非抗性筛选DNA疫苗载体及其沙门氏菌运送系统的研制和原核、真核双表达质粒的构建
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摘要
DNA疫苗被称为第三代疫苗，经过多年来的研究，DNA疫苗已取得很大的进展，它可诱导机体产生细胞免疫和体液免疫，具有制备相对简单，运输保存容易，可方便构建多价疫苗和加入多种免疫佐剂分子等优点。但目前有两个主要限制性因素阻碍了DNA疫苗的发展：第一，安全性问题，DNA疫苗的外源DNA是否会整合进宿主细胞和DNA疫苗载体上的抗生素抗性基因的存在构成了人们对DNA疫苗的担心；第二，免疫效果问题，其根本原因在于目前缺乏有效而简便的免疫方法。DNA疫苗常规免疫方法主要有肌肉注射，皮内(包括基因枪法)、皮下接种，这些方法存在着需特殊的设备，可能传播血源性疾病和不能同时激活粘膜及全身免疫等缺点。本研究从DNA疫苗安全性问题和免疫效果问题入手，首先构建了不含抗生素抗性基因的DNA疫苗通用型载体，然后利用平衡致死系统原理构建了运送该无抗性基因DNA疫苗的减毒沙门氏菌呈递系统，最后基于减毒沙门氏菌可分别表达外源抗原和运送真核表达质粒的特点，成功地构建了用于重组沙门氏菌疫苗研制的集原核和真核表达于一体的通用型双表达质粒载体。
1 非抗性筛选DNA疫苗载体及其沙门氏菌运送系统的研制
将沙门氏菌asd基因引入DNA疫苗载体pVAX1，同时破坏卡那霉素抗性基因，从而将沙门氏菌致死平衡系统引入DNA疫苗，首次构建了无需抗生素筛选的DNA疫苗载体asd-pVAX1。以加强型绿色荧光蛋白(EGFP)为报告基因，构建表达EGFP的真核表达质粒asd-pVAX1-EGFP，将其转染P815细胞后可观察到强荧光。asd-pVAX1-EGFP转化asd基因缺失型大肠杆菌X6212，在无抗生素压力下重组菌可大量扩增；以提取的asd-pVAX1-EGFP质粒肌肉注射途径免疫BALB／c小鼠，可产生抗EGFP抗体，其水平稍高于含卡那霉素抗性基因的EGFP真核表达质粒pVAX1-EGFP所诱生的抗体。
将质粒asd-pVAX1-EGFP转化进asd基因缺失型沙门氏菌X4550，体外和体内稳定性实验表明该质粒可在X4550内稳定存在。将X4550(asd-pVAX1-EGFP)感染COS-7细胞，36小时后荧光显微镜下可看到发绿色荧光的COS-7细胞。
姗如晰
扬州大学硕士论文
本实验结果表明:1.无抗性基因的DNA疫苗载体asd一pVAxl的成功构建解决了
DNA疫苗抗性丛因潜在危险性问题，并且目的质粒可在体外无抗性筛选条件下人}L【
制备;2.应用asd基因缺失型沙门氏菌X4550可运送DNA疫苗至真核细胞，解决了
DNA疫苗载体在细菌载体内的不稳定问题，为进一步研制口)报DNA疫苗捉供了必备
的基础。
2原核、真核双表达质粒载体的构建与鉴定
应用/lg川和行tI从质粒pYA3334上切下原核启动子PtrC及多克!侧立只咬(MC幼
序列，插入真核表达质粒pVAXI的加耐I和反。尸I位点间，构建成只有典核l’clllv
和原核PtrC启动子的双表达质粒载体pVAxD。酶切和测序均证实护t:c一MCS的正确
连入，pVAXD全长3，1 1 5 bp。
为证实pvAXD的有效性，E’GFP基因被置于双启动子的下游构建成pVAXD一巨(;l;!，。
将pVAXD一EGf‘I〕分别转化鼠伤寒沙门氏菌X4550和转染COS一7细)J包。应用流式自IIJJ包
术、可见光谱扫描、SDS一PAGE测得EGFP原核表达，同时应用荧光显微镜观察到比印
在COS一7细胞内的表达。构建的新质粒全长3，823bp，将其转化进鼠伤寒沙门氏菌
得X4550(pVAXD一EGFP)，它表达EGFP的量与仅以原核方式表达EGFp的X吸550
(pYA3334一 EGFP)相当;将质粒pVAXD一EGFP转染COS一7细胞后，EGFI)可钧:COS一7
细胞核和胞浆表达，在荧光显微镜下荧光强度与EGF!〕真核表达质粒pV八XI一I:GI:I’转
染COS一7细胞后发出的荧光强度相似。这些结果表明，成功构建了原核、真核表达
集中于同一质粒的新型质粒pv八XD一EGFP，并且原核、真核!」的蛋自农达虽分别，J单
一原核、真核表达质粒的表达量相当，这不但拓宽了对表达质粒的认识，而且还显
示其在研制新型重组沙门氏菌疫苗方面有着诱人的应用前景。
DNA vaccine is regarded as the third generation vaccine. A lot of progress has been made in the area of DNA vaccine in the past decade. DNA vaccine can elicit both cellular and humoral immunity and has several advantages, such as easy to produce, transport and store, easy to construct multivalent vaccines and easy to incorporate several adjuvant molecules. But two major factors restrict its development. One is the issue of safety. On the one hand, whether DNA vaccine will integrate into somatic cells' chromosome and cause genetic changes is not sure. On the other hand, the existence of antibiotic resistance gene within DNA vaccine is always a concern. The other factor is the immune efficacy is not so satisfactory. The underlying reason for this is the lack of effective and convenient immune methods. The common immune methods include muscle injection, intra-dermal and subcutaneous inoculation. But these methods have such shortcomings as the need of special equipment, the possibility of spreading blood-borne d
