弓形虫GRA6抗原基因的克隆与表达

Cloning and expression of GRA6 gene of Toxoplasma gondi

目的 在大肠杆菌中高效表达弓形虫抗原GRA6。方法 采用聚合酶链反应 (PCR)从弓形虫昆山分离株和RH株总DNA中分别扩增到编码GRA6的基因 ,经DNA序列分析后导入表达载体PGEX - 5X - 3 ,然后在大肠杆菌BL2 1 -CodonPlus(DE3) -RP中进行表达 ,以SDS -PAGE和Westenblotting进行鉴定 ,用GST亲和层析柱纯化表达产物。 结果 1 在我们所比较的 336个碱基中 ,弓形虫昆山分离株与RH株之间碱基基本一致。 2 得到一分子量为 49kDa的融合蛋白 ,占大肠杆菌总蛋白的 2 5 55 % ,通过Westenblotting证实 ,该重组抗原能被弓形虫感染的人特异性IgM和IgG血清所识别。 结论 1 弓形虫昆山分离株和RH株的GRA6基因片段几乎完全一致 ;2 在大肠杆菌中得到了高效表达的融合蛋白 ;3 该重组抗原能被弓形虫感染的人特异性IgM和IgG血清所别 。

Aim To clone the GRA6 of Toxoplasma gondii(T.gondii)and highly express it in Escherichia coli(E coli).Method An DNA fragment of 336 bp encoding the GRA6 antigen of T.gondii was obtained from the total DNA of Kunshan and RH strains by polymerase chain reaction(PCR).The gene was cloned and the DNA sequence was determined subsequently.The gene was introduced into a expression vector pGEX 5X 3,and then BL21 Codon Plus(DE 3) RP strain cells of E.coli were transformed with the recombinant plasmid for expression.The fusion protein was analyzed by SDS polyacrylamide gel eletrophoresis(SDS PAGE)followed by Westerm blotting and was purified by GST affinity chrimatography pillar.Results 1.The aligment chart of the GRA6 DNA sequences between Kunshan strain and RH strain showed that there were almost no different from each other within 336 bp coding sequence.2.The fusion protein with a molecular weight of 49 kDa was detected on SDS PAGE and the expression product accounted for 25 55% of the total protein of cell lysate,the recombinant antigen could be recognized by toxoplasmosis specific human IgM and IgG antibodies and its antigenicity was confirmed by Western blotting Conclusion 1.The GRA6 DNA sequences of Kunshan and RH strains are almost same.2.High efficient expression of GRA6 fusion protein has been achieved in E.coli.3.The recombinant antigen can be recognized by toxoplasmosis specific human IgM and IgG antibodied and the kits constructed by the recombinant antigen are are demonstrated to be highly sensitive and specific for detection of T.gondii infection.