Prokaryotic Expression,Purification And Identification Of Recombinant Chicken Extracellular Fatty Acid Binding Protein

Tibial dyschondroplasia(TD)belongs to a kind of bone disease which was found in the proximal tibiotarsus and tarsometatarsus of rapidly growing broilers.Pathologic characteristic of TD is similar to mammalian skeletal deformities of osteochondrosis in mammals.Extracellular fatty acid binding protein(Ex-FABP)is a kind of apolipoprotein secreted by chicken hypertrophic chondrocytes.Our previous experiment found that the Ex-FABP gene was differentially expressed in the early stages of TD in broilers by using gene chip technology,which suggests that there may be a close relationship between the Ex-FABP and TD in Broilers.In this experiment,the chicken cDNA sequence of Ex-FABP was firstly obtained by designning primers and total RNA was extracted from the broiler tibial growth plate cartilage,afterthat,Ex-FABP gene was amplified by the RT-PCR.The recombinant cloning vector T-Ex-FABP was constructed which was followed by a DNA sequence to verify the length of target gene.After the sequence analysis,the target gene Ex-FABP was cloned into pET-28a(+),and then transferred into the competent cells DH5a to obtain the recombinant expression plasmid pET-28a-Ex-FABP.After the successful identification,pET-28a-Ex-FABP was then transferred to the competent cells BL21(DE3)to induce the expression of the target gene.The recombinant protein was purified by affinity chromatography and detected by Western and Blot SDS-PAGE.The results of this study showed that:(1)The experiment successfully constructed the recombinant cloning vector T-Ex-FABP,and the cloned gene sequenced correctly.(2)The test successfully constructed the recombinant expression vector pET-28a-Ex-FABP,and obtained the high purity recombinant protein Ex-FABP.(3)In this experiment,we used IPTG to induce protein expression.The final induction condition was as follows:the IPTG concentration was 0.2 mM,the temperature was 30?,and the induction time was 21 h.(4)In this study,the molecular weight of fusion proteinC was 24 kDa showed by SDS-PAGE.(5)The test found the purified protein present a clear specific band in film by the Western Blot,indicating that inducible expression of pET-28a-Ex-FABP was correct.In conclusion,high purity fusion protein Ex-FABP was successfully obtained by the recombinant DNA technique in this experiment.This result will lay a basic foundation for the animal experiments in the coming future and fianlly contribute to the study of the pathogenesis and prevention of TD.