【作者】 张秀香；

【导师】 王庆钰；

【作者基本信息】 吉林大学 ， 生物化学与分子生物学， 2009， 博士

【摘要】 为了研制一种廉价、方便的鹦鹉热衣原体口服疫苗,本研究以其主要保护性抗原基因momp基因和大肠杆菌热不稳定肠毒素B亚单位ltb基因为研究对象,分别构建了表达MOMP蛋白和LTB-MOMP融合蛋白的原核表达载体,使其在大肠杆菌中都高效表达。通过小鼠的免疫试验,确定了LTB-MOMP融合蛋白的免疫效果要优于MOMP蛋白单独免疫效果。所以本实验通过前期试验结果,利用分子克隆、农杆菌介导和分子生物学常规检测技术等方法,构建了表达LTB-MOMP融合蛋白的转基因水稻植株,试图探索出一种利用水稻作为生物反应器生产鹦鹉热衣原体口服疫苗的免疫途径和作用模式。通过农杆菌介导法,将ltb-momp融合基因导入水稻植株中,通过抗性筛选及整合性鉴定,证明获得了表达LTB-MOMP融合蛋白的转基因水稻植株。用间接ELISA的方法测定了转基因水稻叶片中融合蛋白的平均含量可占植物总可溶性蛋白的0.0055%,而在种子中的平均含量为0.0069%。实验中以BALB/c小鼠为哺乳类实验动物模型,用获得的表达LTB-MOMP融合蛋白的转基因水稻植株种子在小鼠体内进行了口服免疫实验研究,通过对体液、细胞及粘膜免疫等相关指标的检测,结果表明:口服转基因水稻种子能够诱导小鼠机体产生体液、细胞及粘膜免疫反应,但以细胞和粘膜免疫应答为主。同时,还证明了LTB在转基因水稻口服免疫实验中具有良好的粘膜佐剂效应。通过本实验研究,将为下一步应用表达LTB-MOMP融合蛋白的转基因水稻在禽源动物体内口服免疫实验研究奠定基础,也为禽类鹦鹉热衣原体病的预防提供新思路。更多还原