iseases and the lack of activation of both mucosal and systemic immunity. In this study both safety and efficacy issue of DNA vaccine were dealt with. First, a general DNA vaccine vector asd-pVAX1 containing no antibiotic resistance gene was constructed. Second, a Salmonella DNA vaccine transporting system based on asd-pVAX1 was developed by using the balanced-lethal system. Finally, a prokaryotic-eukaryotic double expression plasmid was successfully constructed for the development of novel recombinant Salmonella vaccines.
1 Characterization of a novel DNA vaccine vector without antibiotic resistance gene and its transporting system based on Salmonella
The antibiotic resistance genes in present DNA vaccine vectors are a great concern. By introducing asd gene from Salmonella typhimurium into DNA vaccine vector pVAX 1 and destroying kanamycin resistance gene at the same time, a new DNA vaccine vector asd-pVAXl which contained no antibiotic resistance gene was constructed. Then enhanced green fluorescence protein (EGFP) gene was inserted into its multiple cloning site (MCS). This new recombinant plasmid asd-pVAX 1-EGFP was harvested from Aasd E. coli X6212 in vitro and strong green fluorescence was observed in transfccted mouse mastocytoma P815 cells, asd-pVAX1-EGFP was used to immunize BALB/c mice intramuscularly and specific serum anti-EGFP antibody was elicited. The liter of anti-EGFP antibody was no lower than the antibody induced by pVAXl-EGFP.
asd-p VAX1-EGFP was transformed into asd S. typhimurium X4550 and in vitro and in vivo tests showed asd-pVAX1-EGFP can be stably maintained within X4550. When X4550(asd-pVAX1 -EGFP) was used to infect COS-7 cells, EGFP positive COS-7 cells can be observed 24 hours post-infection.
Two important conclusions can be made from these results. First, the worry of the antibiotic resistance gene was removed in this novel DNA vaccine vector asd-pVAXl and this vector can be amplified in large amount in vitro without the need of antibiotic pressure. Second, by using the balanced-lethal system, DNA vaccine based on asd-pVAXl can be stably maintained within X4550, thus solving the problem of "instability of DNA vaccine within host strain", which constituted a solid foundation for the further study of oral immunization.
2 Characterization of a prokaryotic-eukaryotic double expression plasmid
Prokaryotic promoter Ptrc and multiple cloning site (MCS) was excised from plasmid pYA3334 and inserted into the downstream of eukaryotic expression promoter Pcmv in DNA vaccine vector pVAX1. Endonucleases digestion and nucleotide sequencing confirmed the Tightness of this ligation. This novel double expression plasmid vector was named as pVAXD and was 3,115 bp in length.
