【作者】 王相清；

【导师】 李一经；

【作者基本信息】 东北农业大学 ， 预防兽医学， 2009， 硕士

【摘要】 以干酪乳杆菌Lactobacillus casei ATCC 393(L.casei 393)作为递呈抗原口服疫苗活菌载体,将猪细小病毒保护性抗原VP2蛋白的基因片段与大肠杆菌不耐热肠毒素B亚单位(LTB)作为目的基因,构建了共表达猪细小病毒主要免疫保护性抗原VP2蛋白与LTB的重组干酪乳杆菌表达系统,经过动物免疫试验,分析比较了共表达VP2-LTB重组干酪乳杆菌与单独表达VP2重组干酪乳杆菌系统免疫效果及细胞表面表达与分泌表达两种不同表达方式的免疫效果。根据目的基因和融合表达载体质粒的特点,应用Primer Premier 5.0软件进行设计引物。以pMD18-T-VP2质粒和pMD18-T-LTB质粒作为模板,通过PCR扩增分别得到大约1750bp的VP2基因和约375bp的LTB基因,PCR产物经酶切后,胶回收连接,并以连接产物为模板,以VP2的上游引物和LTB的下游引物扩增VP2-LTB,经PCR扩增出约2.1kb的目的基因VP2-LTB,将该基因片段克隆到pMD18-T simple载体,并进行了酶切、PCR鉴定和序列测定,重组质粒命名为pMD18-T-VP2-LTB。重组质粒经BamHI和XhoI双酶切,回收目的基因片段VP2-LTB,分别与经同样双酶切的表达载体pPG-1和pPG-2连接,电转化感受态L.casei 393,筛选阳性克隆,并进行酶切、PCR和测序鉴定,重组质粒命名为pPG-1-VP2-LTB和pPG-2-VP2-LTB,重组干酪乳杆菌命名为pPG-1-VP2-LTB/L.casei 393和pPG-2-VP2–LTB /L.casei 393。构建的两种重组菌pPG-1-VP2-LTB/L.casei 393和pPG-2-VP2-LTB/L.casei 393在MRS培养液中进行培养,以2%乳糖为诱导剂进行目的蛋白的诱导表达。重组菌目的蛋白的表达和定位通过SDS-PAGE、Western blot和免疫荧光及免疫胶体金电镜鉴定。细胞表面表达型重组干酪乳杆菌经诱导后的SDS-PAGE检测结果表明,有约80kD的融合蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot结果分析表明,表达的蛋白可被鼠源PPV抗血清所识别,间接免疫荧光及免疫胶体金电镜实验结果表明,所表达的蛋白能够在干酪乳杆菌菌体表面检测到;分泌表达型重组干酪乳杆菌经诱导后,对诱导表达的菌体及培养上清液进行SDS-PAGE检测表明,有约75kD蛋白得到了表达,表达蛋白的大小与理论值相符。Western blot结果分析所表达的蛋白具有与天然病毒蛋白一样的抗原特异性,实验表明,重组的目的蛋白获得了分泌表达。为检验构建的重组菌是否能诱导黏膜和系统免疫应答以及LTB作为粘膜免疫佐剂的作用,本实验以BALB/c小鼠为试验动物,进行免疫学实验。将BALB/c小鼠分为五组,每组10只,通过口服途径分别免疫接种109活菌量pPG-1/L.casei 393、pPG-1-VP2/L.casei 393、pPG-1-VP2-LTB/L.casei 393、pPG-2-VP2/L.casei 393、pPG-2-VP2-LTB/L.casei 393,每隔2w免疫一次,共免疫四次,每次连续免疫3d,一天免疫一次。于免疫前、初次免疫后第7d、21d、35d、49d采集免疫小鼠血液;免疫前、初次免疫后第1d、5d、12d、19d、26d、33d、40d、47d和54d采集免疫口服免疫组小鼠收集粪便;眼洗液和外生殖道洗液是在免疫前、初次免疫后第7d、21d、35d、49d分别以100μl的PBS冲洗眼结膜和冲洗外生殖道获得。所有收集的样品保存于–20℃备用。最后测定了小鼠粪便及眼洗液和外生殖道洗液样品中抗PPV VP2分泌型IgA及小鼠血清样本中抗PPV的特异性IgG水平。通过ELISA方法来检测黏膜样品中特异性分泌型IgA抗体的水平,来评价黏膜免疫反应情况,在粪便、眼洗液和外生殖道洗液中均检测到了高水平的特异性IgA,与对照组相比差异显著。其中,免疫组pPG-1-VP2-LTB/L.casei 393和pPG-2-VP2-LTB/L.casei 393的小鼠粪便、眼洗液和外生殖道样品中IgA抗体水平与免疫组pPG-1-VP2/L.casei 393和pPG-2-VP2/L.casei 393相比,差异显著(P<0.05)或极显著(P<0.01)。初步分析是共表达黏膜免疫佐剂LTB的缘故,表明LTB能加强黏膜系统反应。本研究实验结果初步认为LTB作为一种安全有效的佐剂,具有极大的应用潜力。共表达VP2-LTB重组干酪乳杆菌免疫组与单独表达VP2重组干酪乳杆菌免疫组相比,pPG-1-VP2-LTB/L.casei 393或pPG-2-VP2-LTB/L.casei 393中小鼠血清中IgG的效价高于pPG-1-VP2/L.casei 393或pPG-2-VP2/L.casei 393组,与免疫对照组比较,差异显著,并且分泌型的重组菌免疫性更好。在本研究中,口服免疫表达VP2-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,并且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,而且共表达VP2-LTB组的重组乳酸菌免疫后的sIgA抗体水平高于单独表达VP2组的重组乳酸菌。机体通过分泌性IgA抗体在黏膜表面对病原体进行排斥和清除,这对预防细小病毒感染是至关重要的。中和试验结果表明, pPG-1-VP2/L.casei 393组、pPG-2-VP2/L.casei 393组、pPG-1-VP2-LTB/L.casei 393组、pPG-2-VP2-LTB/L.casei 393组在免疫后49d血清的抗体中和效价分别为1:304、1:317、1:338、1:352。本研究首次构建了重组大肠杆菌不耐热肠毒素B亚单位和猪细小病毒主要免疫保护性抗原VP2蛋白的干酪乳杆菌细胞表面表达和分泌表达系统。本研究所获得的结果表明,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。当VP2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、有效的猪细小病毒口服疫苗奠定了基础。更多还原

