CDNA Cloning, Sequence Analysis And Prokaryotic Expression Of Activin β_A/β_B Mature Peptide DNA Sequences From Guangxi Bama Mini-Pig

Activin(ACT) is one of the transforming growth factor -β(TGF-β)superfamily,which is a dimeric polypeptide linked by one disulfide bond between two maturesubunits. Activin not only can participate in the growth and maturation of mammiferousgerm cells by regulating the action on the pituitary -ovary in the activin/inhibin system.,but also plays a lots of biologic functions in other tissues including liver, kidney andmuscle,ect.In order to get activinrβ_A andβ_B genes, two pairs of primers for each gene weredesigned according their sequences published in the GeneBank. The total RNAs purifiedfrom Guangxi Bama Mini-pig ovary tissue was used as the reverse transcriptiontemplates and the strands of cDNA was synthesized by using AMV reverse transcriptionenzyme. Each segment was amplified by the polymerase chain reaction (PCR), thenligated into pMD18-Tvector after purification. The recombined pMD18-Tvector wastransformed into the coliform DH5a for sequencing. The results showed that the clonedgeneβ_A andβ_B contained 1472bp and 791bp, respectively which all included codingsequences ofβ_A andβ_B mature peptides. The homology analysis showed that bothsequences are highly conservative. The homology rate of the activinβ_A andβ_Bsequence were 99.8％and 99.6％, respectively, between BaMa mini-pig and other pigbreeds reported in Genebank. High conservativeness among mammalian animals(＞90％) was also observed.The mature peptide sequences were inserted into the plasmid pRSETA toconstruct the pRSETA-β_A and pRSETA-β_B recombined vectors after restrictive enzymesdigestion. The recombined prokaryotic expression vectors then respectively transformedinto BL21(DE3)LysS to express theβ_A andβ_B fusion proteins by IPTG inducing afterconfirming the open reading frame was corrected. The proteins were confirmed asβ_Aandβ_B fusion proteins by SDS-PAGE and Western Blotting analysis. The expressingcondition optimization indicated that the best cultivation temperature, IPTGconcentration and inducing time for the fusion protein expression was 37℃, 1 mM and5-8h, respectively.In conclusion, the activinβ_A/β_B gene was successfully cloned and expressed in thisstudy, which supply a firm support for the further study of activin biological functionsin swine.