鹿腐蹄病A型节瘤拟杆菌纤毛蛋白与绵羊IL－2融合基因表达载体的构建与表达
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摘要
A型节瘤拟杆菌是引起鹿、牛、羊等反刍动物腐蹄病的主要菌型之一。菌体纤毛蛋白是其主要保护性抗原。白细胞介素2(IL-2)在特异性免疫应答中起主要作用，能促进T、B淋巴细胞的增殖和分化。已有很多实验证明IL-2作为佐剂能提高体液免疫和细胞介导的免疫反应。本项研究构建了A型节瘤拟杆菌纤毛蛋白和绵羊IL-2融合基因表达载体；以绿脓杆菌为宿主菌，表达了融合基因蛋白，并可在培养上清中提取；为观察绵羊IL-2在融合基因蛋白中对免疫反应的影响，以及研制生产简便、成本低的A型节瘤拟杆菌纤毛蛋白与IL-2融合基因疫苗打下了基础。研究内容如下：
用革兰氏阴性杆菌DNA快速提取法提取A型节瘤拟杆菌染色体DNA，通过PCR技术，扩增出了0.78kb的纤毛蛋白基因。PCR扩增片段与T-Easy载体连接构建了重组TE质粒，并通过EcoRV/SalⅠ双酶切，筛选出正向插入质粒(TE-Pili)进行序列测定。结果正确。
将本实验室构建的pBluescript-IL-2质粒用BamHI/EcoRⅤ双酶切，将切出的0.5kb的IL-2基因片段回收，补平。在TE-Pili载体末端的EcoRⅤ位点，插入IL-2基因，并通过EcoRⅠ酶切鉴定出正向插入质粒。大量扩增融合基因(Pili-IL-2)质粒，回收SacⅡ//SalⅠ酶切出的1.28kb Pilin-IL-2基因片段，补平，插入到HpaⅠ酶切的PPL-λ质粒中，分别用SalⅠ/Xboal，AatⅡ/Xbal双酶切，鉴定出正向插入质粒。扩增此质粒，回收BamHⅠ切出的2.5kb的基因片段，与PME290(BamHⅠ酶切后去磷酸化)载体连接。构建出融合基因表达载体，转化绿脓杆菌，获得了A型节瘤拟杆菌纤毛蛋白与IL-2融合基因工程菌株。
将融合基因工程菌株在营养肉汤中培养，离心后上清用0.1M MgCl_2沉淀，离心，提取表达产物。经SDS-PAGE电泳证明表达产物为纤毛蛋白与IL-2的融合蛋白。对流免疫电泳和Western-blot实验证明融合蛋白具有纤毛蛋白特异性。利用提取的表达产物进行家兔免疫实验，结果表明该产物具有免疫原性。
D.nodosus pili is the main protective immunogen against footrot in ruminants.IL-2 is known to be as an adjuvant in many areas. With the construction of fused Pili and ovine IL-2 genes.a recombinant fused protein vaccine was developed
The pili gene that dominates the main protective immunogen was amplified and cloned from D.nodosus serotype A by PCR.A recombinant plasmid .designated TE-Pilin,was constructed by inserting the Pili gene intoT-Easy which was digested with EcoRV.The ovine IL-2 gene is in pBluescript-IL-2 which is a plasmid constructed by Wang Ke Jian.To construct a fused plasmid,TE-Pili was digested with EcoRV ,which was inserted the IL-2 gene from pBluescript -IL-2 digested with EcoRV and BamHI.the recombinant plasmid was designated TE-Pilin-IL-2.
A fused expression plasmid was constructed by inserting the fused Pilin-IL-2 gene into PME290.The plasmid harbouring Pilin-EL-2 sequence was designated PME290- Pilin-IL-2.The fused protein was expressed by transforming the PME290-Pilin-IL-2 into competent host cell PAK/2pfs.the fused protein was purified by MgCL_(2) from the supernatant of the culture of transformed PAK/2pfs. Specific reaction between fused Pilin-IL-2 protein and the antiserum of D .nodosus serotype A Pilin was observed by cross electrophoresis.The molecular weight of the recombinant fused protein was about 33000 by SDS-PAGE.
Healthy rabbits were inoculated each with vaccine of the recombinant fused Pili-EL-2 protein at 250ul per dose to potentiate the immunogenicity.Rabbits inoculated two times at one week interval. The blood of each rabbit was collected at days 0,7,14,2l,30.The antibody titers were evaluated by k-agglutination test.The results showed that lower agglutination titers was observed at day 7 and up to higher levels over 8000at day 21 induced by the vaccine of recombinant fused protein. These results suggested that the recombinant fused protein is of the good immunogenicity. It also means that the IL-2in the fused Pili-IL-2 construct may also have a potential role in stimulating vaccine antigen.
