【作者】 侯慧丽；

【导师】 傅童生； 还连栋； 钟瑾；

【作者基本信息】 湖南农业大学 ， 临床兽医学， 2008， 硕士

【摘要】 猪链球菌2型（Streptococcus suis type 2,SS2）是我国猪链球菌病的重要病原,不仅可引起猪脑膜炎、关节炎、心内膜炎、败血症、肺炎和突然死亡,还可感染相关人员并致死,是一种重要的人畜共患病病原。目前国内外对SS2的研究主要集中在其毒力相关因子上,公认的毒力相关因子有溶菌酶释放蛋白（muramidase released protein,MRP）、胞外蛋白因子（extracellular factor protein,EF）、溶血素（suilysin,SLY）、荚膜多糖（capsular polysaccharide,CPS）等。乳酸菌是一种公认安全的（generally regards as safe,GRAS）食品级微生物,能作为活疫苗抗原传递载体为探讨SS2的重要毒力因子MRP和EF在乳酸菌中的表达及其用作口服疫苗的可行性,本研究克隆了epf、mrp抗原性较高的部分片段,将其分别与启动子P59和信号肽SP_Usp45连接,最终构建了EF的组成型分泌表达载体pHL301、MRP的组成型分泌表达载体pHL302;分别与诱导型启动子P_nisz和SP_Usp45连接,获得了EF的诱导型表达载体pHL401、MRP的诱导型表达载体pHL402。同时并将epf、mrp基因串联构建了EF-MRP组成型表达载体pHL500和诱导型表达载体pHL600。将构建的组成型表达载体pHL301、pHL302分别转化至Lactococcus lactis MG1363、Lactobacillus casei ATCC27092、Lactobacillus brevis ATCC 8287、Lactobacillus acidophillus HLA、Lactobacillusplantarum NCIMB8826中,诱导型表达载体pHL401、pilL402、pnL600转化至L.lactis NZ9000中,pilL500转化至Lb.plantarum NCIMB8826中,通过Westernblotting检测蛋白的表达情况。EF、MRP在各重组菌中均得到了不同程度的表达,特别是在重组Lb.casei ATCC27092中,EF和MRP蛋白在细菌生长的各阶段都能很好的表达:在重组Lb.plantarum NCIMB8826中,蛋白基本全部分泌至培养基中,且分泌表达时间长,甚至在OD₆₀₀=1.6时仍能很好的表达;在重组L.lactisNZ9000中,蛋白表达量相对更高。将含有表达EF、MRP的重组Lb.casei ATCC27092、Lb.plantarumNCIMB8826、L.lactis NZ9000菌体口服饲喂C57BL/6J小鼠,发现Lb.caseiATCC27092/pHL301能刺激小鼠发生免疫反应产生EF特异性抗体,Lb.caseiATCC27092/pHL302能刺激小鼠产生MRP特异性抗体,而其他重组菌抗体水平很低,或者根本检测不到抗体的产生。上述试验结果说明,表达EF或MRP的Lb.casei ATCC27092均能对小鼠产生保护作用,而重组Lb.plantarumNCIMB8826、L.lactis NZ9000菌却未能达到预期的效果。更多还原

【Abstract】 Streptococcus suis serotype 2（SS2） is the important pathogen of diseases in pigs.It is a major cause of meningitis,septicemia,arthritis and bronchopneumonia in young pigs or even meningitis in humans.Major virulence factors have been recognized in recent years,including extracellular protein factor（EF）,muramidase released protein（MRP）,suilysin（SLY） etc.Since lactic acid bacteria（LAB） are generally regarded as safe（GRAS） and can act as a live delivery vehicle,to develop an oral vaccine to protect from SS2,the EF/MRP antigen fragments were expressed in LAB for the first time in this study.The epf/mrp gene fragments were cloned and ligated with promoter P59 and signal peptide sp_Usp45, thus EF constitutive secretary expressive vector pHL301 and MRP constitutive secretary expressive vector pHL302 were constructed respectively.Similarly,the PCR- amplified epf/mrp gene were ligated with promoter P_nisz and signal peptide sp_Usp45,generating nisin-controlled EF protein expression vector pHL401 and MRP protein expression vector pHL402.At the same time,epf and mrp gene were fused together to get the EF-MRP constitutive expression vector pHL500 and nisin-controlled expression vector pHL600.Then,the constitutive vector pHL301 and pHL302 were transformed into Lactococcus lactis MG1363,Lactobacillus casei ATCC27092,Lactobacillus brevis ATCC8287,Lactobacillus acidophillus HLA and Lactobacillus plantarum NCIMB8826,respectively.The nisin-controlled vector pHL401,pHL402 and pHL600 were transformed into L.lactis NZ9000,respectively. And pHL500 were transformed into Lb.plantarum NCIMB8826.Western blotting analysis demonstrated that the protein products（EF/MRP/EF-MRP） were expressed in the cytoplasm or secreted into the medium.In particular,the EF or MRP protein can be better expressed during the whole growth phase of recombinant Lb.casei ATCC27092.The expression product targeted in supernatant while little found in the cytoplasm in recombinant Lb.plantarum NCIMB8826.Furthermore,the expression product can still be detected in exponential late phase when OD₆₀₀ of incubation reached 1.6.The higher expression level emerged in the recombinant L.lactis NZ9000 by nisin induction.The recombinant strain Lb.casei ATCC27092,Lb.plantarum NCIMB8826 and L. lactis NZ9000 were administered to C57BL/6J mice orally.Results showed that orally delivered recombinant Lb.casei ATCC27092/pHL301 can induce significant EF-specific IgG in the sera and recombinant Lb.casei ATCC27092/pHL302 can also induce significant MRP-specific IgG in the sera.But the specific IgG was lower in recombinant Lb.plantarum NCIMB8826 and L.lactis NZ9000 administered mice. These results suggest that recombinant strain Lb.casei ATCC27092/pHL301 and Lb. casei ATCC27092/pHL302 may be applicable as an oral vaccine to induce protective immunity against SS2 in C57BL/6J mice.更多还原