To verify this novel vector EGFP gene was placed into the downstream of both promoters and consequently transformed into Salmonella typhimurium X4550 and transfected into COS-7 cell. The EGFP expression in X4550 (pVAXD-EGFP) was detected by flow cytometer, visible spectrum scanning and SDS-PAGE and its ex
1 非抗性筛选DNA疫苗载体及其沙门氏菌运送系统的研制
将沙门氏菌asd基因引入DNA疫苗载体pVAX1，同时破坏卡那霉素抗性基因，从而将沙门氏菌致死平衡系统引入DNA疫苗，首次构建了无需抗生素筛选的DNA疫苗载体asd-pVAX1。以加强型绿色荧光蛋白(EGFP)为报告基因，构建表达EGFP的真核表达质粒asd-pVAX1-EGFP，将其转染P815细胞后可观察到强荧光。asd-pVAX1-EGFP转化asd基因缺失型大肠杆菌X6212，在无抗生素压力下重组菌可大量扩增；以提取的asd-pVAX1-EGFP质粒肌肉注射途径免疫BALB／c小鼠，可产生抗EGFP抗体，其水平稍高于含卡那霉素抗性基因的EGFP真核表达质粒pVAX1-EGFP所诱生的抗体。
将质粒asd-pVAX1-EGFP转化进asd基因缺失型沙门氏菌X4550，体外和体内稳定性实验表明该质粒可在X4550内稳定存在。将X4550(asd-pVAX1-EGFP)感染COS-7细胞，36小时后荧光显微镜下可看到发绿色荧光的COS-7细胞。
姗如晰
扬州大学硕士论文
本实验结果表明:1.无抗性基因的DNA疫苗载体asd一pVAxl的成功构建解决了
DNA疫苗抗性丛因潜在危险性问题，并且目的质粒可在体外无抗性筛选条件下人}L【
制备;2.应用asd基因缺失型沙门氏菌X4550可运送DNA疫苗至真核细胞，解决了
DNA疫苗载体在细菌载体内的不稳定问题，为进一步研制口)报DNA疫苗捉供了必备
的基础。
2原核、真核双表达质粒载体的构建与鉴定
应用/lg川和行tI从质粒pYA3334上切下原核启动子PtrC及多克!侧立只咬(MC幼
序列，插入真核表达质粒pVAXI的加耐I和反。尸I位点间，构建成只有典核l’clllv
和原核PtrC启动子的双表达质粒载体pVAxD。酶切和测序均证实护t:c一MCS的正确
连入，pVAXD全长3，1 1 5 bp。
为证实pvAXD的有效性，E’GFP基因被置于双启动子的下游构建成pVAXD一巨(;l;!，。
将pVAXD一EGf‘I〕分别转化鼠伤寒沙门氏菌X4550和转染COS一7细)J包。应用流式自IIJJ包
术、可见光谱扫描、SDS一PAGE测得EGFP原核表达，同时应用荧光显微镜观察到比印
在COS一7细胞内的表达。构建的新质粒全长3，823bp，将其转化进鼠伤寒沙门氏菌
得X4550(pVAXD一EGFP)，它表达EGFP的量与仅以原核方式表达EGFp的X吸550
(pYA3334一 EGFP)相当;将质粒pVAXD一EGFP转染COS一7细胞后，EGFI)可钧:COS一7
细胞核和胞浆表达，在荧光显微镜下荧光强度与EGF!〕真核表达质粒pV八XI一I:GI:I’转
染COS一7细胞后发出的荧光强度相似。这些结果表明，成功构建了原核、真核表达
集中于同一质粒的新型质粒pv八XD一EGFP，并且原核、真核!」的蛋自农达虽分别，J单
一原核、真核表达质粒的表达量相当，这不但拓宽了对表达质粒的认识，而且还显
示其在研制新型重组沙门氏菌疫苗方面有着诱人的应用前景。
DNA vaccine is regarded as the third generation vaccine. A lot of progress has been made in the area of DNA vaccine in the past decade. DNA vaccine can elicit both cellular and humoral immunity and has several advantages, such as easy to produce, transport and store, easy to construct multivalent vaccines and easy to incorporate several adjuvant molecules. But two major factors restrict its development. One is the issue of safety. On the one hand, whether DNA vaccine will integrate into somatic cells' chromosome and cause genetic changes is not sure. On the other hand, the existence of antibiotic resistance gene within DNA vaccine is always a concern. The other factor is the immune efficacy is not so satisfactory. The underlying reason for this is the lack of effective and convenient immune methods. The common immune methods include muscle injection, intra-dermal and subcutaneous inoculation. But these methods have such shortcomings as the need of special equipment, the possibility of spreading blood-borne d
iseases and the lack of activation of both mucosal and systemic immunity. In this study both safety and efficacy issue of DNA vaccine were dealt with. First, a general DNA vaccine vector asd-pVAX1 containing no antibiotic resistance gene was constructed. Second, a Salmonella DNA vaccine transporting system based on asd-pVAX1 was developed by using the balanced-lethal system. Finally, a prokaryotic-eukaryotic double expression plasmid was successfully constructed for the development of novel recombinant Salmonella vaccines.
