表达hCGβ-C3d3融合蛋白的乳酸杆菌活疫苗粘膜免疫增强抗hCGβ免疫效应机制

【作者】 姚晓英；

【导师】 李大金；

【作者基本信息】 复旦大学 ， 妇产科学， 2005， 博士

【摘要】 目的：将含有融合基因hCGβ-C3d3的质粒转化干酪乳酸杆菌，对比表达hCGβ的重组乳酸杆菌活疫苗Lb.hCGβ或Lb.hCGβ-C3d3经不同粘膜途径接种不同品系小鼠，产生抗hCGβ免疫应答，验证C3d3在表达hCGβ重组乳酸杆菌活疫苗粘膜免疫中的佐剂作用；探讨C3d3在表达hCGβ重组乳酸杆菌活疫苗粘膜免疫中增强hCGβ免疫原性的效应机制。 方法：用分子生物学技术构建质粒pIlac.hCGβ-C3d3，电穿孔转化干酪乳酸杆菌（Lactobacillus casei）CECT 5276，挑选转化后的耐药菌落，在含红霉素的MRS培养基中培养，0.5％乳糖诱导后，在不同时间取上清，化学发光法检测hCGβ，Western blot鉴定表达产物，细胞免疫化学鉴定融合蛋白。用10⁸，10⁹，10¹⁰个重组乳酸杆菌Lb.hCGβ或Lb.hCGβ-C3d3分别经消化道、呼吸道和阴道粘膜接种6-8周龄雌性BALB／c和C57BL／6小鼠，3周后用相同剂量加强免疫一次。间接ELISA检测初次接种后2—8周血清和阴道灌洗液中抗hCGβ IgG和IgA抗体滴度；以及抗血清中和hCG抗原的活性。用10¹⁰个重组乳酸杆菌Lb.hCGβ或Lb.hCGβ-C3d3分别经口腔、鼻腔和阴道粘膜接种6-8周龄雌性BALB／c和C57BL／6小鼠，分离免疫小鼠脾、子宮、阴道中的淋巴细胞，流式细胞仪分析其B和T细胞的增殖情况，ELISpot分析其分泌抗hCGβ IgG和IgA抗体的抗体分泌细胞克隆数。 结果：经测序质粒构建成功；转化成功的重组干酪乳酸杆菌在乳糖诱导下可分泌融合蛋白hCGβ-C3d3。在不同品系的小鼠体内10⁹，10¹⁰ Lb.hCGβ可诱导相似的免疫应答，而10⁸诱导的抗体滴度较低。三种粘膜免疫途径诱导的免疫应答相似。加强免疫后10⁹和10¹⁰ Lb.hCGβ诱导的抗血清中和hCG抗原的能力＞100ng／ml。阴道粘膜接种诱导的最高抗体滴度不低于、甚至优于其他两种粘膜接种途径。用Lb.hCGβ-C3d3接种诱导的最高效价比Lb.hCGβ高2—100倍，加强接种后抗血清能有效中和hCG抗原的生物学活性。重组乳酸杆菌至少可在BALB／c小鼠阴道粘膜内存活3周。与Lb.hCGβ相比，Lb.hCGβ-C3d3粘膜接种后T和B细胞增殖能力明显增强（p＜0.05）。脾脏内IgG及IgA抗体分泌细胞个数在Lb.hCGβ或Lb.hCGβ-C3d3各种免疫途径间无显著差异（p＞0.05）；但Lb.hCGβ-C3d3粘膜接种后子宮及阴道内IgG及IgA抗体分泌细胞个数均显著高于Lb.hCGβ（p＜0.05）。 结论：实现了重组乳酸杆菌Lb.hcGβ-C3d3分泌性表达融合蛋白hCGβ-C3d3，培养4小时后0.5％乳糖诱导是重组菌表达hCGβ的优化条件。重组乳酸杆菌活疫苗Lb.hCGβ可经粘膜免疫诱导有效的免疫应答，产生的应答能力与免疫剂量有关。阴道粘膜接种可能是一种诱导生殖道粘膜免疫的较好途径。用表达hCGβ重组乳酸杆菌活疫苗粘膜免疫不同品系小鼠，C3d3均能增强机体对hCGβ抗原的体液免疫应答。显著刺激T和B细胞增殖；显著增加子宫及阴道内抗hCGβ IgG和IgA抗体的抗体分泌细胞克隆数，可能是C3d3在粘膜免疫中增强体液免疫的机制。更多还原

