羊补体分子C3d基因的克隆及羊包虫病基因工程亚单位疫苗的研制
摘要
棘球蚴病(Echinococcosis granulosis)又称包虫病(Hydatidosis) ,是由细粒棘球绦虫(Echinococcus granulosus)的中绦期幼虫──棘球蚴(Echinococcus)寄生于哺乳动物脏器内所引起的一种人兽共患的流行性寄生虫病。免疫预防接种是控制包虫病行之有效的方法,因此研制高效、实用的新型羊包虫病基因工程亚单位疫苗一直是国内外科学家多年来共同追求的目标。
目前,含有重组蛋白EG95的羊包虫病基因工程亚单位疫苗取得了突破性的进展,已经成为预防包虫病最有前景的疫苗。但是由于重组蛋白EG95表达量偏低且不可溶,上述因素都制约了该疫苗的大规模生产应用。近来众多研究表明,作为补体分子C3一个裂解片段的C3d,可大幅度提高与之融合抗原的免疫原性,是一种引起研究人员广泛关注的新型分子佐剂。
基于上述背景,本研究克隆了羊补体分子C3d基因并对羊包虫病基因工程亚单位疫苗进行了的改造。一方面提高重组蛋白EG95的可溶性,并通过串联表达提高其表达量;另一方面,构建含三拷贝羊C3d与EG95的重组质粒,并表达融合蛋白,以图提高重组蛋白EG95的免疫原性。主要研究工作如下:
1.提高重组蛋白EG95的可溶性及其串联表达
通过软件分析,选取EG95基因序列中一段长为350bp、编码高度亲水的优势表位的序列(EG95s),构建了含该基因序列的原核表达载体,并进一步构建了含2拷贝、3拷贝该序列的原核表达载体,表达了重组蛋白GST-EG95s,GST-2EG95s,GST-3EG95s。经western-blotting鉴定证实上述重组蛋白均为正确表达。
2.羊补体分子C3d基因的克隆和序列分析
根据已发表的羊补体分子C3d基因,设计引物,从羊肝脏组织提取总RNA,通过RT-PCR扩增出羊C3d基因,序列测定表明所扩增的基因片断与已公布的羊C3d基因核苷酸序列同源性为99.1%,氨基酸序列完全一致。软件分析表明羊羊C3d基因与大多数哺乳动物C3d基因核苷酸序列同源性为80%左右,其中与牛C3d基因同源性为95%,揭示羊和牛在进化树上最为接近。
3.羊C3d基因与EG95s基因的串联及原核表达
为检验羊C3d对EG95抗原的免疫增强作用,将EG95s基因与3拷贝羊C3d基因串联,在每个串联基因间加入Linker链,构建了原核表达载体pGEX-6P-1-EG95s-3C3d,并表达了重组蛋白GST-EG95s-3C3d。SDS-PAGE电泳和免疫印迹试验证实该重组蛋白与预期的分子量一致,具有EG95蛋白的免疫原性。进一步对以包涵体形式表达的重组蛋白进行变性溶解、透析复性,获得了初步纯化的融合蛋白。
Echinococcosis (Hydatidosis) caused by infection with larval stage of Echinococcus granulosus(Eg), affects both humans and domestic animals, which has been considered as one of the worldwide major zoonoses. This study focus on the cloning of sheep complement component C3d and the development of recombinant hydatids subunit vaccine. At one respect, the subunit vaccine EG95 for the sheep echinococcosis was modified to EG95s to enhance the solubility of the fusion protein, and several copies of Eg95s were connected and expressed to improve the expression of the recombinant protein. At another respect, the gene of sheep C3d was amplified , the plasmid containing the fusion gene EG95s-3C3d was constructed and expressed in order to enhance the immunogenicity of the recombinant protein EG95. The main results are as follows:
1. The improvement of the solubility of recombinant protein EG95 and the tandem expression of EG95
One segment about 350bp encoding highly hydrophilic dominate epitope sequence was selected according to the analysis of the software, the prokaryotic expression vector was construced, also the prokaryotic expression vector containing two or three copies of EG95s gene was construced, the fusion protein GST-EG95s, GST-2EG95s, GST-3EG95s were expressed and identified by western-blotting.