【Abstract】 Lactobacillus casei ATCC 393 (L.casei 393) was selected as an antigen delivery vehicle for the development of live mucosal vaccine. The main protective antigen VP2 of porcine parvovirus and E.coli heat-labile toxin B subunit were selected as the target gene. We constructed recombinant L.casei 393 systems which co-expressing VP2 protein and E.coli heat-labile toxin B subunit protein. The immunogenicity responses induced by oral mucosal immunizations with recombinant strains which with LTB or without LTB were compared and two kinds of recombinant strains expressing interest protein in different cellular locations were also analyzed.We designed primers with Primer Premier 5.0 software according with target gene and considering the characters of fusion expression vector plasmid. About 1750bp gene fragment (VP2) and 375bp gene fragment were amplified by PCR using the plasmid pMD18-T-VP2 and the plasmid pMD18-T-LTB. The PCR products were digested by restriction enzyme, linked by T4 DNA ligase. About 2.1kbp gene fragment (VP2-LTB) was amplified by PCR using the product of linked. The interest gene fragment VP2-LTB was cloned into plasmid pMD18-T simple followed enzyme digestion, PCR identification and sequence analysis, and the recombinant plasmid was named pMD18-T-VP2-LTB. Then the pMD18-T-VP2-LTB plasmid was doubly digested by BamHI and XhoI, and the interest gene fragment VP2-LTB was obtained by agar gel purification, linked with expression vector pPG-1 and pPG-2 doubly digested by BamHI and XhoI, giving rise to pPG-1-VP2-LTB and pPG-2-VP2-LTB. The recombinant plasmids pPG-1-VP2-LTB and pPG-2-VP2-LTB were electroporated into L.casei 393 respectively, generating pPG-1-VP2-LTB/- L.casei 393 and pPG-2-VP2-LTB/L.casei 393 followed enzyme digestion, PCR identification and sequence analysis.The recombinant strains pPG-1-VP2-LTB/L.casei 393 and pPG-2-VP2-LTB/L.casei 393 constructed in this study were induced by 2% lactose in MRS medium to express recombinant interest protein. The recombinant strain pPG-1-VP2-LTB /L.casei 393 of cell surface expression was induced and about 80kD protein was detected with SDS-PAGE according with the theoretic molecule weight. The result of Western blot indicated that the expressed protein possessed the antigenic specificity which could be recognized by mouse anti-PPV serum. The indirect immunofluorescence and immunoelectron microscope test also showed that the expressed protein was displayed on the cell surface of L.casei 393. The recombinant strain pPG-2-VP2-LTB /L.casei 393 of secretion expression was induced by 2% lactose in MRS medium and about 75kD protein was detected via SDS-PAGE in the induced recombinant strain and culture supernatants according with the theoretic molecule weight. The result of Western–blot indicated that the expressed protein possessed the antigenic specificity same as the native virus protein. The experiment indicated that the interest protein was expressed and secreted into the culture supernatant.To identify whether the recombinant strains have the ability to induce systemic and mucosal antibody responses and the