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

发布时间：2018-06-19 00:24

本文选题：重组乳酸杆菌 + 活疫苗　；参考：《复旦大学》2005年博士论文

【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10~8，10~9，10~(10)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10~(10)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[⒁醯乐械牧馨拖赴魇较赴欠治銎銪和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10~9，10~(10) Lb.hCGβ可诱导相似的免疫应答，而10~8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10~9和10~(10) 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[耙醯滥贗gG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。
[Abstract]:Objective : To transform the recombinant lactobacillus live vaccine ( Lb . hCG尾 or Lb . hCG尾 - C3d3 ) containing the fusion gene hCG尾 - C3d3 into different strain mice by different mucosal routes to produce anti - hCG尾 immune response , and to verify the adjuvant role of C3d3 in the expression of hCG尾 recombinant lactobacillus live vaccine mucosal immunity ;
The effect mechanism of C3d3 on enhancing the immunogenicity of hCG尾 in the mucosal immunity of hCG尾 recombinant Lactobacillus live vaccine was investigated .

Methods : The plasmid pIlac . hCG尾 - C3d3 was constructed by molecular biology technique . The transformed drug resistant colonies were selected by electroporation .
The anti - hCG尾 or Lb . hCG尾 - C3d3 were inoculated 6 - 8 weeks old female BALB / c and C57BL / 6 mice respectively via oral cavity , nasal cavity and vaginal mucosa .

Results : The sequencing plasmid was successfully constructed .
There was no significant difference in the number of IgG and IgA antibody - secreting cells in BALB / c mice than that of Lb . hCG尾 - C3d3 , and the number of IgG and IgA antibody - secreting cells in spleen was not significantly different from that of Lb . hCG尾 or Lb . hCG尾 - C3d3 ( p > 0.05 ) .
But Lb . hCG尾 - C3d3 mucosal vaccination was significantly higher than that of Lb . hCG尾 ( p < 0.05 ) .

Conclusion : The recombinant lactobacillus Lb . hcG . beta . - C3d3 secretory expression fusion protein hCG尾 - C3d3 is expressed . The optimal conditions for the expression of hCG尾 in recombinant bacteria after 4 hours of culture . The recombinant Lactobacillus live vaccine Lb . hCG尾 can induce an effective immune response via mucosal immunity .

T and B cell proliferation ;
It is possible to increase the number of antibody secreting cells in uterus and intravaginal anti - hCG beta IgG and IgA antibody , which may be the mechanism of C3d3 to enhance humoral immunity in mucosal immunity .
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R392

【参考文献】