Soluble expression and characterization of mouse lactate dehydrogenase-C4 in Escherichia coli
- VernacularTitle:小鼠乳酸脱氢酶C4在大肠杆菌中的可溶性表达与鉴定
- Author:
Duo ZHANG
;
Yongzhong XIONG
;
Ping CHEN
- Publication Type:Journal Article
- Keywords:
lactate dehydrogenase5;
gene expression
- From:
Medical Journal of Chinese People's Liberation Army
1983;0(02):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective A prokaryotic expression vector was constructed by inserting the coding sequence of mouse sperm specific lactate dehydrogenase C into pET-28a(+)and the recombinant mLDHC44 protein was purified by Ni+-NTA agrose.Methods The cDNA of mouse sperm specific lactate dehydrogenase C was obtained by RT-PCR,with total RNA of mouse testis tissues as templates.The coding sequence of mouse LDHC4 was amplified by PCR with specific primers.This recombinant vector was transformed into Escherichia coli BL21(DE3).The recombinant mLDHC4 protein was induced by isopropy-?-D-thiogalactoside(IPTG)and identified by sodium dodecyl sulfate polyacrylamide electrophoresis and LDH activity determination.After purified with Ni+-NTA agrose,the mLDHC4 protein was probed with antisera from the pVAX1-mLDHC4 vaccine(the eukaryotic expression vector of mouse sperm specific lactate dehydrogenase C)immunized BALB/c mice by Western blot analysis.Results After digested with BamH I-EcoR I,the recombinant plasmids produced right fragment which was about 1000bp.Sequencing showed that the sequence of the cloned fragment was in agreement with sequence in GenBank.This recombinant vector was named as pET-28a(+)-mLDHC4.With induction of IPTG,The recombinant protein with molecular weight of about 35 kD was expressed and the enzyme activity of this protein was high.After purified with Ni+-NTA agrose,this mLDHC4 protein formed a specific band by sodium dodecyl sulfate polyacrylamide electrophoresis and probed with antisera from immunized BALB/c mice and then formed a specific band in the nitrocellulose membrane.Conclusion The coding sequence of mouse lactate dehydrogenase subunit C had been cloned into the prokaryotic expression vector pET-28a(+)and the mLDHC4 protein could be expressed at a high level,the specificity of this protein was high and the activity was strong.
