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2007•Chinese Journal of Veterinary ParasitologyRequires access

CLONING、EXPRESSION AND PURIFICATION OF SAG2 GENE OF TOXOPLASMA GONDII

Yan Huang, XU Mei-qian

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Abstract

Objective In order to clone the SAG2 gene of Toxoplasma gondii and highly express it in E.coli,and then purify it.Methods The SAG2 gene fragment was amplified by PCR,and then the gene fragment was ligated to an expression vector pGEX-4T-2.After digestion with BamHⅠand SalⅠ,screening positive recombinants sequenced were induced for expression.The condition of experiment were optimized.It was purified by affinity chromatography on glutathione-sepharose system.Results SDS-PAGE indicated that the target protein was around 41KD.Western-blotting showed it could react with rabbit-anti Toxoplasma gondii serum.The higher yield could be gotten by 2×YTA,0.1 mmol/L IPTG,220 r/min,till 3 hours at 30℃.Conclusion The soluble protein of SAG2 was highly expressed in E.coli, and the research foundation was laid for developing the new genetic engineering diagnostic reagents.

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Objective In order to clone the SAG2 gene of Toxoplasma gondii and highly express it in E.coli,and then purify it.Methods The SAG2 gene fragment was amplified by PCR,and then the gene fragment was ligated to an expression vector pGEX-4T-2.After digestion with BamHⅠand SalⅠ,screening positive recombinants sequenced were induced for expression.The condition of experiment were optimized.It was purified by affinity chromatography on glutathione-sepharose system.Results SDS-PAGE indicated that the target protein was around 41KD.Western-blotting showed it could react with rabbit-anti Toxoplasma gondii serum.The higher yield could be gotten by 2×YTA,0.1 mmol/L IPTG,220 r/min,till 3 hours at 30℃.Conclusion The soluble protein of SAG2 was highly expressed in E.coli, and the research foundation was laid for developing the new genetic engineering diagnostic reagents.

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Available abstract

Objective In order to clone the SAG2 gene of Toxoplasma gondii and highly express it in E.coli,and then purify it.Methods The SAG2 gene fragment was amplified by PCR,and then the gene fragment was ligated to an expression vector pGEX-4T-2.After digestion with BamHⅠand SalⅠ,screening positive recombinants sequenced were induced for expression.The condition of experiment were optimized.It was purified by affinity chromatography on glutathione-sepharose system.Results SDS-PAGE indicated that the target protein was around 41KD.Western-blotting showed it could react with rabbit-anti Toxoplasma gondii serum.The higher yield could be gotten by 2×YTA,0.1 mmol/L IPTG,220 r/min,till 3 hours at 30℃.Conclusion The soluble protein of SAG2 was highly expressed in E.coli, and the research foundation was laid for developing the new genetic engineering diagnostic reagents.

Key concepts: Molecular biology, Toxoplasma gondii, Cloning (programming), lac operon, clone (Java method), Gene, Biology, Blot

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