Construction of Prokaryotic Expression Vectors for Livin Alpha and Livin Beta

Objective 　To const ruct prokaryotic expression vectors for Livinαand Livinβand to obtained the fusion protein of p ET32a ( + )-livinα and p ET32a ( + )2livinβ. Methods 　Total RNA of Hela cell was extracted. The full-length cDNA of Livin isoforms was gained by RT-PCR. Then inserted into p ET32a ( + ) vector and const ructed the recombinant plasmids p ET32a ( + )2livinα/ DH5α and p ET32a ( + ) / livinβ/DH5α, sequencing was performed to guarantee correct sequence insertion for the isoforms Livinα and Livinβ. Reconst ructed the recombinant plasmids p ET32a ( + )-livinα/ BL21 and p ET32a ( + ) / livinβ/BL21, the p ET32a ( + )-livinα/ BL21 and p ET32a ( + )-livinβ/ BL21 plasmids was induced af ter 4. 5 hours by IPTG, and the value of A600nmwas 0. 6 in LB medium. Expression of the p ET32a ( + )-livinα/ BL21 and p ET32a ( + )-livinβ/ BL21 were analyzed by SDS-PA GE . Results 　Full-length cDNA of Livinαand Livinβ was cloned respectively and subcloned into p ET32a ( + ) successfully. Fusion protein of positive recombinants were gained by p ET32a ( + ) prokaryotic expression system. Conclusion 　The construction of prokaryotic expression vector for Livinαand Livinβand validation of expression in cells provide basis for further research on functions of Livinαand Livinβand their anti-apoptosis effect in cancer cell.