value of the LTB as mucosa adjuvant, five groups of ten female mice were immunized via oral route with pPG-1/L.casei 393, pPG-1-VP2/L.casei 393 pPG-1-VP2-LTB /L.casei 393, pPG-2-VP2/L.casei 393, pPG-2-VP2-LTB/L.casei 393 respectively. The immune pro- tocol was administered on three consecutive days at days 0, 1and 2. A booster immunization was given at days 14, 15 and 16 and a second booster was given at days 28, 29 and 30, the third boost- er was given at days 42, 43 and 44, the mice were fed with 109 recombinant strains. Serum of mice were collected before immunization and 7, 21, 35, 49 days after immunization. Fecal pellets were collected before immunization and 1, 5, 12, 19, 26, 33, 40, 47, 54 days after immunization. Ophth- almic wash and vaginal samples were obtained by washing the eyes and the vagina with 100μl phosphate-buffered saline (PBS) before immunization and 7, 21, 35, 49 days after immunization respectively. All samples were stored at–20℃until required. Finally, we determined the level of sIgA and IgG in those samples.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-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/ L.casei 393 are higher than those administered with pPG-1-VP2/L.casei 393 or pPG-2-VP2/ L.casei 393 in fecal, ophthalmic and vaginal samples. This is due to co-expressing of mucosal immunoadjuvant LTB. This indicates that LTB can enhance the mucosal system response. This study has highlighted the potential of LTB as a safe and effective adjuvant.IgG titers of serum in mice given pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/ L.casei 393 were higher than pPG-1-VP2/L.casei 393 or pPG-2-VP2/L.casei 393. It is prominence when compare with given pPG-1/L.casei 393 and the recombinant L.casei 393 of secretion expression type could elicit higher immune response level than that induced by cell surface expression type. In this study, oral administration of recombinant strains displaying VP2-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. And the sIgA titers in mice given pPG-1-VP2-LTB/L.casei 393 or pPG-2-VP2-LTB/L.casei 393 were higher than given pPG-1-VP2/L.casei 393 or pPG-2-VP2/L.casei 393. The immune exclusion and elimination of the pathogen at the mucosal surfaces by secretory IgA is crucial in preventing porcine parvovirus.The result of neutralization ability test showed that the neutralization ability of serum antibody was 1:304、1:317、1:338、1:352 which was induced by pPG-1-VP2/L.casei393、pPG-2-VP2/L.casei 393、pPG-1-VP2-LTB/L.casei 393、pPG-2-VP2-LTB/L.casei 393 respectively after 49d oral administration.This is the first report on the cloning and expression of recombinant LTB and PPV antigen in Lactobacillus. The results obtained so far demonstrate that lactobacillus are capable of delivering antigen to the mucosal and systemic immune systems following oral immunization. When VP2 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 parvovirus.更多还原