunological methods and indicators detected in common with the second part of the test. The results clearly show that CpG ODN promoted the induction of IgG antibody in serum and s-IgA antibody in intestinal washes and lung lavage fluid. And the production of the s-IgA antibody in lung lavage fluid of immunized mice was higher than that in intestinal washes; the activity of cytokines IL-2and IFN-γ in splenocytes on day37were investigated, in compared with the control group Ⅰ, the activity of cytokines IL-2and IFN-γ in CpG ODN plus recombinant L.1actis were increased, and higher significantly than that of the control groupⅢ(p<0.01). Results from this study indicate that stimulatory CpG ODN may be effective as a mucosal adjuvant with the recombinant L.1actis expressing ORF6gene of PRRSV antigen. The study is expected to lay a theoretical foundation on the adjuvant about recombinant strain vaccine.更多还原