Study On Construction Of Prokaryotic And Eukaryn Vectors For Expression Of Apoptin Gene

Objective To conrtuct a prokaryotic vector for expression of apoptin to purify it and produce its monoantibody; to construct a eukaryn vector for apoptin gene(VP3) which has the Kozak sequence to increase efficiency in expression of apoptin gene(VP3) and study its mechanism of inducing apoptosis of tumor cells.Methods The apoptin genes(VP3) were amplified from the template of plasmid pcDNA-VP3 by means of once and twice PCR. The VP3 amplified by once PCR was subcloned into the multiple clone site(MCS) of plasmid pET-DsbA and the other recombined with Kozak sequence was subcloned into the multiple clone site of plasmid pRevTRE to obtain plasmids pET-DsbA-VP3 and pRevTRE-VP3, which were identified by restriction endonuclease cutting and sequence analyzing. The plasmid pET-DsbA-VP3, which was subcloned with correct sequence of apoptin gene, was transformed into E.coli BL21(DE3)plysS. Expression of E.coli BL21(DE3)plysS was induced by IPTG(isopropylthio-D-galactoside, IPTG). The fusion protein with apoptin and DsbA expressed in E.coli BL21(DE3)plysS was purified through Ni-NTA His Bind Resins and several classes of protein were partitioned by polyacrylamide gel electrophoresis.Results The sequences of apoptin gene in the prokaryotic and eukaryn vectors were identical with that reported by Noteborn et al. The Kozak sequence was added to the front of ATG in apoptin gene in the vector pRevTRE. The cracked bacteria E.coli BL21(DE3)plysS which induced by IPTG was purified and partitioned by polyacrylamide gel electrophoresis. The protein with 38.3KD, a fusion protein of DsbA and VP3, was separated.Conclusion (l)The apoptin expression system with pET-DsbA-VP3 can effectively express apoptin fusion protein; (2)The eukaryn ribosome locus may be added to the front of initiation codon of apoptin gene by twice PCR and the eukaryn vector pRevTRE-VP3 can be constructed.