1 Characterization of a novel DNA vaccine vector without antibiotic resistance gene and its transporting system based on Salmonella
The antibiotic resistance genes in present DNA vaccine vectors are a great concern. By introducing asd gene from Salmonella typhimurium into DNA vaccine vector pVAX 1 and destroying kanamycin resistance gene at the same time, a new DNA vaccine vector asd-pVAXl which contained no antibiotic resistance gene was constructed. Then enhanced green fluorescence protein (EGFP) gene was inserted into its multiple cloning site (MCS). This new recombinant plasmid asd-pVAX 1-EGFP was harvested from Aasd E. coli X6212 in vitro and strong green fluorescence was observed in transfccted mouse mastocytoma P815 cells, asd-pVAX1-EGFP was used to immunize BALB/c mice intramuscularly and specific serum anti-EGFP antibody was elicited. The liter of anti-EGFP antibody was no lower than the antibody induced by pVAXl-EGFP.
asd-p VAX1-EGFP was transformed into asd S. typhimurium X4550 and in vitro and in vivo tests showed asd-pVAX1-EGFP can be stably maintained within X4550. When X4550(asd-pVAX1 -EGFP) was used to infect COS-7 cells, EGFP positive COS-7 cells can be observed 24 hours post-infection.
Two important conclusions can be made from these results. First, the worry of the antibiotic resistance gene was removed in this novel DNA vaccine vector asd-pVAXl and this vector can be amplified in large amount in vitro without the need of antibiotic pressure. Second, by using the balanced-lethal system, DNA vaccine based on asd-pVAXl can be stably maintained within X4550, thus solving the problem of "instability of DNA vaccine within host strain", which constituted a solid foundation for the further study of oral immunization.
2 Characterization of a prokaryotic-eukaryotic double expression plasmid
Prokaryotic promoter Ptrc and multiple cloning site (MCS) was excised from plasmid pYA3334 and inserted into the downstream of eukaryotic expression promoter Pcmv in DNA vaccine vector pVAX1. Endonucleases digestion and nucleotide sequencing confirmed the Tightness of this ligation. This novel double expression plasmid vector was named as pVAXD and was 3,115 bp in length.
To verify this novel vector EGFP gene was placed into the downstream of both promoters and consequently transformed into Salmonella typhimurium X4550 and transfected into COS-7 cell. The EGFP expression in X4550 (pVAXD-EGFP) was detected by flow cytometer, visible spectrum scanning and SDS-PAGE and its ex
引文
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2 Sirard J C, Niedergang F, Kraehenbuhl J P. Live attenuated Salmonella: a paradigm of mucosal vaccines. Immunol Rev, 1999,171: 5～26
3 Hopkins S A, Niedergang F, Corthesy-Theulaz I E, et al. A recombinant Salmonella typhimurium vaccine strain is taken up and survives within murine Peyer's patch dendritic cells. Cell Microbiol, 2000,2(1): 59～68
4 Vazquez-Torres A, Jones-Carson J, Baumler A J, et al. Extraintestinal dissemination of Salmonella by CD18-expressing phagocytes. Nature, 1999,401 (6755): 804～808
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