用革兰氏阴性杆菌DNA快速提取法提取A型节瘤拟杆菌染色体DNA，通过PCR技术，扩增出了0.78kb的纤毛蛋白基因。PCR扩增片段与T-Easy载体连接构建了重组TE质粒，并通过EcoRV/SalⅠ双酶切，筛选出正向插入质粒(TE-Pili)进行序列测定。结果正确。
将本实验室构建的pBluescript-IL-2质粒用BamHI/EcoRⅤ双酶切，将切出的0.5kb的IL-2基因片段回收，补平。在TE-Pili载体末端的EcoRⅤ位点，插入IL-2基因，并通过EcoRⅠ酶切鉴定出正向插入质粒。大量扩增融合基因(Pili-IL-2)质粒，回收SacⅡ//SalⅠ酶切出的1.28kb Pilin-IL-2基因片段，补平，插入到HpaⅠ酶切的PPL-λ质粒中，分别用SalⅠ/Xboal，AatⅡ/Xbal双酶切，鉴定出正向插入质粒。扩增此质粒，回收BamHⅠ切出的2.5kb的基因片段，与PME290(BamHⅠ酶切后去磷酸化)载体连接。构建出融合基因表达载体，转化绿脓杆菌，获得了A型节瘤拟杆菌纤毛蛋白与IL-2融合基因工程菌株。
将融合基因工程菌株在营养肉汤中培养，离心后上清用0.1M MgCl_2沉淀，离心，提取表达产物。经SDS-PAGE电泳证明表达产物为纤毛蛋白与IL-2的融合蛋白。对流免疫电泳和Western-blot实验证明融合蛋白具有纤毛蛋白特异性。利用提取的表达产物进行家兔免疫实验，结果表明该产物具有免疫原性。
D.nodosus pili is the main protective immunogen against footrot in ruminants.IL-2 is known to be as an adjuvant in many areas. With the construction of fused Pili and ovine IL-2 genes.a recombinant fused protein vaccine was developed
The pili gene that dominates the main protective immunogen was amplified and cloned from D.nodosus serotype A by PCR.A recombinant plasmid .designated TE-Pilin,was constructed by inserting the Pili gene intoT-Easy which was digested with EcoRV.The ovine IL-2 gene is in pBluescript-IL-2 which is a plasmid constructed by Wang Ke Jian.To construct a fused plasmid,TE-Pili was digested with EcoRV ,which was inserted the IL-2 gene from pBluescript -IL-2 digested with EcoRV and BamHI.the recombinant plasmid was designated TE-Pilin-IL-2.
A fused expression plasmid was constructed by inserting the fused Pilin-IL-2 gene into PME290.The plasmid harbouring Pilin-EL-2 sequence was designated PME290- Pilin-IL-2.The fused protein was expressed by transforming the PME290-Pilin-IL-2 into competent host cell PAK/2pfs.the fused protein was purified by MgCL_(2) from the supernatant of the culture of transformed PAK/2pfs. Specific reaction between fused Pilin-IL-2 protein and the antiserum of D .nodosus serotype A Pilin was observed by cross electrophoresis.The molecular weight of the recombinant fused protein was about 33000 by SDS-PAGE.
Healthy rabbits were inoculated each with vaccine of the recombinant fused Pili-EL-2 protein at 250ul per dose to potentiate the immunogenicity.Rabbits inoculated two times at one week interval. The blood of each rabbit was collected at days 0,7,14,2l,30.The antibody titers were evaluated by k-agglutination test.The results showed that lower agglutination titers was observed at day 7 and up to higher levels over 8000at day 21 induced by the vaccine of recombinant fused protein. These results suggested that the recombinant fused protein is of the good immunogenicity. It also means that the IL-2in the fused Pili-IL-2 construct may also have a potential role in stimulating vaccine antigen.
引文
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7．刘晓颖等，一种适宜节瘤拟杆菌生长的改良液体培养基，特产研究[J]，2000，1(1)：48-49
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2．甘肃农业大学主编，兽医微生物学[M](第二版)，农业出版社，北京：239
3．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯，分子克隆实验指南[M]，科学出版社，1996，北京：19-20
4．J．萨姆布鲁克，E．F．弗里奇，T．曼尼阿蒂斯，分子克隆实验指南[M]，科学出版社，1996，北京：55
5．J．萨姆布鲁克等著，E．F．弗里奇，T．曼尼阿蒂斯，分子克隆实验指南[M]，科学出版社，1996，北京：40-41
6．J．萨姆布鲁克等著，E．F．弗里奇，T．曼尼阿蒂斯，分子克隆实验指南[M]，科学出版社，1996，北京：888
7．刘晓颖等，一种适宜节瘤拟杆菌生长的改良液体培养基，特产研究[J]，2000，1(1)：48-49
8．卢圣栋主编，现代分子生物学实验技术[M]，教育出版社，1993：71，107
9．卢圣栋主编，现代分子生物学实验技术[M]，1993，北京，264．
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