弓形虫昆山分离株P30抗原基因的克隆与表达

CLONING AND EXPRESSION OF P30 GENE OF TOXOPLASMA GONDII KUNSHAN STRAIN

目的 在大肠杆菌中高效表达P30抗原。方法 采用聚合酶链反应 (PCR)从弓形虫昆山分离株cDNA文库中扩增得到编码P30抗原的基因 ,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中进行表达 ,用亲和层析柱纯化表达产物 ,并以SDS -PAGE和Westernblotting进行鉴定。 结果 1、在我们比较的 783个碱基中 ,弓形虫昆山分离株与RH株之间只有两个碱基不同 ;2、得到一分子量为 5 4kDa的融合蛋白 ,占大肠杆菌总蛋白的 38%。结论 1、弓形虫昆山分离株与RH株的P30基因没有大的差异 ;2、在大肠杆菌中得到了P30融合蛋白的高效表达。

Aims To clone the P30 gene of Toxoplasma gondii(T.gondii)Kunshan strain and express it in Escherichia coli(E.coli).Methods An DNA fragment of 795 bp encoding the P30 antigen of T.gondii was obtained from the cDNA of Kunshan strain by polymerase chain reaction(PCR).The gene was cloned and the DNA sequence was determined subsequently.The gene was then introduced into a fusion protein expression vector pGEX-5x-3,and was transformed into BL21 strain cells of E.coli for expression.The fusion protein with a molecular weight of 54 kDa was purified by affinity chromatography,and was analyzed by SDS-polyacrylamide-gel-eletrophoresis(SDS-PAGE)followed by Western blotting and immuno-staining.Results 1.Comparison of the P30 cDNA sequences between Kunshan strain and RH strain showed that there were only two base pairs different from each other within the 783 bp coding sequence.2.Distinct protein band with a molecular weight of 54 kDa was detected on SDS-PAGE and its antigenicity was confirmed by immuno-staining.Conclusion 1.The P30 cDNA sequence of Kunshan and RH strain are almost identical.2.Efficient expression of P30 fusion protein was achieved in E.coli.