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Prokaryoti c Expression Plasmid Construction and Expression/Purification of Anti-a poptot ic Fu sion Protein PTD-Bcl-xL
Pages: 1-7
Year: Issue:  1
Journal: Acta Agriculturae Boreali-Sinica

Keyword:  Bcl-xL proteinProkaryotic expressionProtein purification;
Abstract: Artificial anti-apoptotic protein PTD-Bcl-xL can control abnormal apoptosis induced by a variety of factors.The present study was to obtain a high-purity Bcl-xL and PTD (Protein transduction domains) fusion pro-tein.SD rat liver total RNA was extracted by TRIzol and transcribed into cDNA.Bcl-xL gene was amplified by PCR with cDNA as a template and was cloned into pUM19-T vector to construct pUM19-T-Bcl-xL plasmid,which was I-dentified by restriction enzyme digestion and sequencing and the pUM19-T-Bcl-xL plasmid was used as a template to amplify PTD-Bcl-xL fragment which was cloned into vector pET28a to construct recombinant plasmid pET28a-PTD-Bcl-xL and PTD sequence were designed to be placed before the Bcl-xL by designing primers.Then the recom-binant plasmid was identified by restriction enzyme and was transformed into E.coli BL21(DE3),PTD-Bcl-xL fu-sion protein was induced to express with different IPTG concentration and induction time.Then the expression cul-ture was analyzed for it′s solubility and was prepared to purify PTD-Bcl-xL fusion protein with Ni-NTA agarose un-der denaturing condition.Finally,the expressing culture and purified protein was identified with SDS-PAGE analy-sis,Western Blot and MS.The results showed that detected by sequencing and enzyme digestion plasmid pUM19-T-Bcl-xL was constructed;prokaryotic expression vector pET28a-PTD-Bcl-xL was constructed with confirmed by se-quencing and enzyme digestion;The fused protein PTD-Bcl-xL could be expressed by IPTG induction with 0.1 mmol/L IPTG induction 6 hours for well expression;The fusion protein expressed in an insoluble form of inclusion bodies and a high-purity fused protein was obtained with Ni-NTA agarose purification;the expressing culture and purified protein were proved to be the PTD-Bcl-xL fusion protein with SDS-PAGE,Western Blot and MS analysis. This study obtains purified PTD-BcL-xL fusion protein and promotes the application process PTD-Bcl-xL protein in pork,beef and other livestock semen cryopreservation.
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