【作者】 赵丹；

【导师】 王春凤；

【作者基本信息】 吉林农业大学 ， 预防兽医学， 2007， 硕士

【摘要】 目前基因工程疫苗在疾病防制中发挥越来越大的作用，也是生命科学研究的热点。然而在基因工程操作中迄今为止报道的表达载体系统，多是以抗生素抗性作为外源基因稳定表达的压力。以抗生素为选择压力，虽然对克隆子容易进行筛选，但是由于抗药性基因不断向环境中漂移扩散，可能造成对环境微生态的破坏，带来严重后果，因而这类表达系统离“食品级”还有很大的距离，不能直接用于人和动物。目前已报道的人和动物消化道共生乳酸菌的表达载体系统，均是以抗生素抗性作为外源基因稳定表达的压力，不可能直接进入体内。所以，构建以非抗生素抗性为标记和选择压力的“可食性”乳酸杆菌表达系统就显得十分必要。非抗生素抗性为选择压力的表达系统就是以细菌的管家基因缺陷菌株为受体菌，在质粒上克隆入同源或异源完整的受体缺陷基因，作为外源性表达载体稳定的压力。自1998年Nakayama用Asd基因为选择压力构建鼠伤寒沙门氏菌非抗性表达系统以来，发展很快。近年来，在乳酸杆菌中建立了一系列不同用途的载体受体系统(Leer，1996；Savijoki，1997；Pouwels，1993)。为了在食品和医药中应用的安全性，“食品级”表达系统的研究则是该研究领域的前言和热点。乳酸菌食品级表达系统构建的一般策略是：宿主为食品级微生物，载体采用食品级选择标记，即不能有抗生素标记存在。通常以缺陷型菌株作为受体菌，载体上携带有受体菌所缺陷的基因与受体菌互补，作为选择标记。目前比较成熟的系统包括：胸苷酸合成酶基因(Thymidylate synthase，ThyA)(Morona，1991)、Asd基因(Nakayama，1998)、β-半乳糖苷酶(Hashiba，1992)、SSB基因(Porter，1990)、D-木糖异构酶基因(Prickly，1995)、琥珀突变体(Alison，1996)、赭石突变体(Chaillou，1998)、乳链菌肽(Sorensen，1999)和其它的营养生长因子基因等。胸苷酸合成酶ThyA，在体内DNA合成中起关键作用，它催化dUMP转变为dTMP的甲基化，同时使5，10-二甲基四氢叶酸转变为7，8-二氢叶酸。胸苷酸合成酶缺陷的突变株依靠外源性胸苷酸或胸腺嘧啶核苷进行DNA的生物合成，如果突变株生存的环境不能提供足够浓度的胸苷酸或胸腺嘧啶核苷，突变株就会死亡。在许多情况下，ThyA突变株可以直接从抗叶酸药物的菌株中筛选出来。Pinter(1998)等从干酪乳酸杆菌中克隆了ThyA基因，并且对之进行了测序。以ThyA基因为选择压力重新构建了质粒PUC9、PUC18和PKGS，以这些衍生质粒为载体使许多可溶性蛋白在ThyA~—的E．coli中得到了表达。Ross等(1990)从乳酸球菌中也克隆了ThyA基因并以此为选择压力构建了质粒pPR101、pPR102以及其他一系列衍生质粒，并导入ThyA的大肠杆菌中进行表达。乳酸菌“食品级”载体表达系统构建的成功，就可以把有利于人和动物的各种外源性基因或相关基因克隆入这种载体中，再导入乳酸菌受体菌中，得到的这种重组工程乳酸菌经口服后，重组菌在鸡肠道内稳定地定植和繁衍，使重组抗原和细胞因子持续地、稳定地、高效地表达并释放，从而刺激机体产生持续有效的免疫保护作用，这样就将集益生菌和基因产物的功能于一体，其科学意义和应用前景将十分广阔。本研究旨在国内外基因工程疫苗研究的基础上，采用现代分子生物学技术，克隆绿色荧光蛋白(GFP)基因，以球虫SO7基因为报告基因构建非抗生素抗性表达载体。通过电转化方法转化至thyA基因缺陷的嗜酸性乳酸杆菌中，制备非抗性基因工程乳酸菌，进一步检测该基因工程乳酸菌对柔嫩艾美耳球虫的保护效果。最后，以GFP基因为示踪基因，将基因工程乳酸菌回归雏鸡消化道，就其在雏鸡盲肠定植进行研究，为开发乳酸菌活载体疫苗打下坚实基础。更多还原

