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The Prokaryotic Expression of Melittin Gene and Its Targeting Transcription in Hela Cells

Author: ChenHuaWei
Tutor: NiuLiXi
School: Shanxi University
Course: Biochemistry and Molecular Biology
Keywords: Melittin Prokaryotic Expression hTERT core promoter Hela cell Targeting property
CLC: R346
Type: Master's thesis
Year: 2011
Downloads: 2
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Abstract

To expressing the fusion protein of melittin and oleosin in E. coli cells, the recombinat expression vector of pET-3a-ole-mel、pET-28a-ole-mel、pExSecI-ole-mel、pET-3a-ole-pmel、pExSecI-ole-pmel、pRSET-ole-pmel has been successfully constructed by inserted fusion genes of ole-mel or ole-pmel into prokaryotic expression vectors respectively. The hydroxylamine cleavage site has been designed between oleosin gene sequences and gene sequences of melittin. Then the expression conditions of engineered strains were investigated. E. coli BL21(DE3) or E. coli BL21(DE3)plysS transformed with anyone of pET-3a-ole-mel, pET-28a-ole-mel, pET-3a-ole-pmel failed to express the target protein after induction, and significant mortality was observed after pExSecl-ole-mel transformed into E. coli BL21(DE3)plysS. Slow cell growth was observed after the induction and SDS-PAGE analysis showed no abvious target protein band was found when pExSecI-ole-pmel or pRSET-ole-pmel was transformed into E. coli BL21 or E. coli BL21(DE3)plysS.To construct and identify a eukaryotic expression vector which contains an EGFP gene driven by human telomerase reverse transcriptase (hTERT) gene core promoter. The core sequence of hTERT gene promoter was cloned using PCR from genomic DNA extracted from cultured HepG2 cell of human. The EGFP gene was amplified using a pair of designed primers from plasmid pEGFP-N1. The gene fragments of hTERT gene core promoter (hTERTp) and EGFP were ligated by overlap PCR and then the fusion gene hTERTp-EGFP was insterted into the vector pGL3-Enhancer at sites of Sac I and Xba I. Furthermore, the recombinant plasmid pGL3-hTERTp-EGFP was transiently transfected into Hela and HELF cells and expression of EGFP was detected by using laser confocal microscope. The inserted fragment was confirmed by DNA sequencing. A large number of EGPF was appeared in all the infected Hela cells, but not in infected HELF cells, within 48 h after transient transfection with pGL3-hTERTp-EGFP. We successfully constructed the recombinant expression vector pGL3-hTERTp-EGFP which could specifically express the therapeutic gene under the driving of hTERT core promoter.Based on successfully constructed targeting transcriptional vector driven by hTERTp to restrict to tumors, therapeutic gene melittin was introduced into this system, and then the recombinat plasmids pGL3-hTERTp-EGFP-mel and pGL3-mel were constructed and identified by PCR, enzyme digestion and gene sequencing. Under the same conditions, respectively, the recombinant plasmid pGL3-hTERTp-EGFP, pGL3-hTERTp-EGFP-mel and pGL3-mel were transiently transfected into Hela cells and then EGFP protein expression and cell morphology were observed under confocal laser scanning microscope, using the MTT method Detecting apoptosis. After transient transfection for 48h, EGFP was overexpressed in Hela cells transfected with recombinant plasmid pGL3-hTERTp-EGFP and cell morphology was normal without any signs of apoptosis. On the contury, Hela cells transfected with recombinant plasmid pGL3-hTERTp-EGFP-mel or pGL3-mel showed apoptosis, in which a large number of EGFP protein expressed. This result was also verified by MTT method. Further research showed that both melittin and its fusion protein EGFP-Mel had their role in apoptosis of Hela cells and no significant difference was observed in the effects of apoptosis.

The Prokaryotic Expression of Melittin Gene and Its Targeting Transcription in Hela Cells

Abstract

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