TI:Cloning and Prokaryotic Expression of Transcriptional Co2activator Gene of Clonorchis sinensis and Functional Analysis of the Expressed Protein

AU:ZHAN G Yongli , YU Xinbing, WU De , WU Zhongdao , BI Huixiang

AD:( Depa rtment of Pa rasitology , Sun Yat2Sen University of Medical Sciences , Guangzhou 510089 , China)

【Abstract】Objective To construct prokaryotic recombinant plasmids of transcriptional co2activator ( TC) gene of Clonorchis sinensis , express and purify the recombinant protein and analyze its biological function. 　Methods 　A pair of primers was designed according to the known sequence of TC gene. The TC gene fragment was amplified by PCR. After purifi2 cation and digestion with Bam H Ⅰand S al Ⅰ, the TC gene was connected to the prokaryotic expression vectors , p GEX24T21 and p ET30a ( + ) . By cloning target gene into these vectors , p GEX24T21 and p ET30a ( + ) , prokaryotic recombinant plasmids of TC gene were constructed and transferred into E. coli BL21. The positive expressed recombinants were detected by SDS2 PAGE and Western blotting. Immobilized metal (Ni2 + ) chelation affinity chromatography was used to purify His2TC produced by the expression of the recombinant protein p ET30a ( + )2TC. 　Results 　The recombinant plasmids , p GEX24T212TC and p ET30a ( + )2TC , were constructed successfully. SDS2PAGE testified that the molecular weight of the recombinant protein was correct . Western blot analysis of GST2TC recombinant protein testified that the recombinant protein could be recognized by im2 munized rabbit serum , which means the protein is GST2immune active and the clone can express recombinant Clonorchis sinen2 sis antigen. After affinity chromatography of the p ET2TC protein , there was only one protein band with expected size on the SDS2PAGE gel. 　Conclusion 　The TC gene was screened from cDNA library of adult Clonorchis sinensis , cloned , expressed and purified. The purified protein of TC gene will be of importance for further research on the biological function of the gene.