【Abstract】 Present, the gene engineering vaccine is brought into full play more and more effect in presence and control diseases .Which is also the warm spots of life science research. But the expression vector systems that were reported in gene engineering operating are used antibiotic resistance as the stable expressive pressure of exogenous gene. It is easy to screen the cloning when antibiotics are the selection pressure. But the chemical resistant genes drift and scatter to environment uninterrupted, this phenomenon causes the micro-ecology is destroyed. This expression vector systems are not used on human and animals directly, they are far from the food grade. So we have to construct the new expression vector system which is symbol of non-antibiotic resistance and selection pressure. The expression vector system of non-antibiotic resistance is used a bacterial who is defect the house-keeping gene for receptor, cloned homologisation or heterogenesis integrated receptor defects gene in plasmid as the exogenous and stable pressure. They are developed quickly since Nakayama had constructed the Salmonella typhimurium expression vector system of non-antibiotic resistance that was used Asd gene as the selection pressure in 1998. Study on expression vector of food grade is the previous remarks and hot spot. The requirement of stratagem constructions are: the host is micro-organism; the vector adopts food grade selective marker. Now, more full-fledged systems are : Thymidylate synthase, ThyA, (Morona, 1991); Asd Gene (Nakayama, 1998);β-galactosidase (Hashiba, 1992); SSB Gene (Porter, 1990); D-xylose isomerase Gene (Prickly, 1995); Amber mutant (Alison, 1996); Ochre mutant (Chaillou, 1998); Nisin (Sorensen, 1999) and other vegetable factors.Thymidylate synthase, ThyA has the important effect in synthesis of DNA in vivo. It catalysis’s dUMP changing into methylation dTMP, and it turns 5, 10-dimethyl tetrahydrofolic acid into 7, 8-dihydrofolic acid. The vicious saltant of Thymidylate synthase undertakes biosynthesis of DNA depending on exogenous thymidylate or exogenous thymidine. If the environment that saltant lives cannot provide enough thymidylate or thymidine, the saltant will dead. Under many circumstances, ThyA vicious saltant could drectly screen from antifolic. Pinter (1998) cloned ThyA gene from fondue lactobacilli and sequenced. R-constructed plasmids of PUC9、PUC18 and PKGS made expression of solubleness protein in ThyAˉE.coli. Ross (1990) cloned ThyA gene, constructed plasmids of pPR101、pPR102, expressed in E. coli.For the success of constructed food grade expression vector systems of lactobacilli, we could clone exogenous gene or related gene that makes for human being and animal into thisv-ector, and introduce into receptor of lactobacilli. Chicken take orally recombine engineering lactobacilli, recombine lactobacilli plant and multiply in chick’s intestinal tract stably, recombine antigenand cytokine expressed and realest persistently. That stimulates organism to produce effective immunoprotection. In this experiment, base on the national and international experiments on gene engineering vaccine, we cloned GFP gene with molecular biology and constructed non-antibiotic resistance expression vector of SO7 gene. We translated recombine plasmid in ThyA gene defects acidophilia lactobacilli by electrotransfo-rmation, further detected the protective affection of E.tenella. In the end, we put gene engineering lactobacilli regressed chick’s enteron, studied the colonization in chick’s cecum. So there results could serve as a basis for further studies on the vivi-vector vaccine of lactobaeilli.更多还原