【Abstract】 Chlamydia psittaci, (Cps) is an important pathogen that causes several kinds of zoonosis. It has highly infectivity, and can tolerance many antibiosis medicince. It has caused enormous economic loss for the livestock farming, especially the rate of abortion, dead foetus and death. The study indicated the major outer membrane protein (MOMP) of Cps was the main structural protein had highly immunogenicity and reactionogenicity, and momp gene of different Cps strains had highly homology. For this reason, momp gene had highly study value. Since the portal of entry and primary site of infection of Cps is the respiratory tack, mucosal vaccination should have inherent advantage to induce a protective immune response. Unfortunately, parenteral immunization, the traditional means of vaccine delivery, is a poor inducer of mucosal immunity. To improve the efficacy of oral vaccination, antigens can be co-administered, of fused with a strong mucosal adjuvant. Heat-labile toxin (LT) is an immunogenic molecule that stimulates systemic and mucosal immune system responses, B subunit of LT (LTB) has the merits of avirulence and strong immunoadjuvant. Vaccines are still the main means to prevent Cps. But traditional vaccines can’t play an efficient role to prevent the Cps for the mucosal infection. Traditional vaccine injection needs special instruments and worker and also many times. And it is unrealistic idea to vaccinate all nature hosts against Cps infection. It is an urgent question how to design and develop novel vaccines, especially vaccinated through mucosal route.Mucosal immune system which is one of main important ingredient of the system immunological network play a vital role in anti-infection. The surface of mucosal contact with outside antigen directly, is the first screen of defense. Lots of researches have been proved that vaccines inoculated by mucosal tract were effective against mucosal infection disease.By inserting the momp gene and ltb-momp gene into prokaryotic expression vector pET-28a(+), respectively. The plasmids of pET-28a-MOMP and pET-28a-LTB-pep-MOMP were constructed and transformed into E.coli strain BL21, respectively. After IPTG induction, MOMP protein and LTB-MOMP fused protein were expressed in E.coli by high level. SDS-PAGE of MOMP and LTB-MOMP accumulated to the level of 30% and 42% of total bacterial proteins, respectively. The results of Western blot analysis also demonstrated that the fusion protein could be recognized by the anti-Cps and anti-LT antibody, respectively. By means of affinity chromatography, the MOMP protein and LTB-MOMP fusion protein were purified.After inclusion bodies were denaturalized and refolded in vitro, the result of the fusion protein-GM1 binding assay, that the two proteins could bind to monosialoganglioside specifically, showed it possesed biological activity in vitro. The mice were inoculated with the two kinds fusion protein that were denaturalized and refolded in vitro, respectively. The levels of humoral, cellular and mucosal immune responses were detected. The proliferation ability of splenic lymphocyte stimulated by Cps and ConA was detected, the result showed that lymphocyte Cps stimulating index in group immunized with MOMP and LTB-MOMP are higher than that control group. The results of ELISPOT detection for number of IFN-γblot showed that the number of MOMP and LTB-MOMP groups were higher than the control group. The titer of anti-MOMP IgG and IgA antibody in mice serum from group LTB-MOMP was higher than MOMP and control groups. The sIgA level in gut and lung lavage fluid in LTB-MOMP group was higher than other groups. These results showed the fusion protein of LTB-MOMP was able to evoke strong humoral immune responses. This indicated that LTB as mucosal adjuvant could improve the level of mucosal immunity.The plant genetic engineering has been greatly developed since the first transgenic plant came out in 1983. The transgenic plant has showed great potential as a cheap bioreactor to produce various medicine material needed by mankind. The transgenic technology has an important breakthrough of gramineae with rice as a typical grass, and it is possible for rice to carry heterogeneity protein. The rice is taken as the expression system to produce safe and cheap transgenic vaccine, antibody and cytokines by the transgenic technology, which is not only the improvement of the research of medical genetic engineering, but also an important method for rice from a simple food plant to a high attached-value functional food. Based on the above reseaches, LTB-MOMP fusion protein had a better immunity than MOMP protein in mice experiment. So, the plant expression vectors of pPCV701-LTB-pep-MOMP were constructed for transformation of rice. The competent Agrobacterium tumefacien was transformed by rice expression vector pPCV701-LTB-pep-MOMP, and obtain Agrobacterium tumefacien transformant after identified with PCR and enzymes digestion. Embryogenic calli was induced from rice mature embryos. Rice embryogenic calli was transformed with pPCV701-LTB-pep-MOMP by Agrobacterium tumefacien. Finally we obtained transgenic plant after resistance screen, differentiation and root culture. The plantlets were regenarted on antibiotic selective media and the integration, transcription and expression of the transgene was identified Southern blot, RT-PCR and Western blot. The recombinant protein was demonstrated to acquire the high performance and have perfect reactionogenicity by SDS-PAGE, and further quantified by indirect ELISA. The results indicated that LTB-MOMP fusion protein made up 0.0055% of the total soluble leaf protein, Total soluble plant protein of MOMP was approximately 0.0069% in the plant seed extract.BALB/c mice were used as mammal experimental model, were inoculated by the transgenic rice. The immunity levels of humoral, cell and mucosal were detected. Systemic and mucosal MOMP-specific antibody in sera and feces from immunized mice were then determined by ELISA analysis. Antibodies from experimentally immunized mice showed a strong response against MOMP as shown by the ELISA. Significant IgA levels were observed in the feces from the mice immunized with transgenic plant and transgenic plant plus saponin. Furthermore, the memory immunoglobulin B cells were formed in the mice orally immunized with MOMP protein because the strongest IgG and IgA responses during 2-4 weeks were found after the final booster.The proliferation ability of splenic lymphocyte and profiles of cytokine were detected and the results show that the special and non-special stimulate indexes of each group. Mice inoculated with transgenic plant, non-transgenic plant, 6BC inactivated vaccine plus LTB and nonthing showed a strong increase of the splenocyte proliferative response, compared with the groups immunized with non-transgenic plant and nonthing (P≤0.01). The splenocytes from mice immunized with non-transgenic plant showed a slight proliferative response to MOMP (P>0.05), compared with that of mice immunized with nothing.Profiles of cytokine released from MOMP-stimulated spleen cells were examined to determine the primed pathway of immune response. Cytokines IFN-γ, IL-2, IL-4, IL-5, IL-10 and TGF-βlevels were measured from supernatants of splenocyte cultures from all groups of mice. significant levels of IL-2, IFN-γ, and TGF-βwere produced in mice vaccinated with transgenic plant and 6BC inactivated vaccine plus LTB when stimulated with MOMP (P≤0.01), compared with mice vaccinated with non-transgenic rice. None of the groups differed significantly in production of IL-5. Meanwhile, to determine whether MOMP-transformed rice stimulate a cytotoxic T lymphocyte (CTL) response, splenocytes obtained from Balb/C mice inoculated with transgenic plant and nonthing. MOMP-specific target cells lysis was detect, it might approxiamately achieve 50% administered with transgenic plant at 4 weeks in post-inoculation, it was significant difference compared with the other two groups vaccinated with non-transgenic plant and nonthing (P<0.05). The micro-neutralization test was performed, the results of neutralizing antibodies (NA) showed that the NA were not elicited in all groups during period of vaccinating. And, transgenic plant immunization induced partial protection (53.3%) against a lethal challenge by the aerosol route with the highly virulent Cps 6BC strain. At the same time, all of the control mice were died during 10-21d after challenge.The resulults above indicated that the expression vector pPCV701 which ware constructed above can express exogenous gene in rice. The transgenic rice could express the ltb-momp gene, and the expressing protein can induce more cellular and mucosal immunity in test mice, and also with low humoral immunity which compared with control group. The research had estabilised the foundation for the plant oral vaccine of Cps.更多还原