牛环形泰勒虫裂殖子/梨浆虫表面抗原基因非疏水区的克隆及GST-P27重组蛋白的高效表达
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摘要
试验据GenBank登载的牛环形泰勒虫裂殖子/梨浆虫表面抗原(the merozoite/piroplasm surface antigen,Tams1)基因序列(登录号:AF214842),设计1对特异性引物,经PCR扩增出696bp的Tams1基因片段,将其插入pGEX-4T-2表达载体,提取质粒进行双酶切鉴定并测序,同源性达98%;将插入阳性质粒的BL21(DE3)菌种诱导表达重组蛋白,经优化后在37℃、0.5mmol/L IPTG诱导4h的表达条件下获得了表达量达1.39mg/mL的可溶性融合蛋白,大小为53ku;表达产物在Glutathione Sepharose 4B柱上纯化后,免疫印迹检测结果表明重组蛋白GST-P27具有良好的反应原性。
A fragment about 696bp was amplified by PCR technique with specific primers based on Tams1gene sequence(AF214842)of Theileria annulata reported in GenBank.Then the target fragment was directionally cloned into pGEX-4T-2 expression vector,the recombinant plasmid DNA was cut by enzymes,and then sequenced.The result showed that the homology of the cloned Tams1gene was 98%.The positive plasmid was transformed into BL21(DE3),and then induced by IPTG. Soluble fusion protein whose expression level reached 1.39mg/mL was obtained when it was induced with 0.5mmol/L IPTG for 4hat 37℃,and it was 53ku.Western blotting showed that recombinant protein GST-P27which was purified by Glutathione Sepharose 4Bhad the favorable reactionogenicity.
A fragment about 696bp was amplified by PCR technique with specific primers based on Tams1gene sequence(AF214842)of Theileria annulata reported in GenBank.Then the target fragment was directionally cloned into pGEX-4T-2 expression vector,the recombinant plasmid DNA was cut by enzymes,and then sequenced.The result showed that the homology of the cloned Tams1gene was 98%.The positive plasmid was transformed into BL21(DE3),and then induced by IPTG. Soluble fusion protein whose expression level reached 1.39mg/mL was obtained when it was induced with 0.5mmol/L IPTG for 4hat 37℃,and it was 53ku.Western blotting showed that recombinant protein GST-P27which was purified by Glutathione Sepharose 4Bhad the favorable reactionogenicity.
引文
1 王振宝,刘启生,哈森,等.牛环形泰勒虫病二温式PCR诊断方法的建立及初步应用[J].动物医学进展,2012,33(5):59 ~63.
2 吴永红,刘玲,王真,等.南疆部分散养户牛场梨形虫及其媒介蜱感染情况的调查[J].新疆农业科学,2011,48(10):1918~1922.
3 李明祯,毕可东,赵忠玲,等.环形泰勒焦虫对奶牛免疫水平的影响[J].中国畜牧兽医,2009,36(7):141~142.
4 简子健,黄家雨,马素贞,等.牛环形泰勒虫巢式PCR诊断方法的建立[J].新疆农业大学学报,2008,31(6):59~62.
5 d′Olivcira C,vander Weide M,Jacquiet P,et al.Detection of Theileria annulata by the PCR in ticks(Acari:Ixodidae)collected from cattle in Mauritania[J].Exp Appl Acarol,1997,21(5):279 ~291.
6 Glascodine J,Tetley L,Tait A,et al.Developmental expression of a Theileria annulata merozoite surface antigen[J].Molecular and Biochemical Parasitology,1990,40:105~112.
7 Schnittger L,Katzer F,Biermann R,et al.Characterization of a polymorphic Theileria annulata surface protein(TaSP)closely related to PIM of Theileria parva:Implications for use in diagnostic tests and subunit vaccines[J].Mol Biochem Parasitol,2002,120(2):247~256.
8 Tanaka M,Onoe S,Matsuba T,et al.Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA[J].J Clin Microbiol,1993,31:2565~2569.
2 吴永红,刘玲,王真,等.南疆部分散养户牛场梨形虫及其媒介蜱感染情况的调查[J].新疆农业科学,2011,48(10):1918~1922.
3 李明祯,毕可东,赵忠玲,等.环形泰勒焦虫对奶牛免疫水平的影响[J].中国畜牧兽医,2009,36(7):141~142.
4 简子健,黄家雨,马素贞,等.牛环形泰勒虫巢式PCR诊断方法的建立[J].新疆农业大学学报,2008,31(6):59~62.
5 d′Olivcira C,vander Weide M,Jacquiet P,et al.Detection of Theileria annulata by the PCR in ticks(Acari:Ixodidae)collected from cattle in Mauritania[J].Exp Appl Acarol,1997,21(5):279 ~291.
6 Glascodine J,Tetley L,Tait A,et al.Developmental expression of a Theileria annulata merozoite surface antigen[J].Molecular and Biochemical Parasitology,1990,40:105~112.
7 Schnittger L,Katzer F,Biermann R,et al.Characterization of a polymorphic Theileria annulata surface protein(TaSP)closely related to PIM of Theileria parva:Implications for use in diagnostic tests and subunit vaccines[J].Mol Biochem Parasitol,2002,120(2):247~256.
8 Tanaka M,Onoe S,Matsuba T,et al.Detection of Theileria sergenti infection in cattle by polymerase chain reaction amplification of parasite-specific DNA[J].J Clin Microbiol,1993,31:2565~2569.