【Abstract】 Objective: To construct a recombinant lactobacillus excreting hCGβ-C3d3, Lb.hCGβ-C3d3, which can be used as a live vaccine via mucosal immunization. To compare anti-hCGβ antibody response induced by reconbinant Lb. expressing hCGβ through different mucosal immunization pathways in different strain mice so as to check the adjuvant effect and mechanisms of C3d3 in mucosal immunization of Lactobacillus live vaccine.Methods: Molecular donning was used to construct plasmids pIlac.hCGβ-C3d3, and then transfected into Lactobacillus casei CECT5276 by electroporation, which could stably express hCGβ-C3d3 protein. Chemoluminescence was used to determine the level of hCGβ in the culture supernatant. The expressed products by the recombinant lactobacillus were analyzed by western blot. The fusion protein hCGβ-C3d3 level was assayed by immunocytochemistry. The 6-8-week-old female BALB/c and C57BL/6 mice were immunized with 10⁸,10⁹,10¹⁰ Lb.hCGβ or Lb.hCGβ-C3d3 via oral, nose and vagina, respectively, and boosted with the same dosage 3 weeks later. An indirect ELISA was used to determine anti-hCGβ IgG and IgA antibody titer in vaginal lavage fluid and serum, which were got respectively at the end of 2-8 weeks after the prime immunization. Lymphocytes of were separated. The proliferation of B and T cells in spleen, uterus and vagina were analyzed by flow cytometry. The numbers of antibody-secreting cells of anti-hCGβ IgG or IgA were evaluated by ELI Spot.Results: The plasmid pIlac.hCGβ-C3d3 construction was successful by sequencing. Transfection of the pIlac.hCGβ appeared a higher efficiency than that of pIlac.hCGβ-C3d3, and the highest transfecting efficiency of the both plasmids were got at 1200V and 1000V of the pulse voltage, respectively, i.e. about 1.5×l0³CFU/μg DNA and 10³CFU/μg DNA. The recombinant Lb. hCGβ and Lb. hCGβ-C3d3 could secrete the protein of interest after 0.5% lactose induction, and the highest level was 31.4 mIU/10¹⁰Lb.hCGβ and 39.0 mIU/10¹⁰Lb.hCGβ-C3d3. Western blot analysis revealed that the recombinant strains could express the proteins hCGβ or hCGβ-C3d3 with expected molecule sizes. Immunocytochemistry analysis showed that the supernatant obtained from Lb.hCGβ-C3d3 appeared positive staining on Raji cells, while that of Lb.hCGβ was negative staining. The mucosal inoculation by 10⁹,10¹⁰ Lb.hCGβ could induce resemble response, while that of 10⁸ only induce lower response. Three differentmucosal immunization pathways could induce likely response. The 10⁹ & 10¹⁰ Lb. hCGβ-immunized antiserum after booster could neutralize more than 100ng/ml hCG antigen both in BALB/c and C57BL/6 mice. The highest titers induced by vaginal mucosal immunization were stronger than other pathway in certain conditions. The highest titers induced by Lb. hCGβ-C3d3 were 2-100 times higher than Lb.hCGβ. Antiserum after booster appeared to the biological role of neutralizing hCG antigen. The recombinant Lactobacillus could survival in vagina at least 3 weeks after immunization. Compared to Lb.hCGβ, more proliferation of T and B cells was obtained after mucosal immunization with Lb.hCGβ-C3d3 （p<0.05）. The numbers of IgG or IgA antibody-secreting cells in spleen were not significant deviation （p>0.05）. While the numbers of IgG or IgA antibody-secreting cells in uterus and vagina were significantly higher induced by Lb.hCGβ-C3d3 than Lb.hCGβ （p<0.05）.Conclusions: We have got stable and efficient hCGβ-C3d3 excretion in Lb. casei, which may be induced by 0.5% lactose. Lb.hCGβ and Lb.hCGβ-C3d3 used as live vaccines can induce efficient response through mucosal immunization. Vaginal mucosal immunization may be a better immunization pathway to induce reproductive mucosal anti-hCGβ immunity. C3d3 can enhance humoral immune response in mucosal immunization of Lactobacillus expressing hCGβ, even in different strain mice. Strikingly stimulating the proliferation of T and B cells, increasing the numbers of IgG and IgA antibody-secreting cells might be one of the mechanism of C3d3 enhancing the anti-hCGβ humoral immunity in mucosal immunization.更多还原