2. cloning and sequence analysis of the complement molecular C3d of sheep
C3d gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) using the total RNA extracted from the liver tissue of the sheep and the primer pairs designed according to the published sheep C3d sequence and cloned into pUC-18 vector. Then the recombinant plasmid was sequenced and analysed. Sequencing result reveal the sheep C3d has a similarity of 99.1% with published data, and the amino acids sequences encoded by these two sequence were the same. The similarity between sheep C3d and other mammal animals are nearly 80%, and the bovine C3d has the highest similarity of 95% with sheep C3d, this result revealed sheep and bovine are close in the cladogram.
3. the connection and expression of sheep C3d gene and EG95s gene
In order to analysis the immunogenicity of EG95s fused with C3d, the EG95s gene was connected to three tandem copies of sheep C3d, and there was peptide chain adaptor (Linker) between each gene. Then the prokaryotic expression vector pGEX-6P-1-EG95s-3C3d was construced , fusion protein GST-EG95s-3C3d was expressed and identified by SDS-PAGE and western-blotting. The fusion protein expressed as inclusion body was denaturation dissolved and renaturation by dialyse, the fusion protein after intial purification was obtained.
目前,含有重组蛋白EG95的羊包虫病基因工程亚单位疫苗取得了突破性的进展,已经成为预防包虫病最有前景的疫苗。但是由于重组蛋白EG95表达量偏低且不可溶,上述因素都制约了该疫苗的大规模生产应用。近来众多研究表明,作为补体分子C3一个裂解片段的C3d,可大幅度提高与之融合抗原的免疫原性,是一种引起研究人员广泛关注的新型分子佐剂。
基于上述背景,本研究克隆了羊补体分子C3d基因并对羊包虫病基因工程亚单位疫苗进行了的改造。一方面提高重组蛋白EG95的可溶性,并通过串联表达提高其表达量;另一方面,构建含三拷贝羊C3d与EG95的重组质粒,并表达融合蛋白,以图提高重组蛋白EG95的免疫原性。主要研究工作如下:
1.提高重组蛋白EG95的可溶性及其串联表达
通过软件分析,选取EG95基因序列中一段长为350bp、编码高度亲水的优势表位的序列(EG95s),构建了含该基因序列的原核表达载体,并进一步构建了含2拷贝、3拷贝该序列的原核表达载体,表达了重组蛋白GST-EG95s,GST-2EG95s,GST-3EG95s。经western-blotting鉴定证实上述重组蛋白均为正确表达。
2.羊补体分子C3d基因的克隆和序列分析
根据已发表的羊补体分子C3d基因,设计引物,从羊肝脏组织提取总RNA,通过RT-PCR扩增出羊C3d基因,序列测定表明所扩增的基因片断与已公布的羊C3d基因核苷酸序列同源性为99.1%,氨基酸序列完全一致。软件分析表明羊羊C3d基因与大多数哺乳动物C3d基因核苷酸序列同源性为80%左右,其中与牛C3d基因同源性为95%,揭示羊和牛在进化树上最为接近。
3.羊C3d基因与EG95s基因的串联及原核表达
为检验羊C3d对EG95抗原的免疫增强作用,将EG95s基因与3拷贝羊C3d基因串联,在每个串联基因间加入Linker链,构建了原核表达载体pGEX-6P-1-EG95s-3C3d,并表达了重组蛋白GST-EG95s-3C3d。SDS-PAGE电泳和免疫印迹试验证实该重组蛋白与预期的分子量一致,具有EG95蛋白的免疫原性。进一步对以包涵体形式表达的重组蛋白进行变性溶解、透析复性,获得了初步纯化的融合蛋白。
Echinococcosis (Hydatidosis) caused by infection with larval stage of Echinococcus granulosus(Eg), affects both humans and domestic animals, which has been considered as one of the worldwide major zoonoses. This study focus on the cloning of sheep complement component C3d and the development of recombinant hydatids subunit vaccine. At one respect, the subunit vaccine EG95 for the sheep echinococcosis was modified to EG95s to enhance the solubility of the fusion protein, and several copies of Eg95s were connected and expressed to improve the expression of the recombinant protein. At another respect, the gene of sheep C3d was amplified , the plasmid containing the fusion gene EG95s-3C3d was constructed and expressed in order to enhance the immunogenicity of the recombinant protein EG95. The main results are as follows:
1. The improvement of the solubility of recombinant protein EG95 and the tandem expression of EG95
One segment about 350bp encoding highly hydrophilic dominate epitope sequence was selected according to the analysis of the software, the prokaryotic expression vector was construced, also the prokaryotic expression vector containing two or three copies of EG95s gene was construced, the fusion protein GST-EG95s, GST-2EG95s, GST-3EG95s were expressed and identified by western-blotting.
