Cloning,Expression And Purification Of Broilers Glutathione S-Transferase A3 In Escherichia Coli

Tibial dyschondroplasia(TD)is a kind of common bone diseases which mainly happens in Broilers and could lead to huge economic losses to poultry industry every year.In previous study,we selected the differentially expressed genes in TD broilers using gene chip technology,and found that the glutathione S-transferase A3(GSTA3)was significantly differently expressed.GSTA3 is a member of alpha family of the GSTs superfamily,which has alexipharmic function.It is also essential for the regulation of signal pathways involved in oxidative stress and in the synthesis of steroids and prostaglandin.In order to fully understand the role of GSTA3 in TD,this experiment was to acquire GSTA3 in vitro used prokaryotic expression technique,and then purify and identify the recombinant GSTA3.The GSTA3 gene was amplified by RT-PCR method and the amplified products were connected to the pZeroBack/blunk cloning vector which was followed by the transferation of connected product into E.coli DH5a.In this study,the cloned products were identified by PCR technique and sequence determination.The correct GSTA3 gene was connected to the pET-28a,then the connected product was transferred into E.coli DH5 to obtain recombinant expression plasmid pET-28a-GSTA3.After pET-28a-GSTA3 was identified again,the correct pET-28a-GSTA3 was transferred into E.coli BL21 and IPTG was choosed to induce the expression of recombinant protein.The soluble GSTA3 fusion protein was purified by nickel column affinity chromatography,and the purified protein was identified by Western Blot and SDS-PAGE.Our results showed that:(1)The recombinant cloning vector pZeroBack/Blunk-GSTA3 was successfully constructed in the experiment.Compared to the GSTA3 sequencing from NCBI,gene homology of GSTA3 was 99%,the amino acid homology was 100%.Length of GSTA3 gene was 672 bp and had a complete reading frame which encoding 224 amino acids.(2)The test successfully constructed the recombinant expression vector pET-28a-GSTA3 and the connected product was transformed into E.coli BL21(DE3).When the final concentration of IPTG was 1.0 mM,the inducted temperature was 30?,the inducted time was 12 h,the soluble GSTA3 fusion protein could be obtained efficiently.(3)After Western and Blot SDS-PAGE identification,the soluble GSTA3 fusion protein with high purity was successfully obtained by nickel column affinity chromatography.The size of the fusion GSTA3 protein was 29.6 kDa.The best purification conditions was the soluble binding buffer containing 30 mM iminazole,the soluble elution buffer containing 500 mM iminazole.In conclusion,the soluble fusion protein of high purity GSTA3 was obtained successfully in this experiment.The research is expected to provide effective protection for the molecular mechanism and prevention study of TD.