摘要
目的构建弓形虫GRA8原核重组表达质粒,分析其表达状况。方法采用PCR技术扩增GRA8及其截短型片段的基因序列,经克隆后,亚克隆至原核表达载体中,构建GRA8及其截短型片段的重组表达质粒,分析GRA8的表达;将各重组菌进行诱导,将裂解上清用谷胱甘肽-琼脂糖亲合层析法和SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)纯化目的蛋白,免疫印迹法(Westernblotting)分析纯化蛋白的活性。结果GRA8基因被正确插入原核表达质粒中,原核重组表达质粒在大肠埃希菌JM109中表达GRA8的水平低,几乎无完整GRA8的表达,截短型GRA8经谷胱甘肽-琼脂糖亲合层析法和SDS-PAGE获得纯化。纯化的截短型GRA8能被弓形虫感染兔血清识别。结论GRA8的原核重组表达质粒表达GRA8的水平低,纯化的截短型GRA8具一定的抗原反应性。
Objective To construct the prokaryotic recombinant expression plasmids of Toxoplasma gondii GRA8 and analyze their expression in E. coli containing the prokaryotic recombinant plasmids. Methods The full gene and its truncated fragment of GRA8 were amplified by PCR from T. gondii genomic DNA, and cloned into pMD18-T vector. The right genes in positive clones sequenced with ABI PRISMTM 377XL DNA Sequencer were digested with restrictive endonucleases and subcloned into pGEX-4T-2. The recombinant plasmids were transformed into E. coli JM109. The recombinant clones were characterized by PCR and digested with restriction endonucleases. Positive clones were induced with IPTG to express target protein and characterized by SDS-PAGE. The recombinant protein was purified from E. coli lysate by affinity chromatography and SDS-PAGE. The immunological activity of this protein was analyzed by Western blotting. Results The gene fragments encoding GRA8 were amplified by PCR from T. gondii genomic DNA. The inserts of GRA8 in positive clones were coincident with the original sequence of GRA8 gene from GenBank. The recombinant expression plasmids were contructed through subcloning the right inserts of GRA8 into pGEX-4T-2. The expression level of GRA8 from the recombinant expression plasmids in E. coli was low, and there was almost no full-length GRA8 expressed in E. coli. The purified protein reacted with the sera from rabbits infected with T. gondii RH strain. Conclusion The expression level of GRA8 from the recombinant expression plasmids in E. coli is low and the purified truncated GRA8 shows certain antigenicity.[
出处
《中国寄生虫学与寄生虫病杂志》
CAS
CSCD
北大核心
2005年第3期129-134,共6页
Chinese Journal of Parasitology and Parasitic Diseases
关键词
弓形虫
致密颗粒蛋白
GRA8
表达
Toxoplasma gondii
Dense granule antigen
GRA8
Expression
