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Studies On The Construction Of The Chimeric Gene "mEGF-Melittin" And The Recombinant Expressions Of Its Relative Genes

Posted on:2004-12-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:H RuanFull Text:PDF
GTID:1100360092470954Subject:Food Science
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Expressing bioactive proteins or peptides in microorganisms has been a hot spot of biotechnology all the time. Cationic antimicrobial peptides (CAP) are becoming the focus of people owing to its special cytotoxic mechanism. Melittin,which is taken as a typical CAP,has many kinds of bioactivities. Mouse epidermal growth factor,mEGF,as a factor to stimulate cell proliferation,also has been applied widely in clinical practice. This project is aimed at:(1) Expressing Melittin and mEGF in E.coli respectively;(2) Constructing a membrane-toxic immunotoxin by using Melittin as toxic part and mEGF as target part;(3) Measuring the mEGF mRNA expression abundance by quantitative competitive RT-PCR;(4) Checking the genetic stability of the recombinant strains constructed in the project;(5) Optimizing the fermentation conditions of the target strains by flask shake.The main results are shown as following:The mature region of Melittin from Apis Cerana and mEGF were cloned by using Tag DNA polymerase to do RT-PCR. Comparing with using MulV reverse transcriptase or AMV reverse transcriptase to do reverse transcription at 3 7-42 "C,more accurate and complete DNA could be gotten by using Tag DNA polymerase at 72 C since dimeric structure of mRNA was opened fully at high temperature. The mature region cDNA of Melittin of Apis cerana was cloned by using pUCT-vector developed from pUCIS vector. The sequence was compared with the corresponding region of Melittin of Apis mellifera reported in GenBank,the mutant site was found in 33rd base (A:Apis mellifera;G:Apis cerana),but the primary structure of the two proteins were identical,which suggested Melittin,as an important defensive gene,was very conservative in evolution.The fusion protein of Melittin of Apis cerana was expressed by using pET30a as expression vector and E.coli as expression strain. Activity determination showed the fusion protein of Melittin had very weak cytotoxicity,only in very high concentration it had some effect of cytotoxicity. But the naturalMelittin,which is formed by cutting the original fusion protein using CNBr,had significant cytotoxicity. The LDso value of the crude extraction to Rcoli DH5 a was between 1.6ug/mL (killing activity 55.77+6.52%) and 1.4ug/mL (killing activity 47.14+5.16%). The results indicated the spatial structure of the expressed product was correct,and the cationic region formed by two Lys and two Arg was necessary for keeping the activity of Melittin.The mature region cDNA of mEGF was cloned by using pGEMT-vector,and the mEGF mRNA expression abundance was also measured by using quantitative competitive RT-PCR technique. The mRNA expression of mEGF was the highest in submaxillary gland,the mean relative content for parotid gland,kidney,lung and liver was 0.86+0.14,0.62+0.18,0.14+0.03,and 0.09+0.02,respectively,compared to a mean relative content of 1.00 for submaxillary gland. No mRNA expression was detected in heart and muscle. Owing to the common expression of EGF in various organs of mouse,it could be deduced that the role of EGF was mainly autocrine and paracrine actions,and its endocrine role was also not excluded owing to its high expression abundance in submaxillary gland. This result could offer basic informations for estimating the safety of anti-tumor immunotoxin aimed at EGFR as target binding site.The fusion protein of mEGF was expressed by using pET30a as expression vector and E.coli as expression strain. The fusion protein product had significant activity,the activity of crude extraction corresponded to 22.5% of that of standard mEGF. It could be expected the activity would be improved further after cutting the fusion partner and purifying.mEGF and Melittin was linked together by a linker into an chimeric DNA,mEGFMel,and the chimeric DNA was expressed by using pET30a as expression vector and E.coli as expression strain. And the expression product was an membrane-toxic immunotoxin. The activity determination in vitro indicated the product had significant killing effect to A43i cell of squamous epith...
Keywords/Search Tags:Melittin, Epidermal growth factor, mRNA expression abundance, Membrane-toxic immunotoxin, Genetic stability, Fermentation
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