2. cloning and sequence analysis of the complement molecular C3d of sheep
C3d gene was amplified by reverse transcription polymerase chain reaction(RT-PCR) using the total RNA extracted from the liver tissue of the sheep and the primer pairs designed according to the published sheep C3d sequence and cloned into pUC-18 vector. Then the recombinant plasmid was sequenced and analysed. Sequencing result reveal the sheep C3d has a similarity of 99.1% with published data, and the amino acids sequences encoded by these two sequence were the same. The similarity between sheep C3d and other mammal animals are nearly 80%, and the bovine C3d has the highest similarity of 95% with sheep C3d, this result revealed sheep and bovine are close in the cladogram.
3. the connection and expression of sheep C3d gene and EG95s gene
In order to analysis the immunogenicity of EG95s fused with C3d, the EG95s gene was connected to three tandem copies of sheep C3d, and there was peptide chain adaptor (Linker) between each gene. Then the prokaryotic expression vector pGEX-6P-1-EG95s-3C3d was construced , fusion protein GST-EG95s-3C3d was expressed and identified by SDS-PAGE and western-blotting. The fusion protein expressed as inclusion body was denaturation dissolved and renaturation by dialyse, the fusion protein after intial purification was obtained.
引文
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2. Joe sambrook, David russell. 分子克隆(中译版),科学出版社,第三版,1256~1259
3. Joe sambrook, David russell. 分子克隆(中译版),科学出版社,第三版,1717~1719.
4. 丁剑冰,林仁勇,温浩等.细粒棘球蚴95抗原基因的克隆及原核表达质粒的构建. 新疆医科大学学报,2002,25(4):356~358.
5. 丁剑冰,林仁勇,温浩等.细粒棘球蚴95抗原基因的克隆与真核表达质粒的构建.中国人兽共患病杂志,2003,19(1):42~44.
6. 丁剑冰,马秀敏,魏晓丽等.细粒棘球绦虫Eg95基因疫苗和重组抗原诱导小鼠免疫应答的比较研究.中国人兽共患病杂志,2006,22(4):347~351.
7. 丁剑冰,魏晓丽,林仁勇等.Eg95基因疫苗不同免疫途径的体液免疫应答比较.新疆医科大学学报,2003,26(3):219~221.
8. 关庆东,王立新,郭强等. C3d增强基因免疫诱导的小鼠抗乙型肝炎病毒特异性免疫应答. 中华医学杂志,2005,85(2):101~105.
9. 韩秀敏,史大中,贾万忠等.青海省羊源细粒棘球蚴EG95基因的克隆与序列分析.中国兽医杂志,2004,34(1):17~21.
10. 胡旭初,徐劲,陆家海等.细粒棘球蚴EG95全长基因的克隆与在甲醇酵母中的表达.热带医学杂志,2003,3(1):28~31.
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