摘要
目的构建小鼠精子特异性乳酸脱氢酶C4(mLDHC4)的原核表达载体并进行重组蛋白的纯化。方法提取小鼠睾丸组织总RNA,反转录合成cDNA。自行设计引物,PCR扩增mLDHC4的编码序列,产物经EcoRⅠ/BamHⅠ双酶切后,克隆至表达载体pET-28a(+)中,获得重组载体。将重组载体转化大肠杆菌E.coliBL21(DE3),用异丙基-β-D-硫代半乳糖苷(IPTG)诱导mLDHC4表达,采用聚丙烯酰胺凝胶电泳和乳酸脱氢酶活性测定方法对重组mLDHC4进行鉴定,Ni+-NTA亲和层析法纯化mLDHC4,并将其与pVAX1-mLDHC4(小鼠mLDHC4的真核表达载体)免疫小鼠的抗血清进行免疫印迹反应。结果重组载体经EcoRⅠ/BamHⅠ双酶切后,可释放大小约1000bp的插入片段,序列分析表明,克隆的DNA片段与GenBank登录的mLDHC4编码序列完全一致,将此重组质粒命名为pET-28a(+)-mLDHC4。在IPTG的诱导下,重组菌可表达大小约35kD的蛋白质,且其裂解液具有很高的乳酸脱氢酶活性,经亲和层析纯化后在聚丙烯酰胺凝肢中仅显示单一蛋白区带,与抗血清反应后在硝酸纤维素膜上形成特异性条带。结论小鼠mLDHC4的全长编码序列已被克隆至原核表达载体pET-28a(+)质粒,并在E.coliBL21(DE3)中获得高效表达,纯化后的蛋白特异性高、活性强。
Objective A prokaryotic expression vector was constructed by inserting the coding sequence of mouse sperm specific lactate dehydrogenase C imo pET-28a （＋） and the recombinant mLDHCA4 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 LDHCA was amplified by PCR with specific primers. This recombinant vector was transformed into Escherichia coli BL21（DE3）. The recombinant mLDHCA protein was induced by isoprop^-D-thiogalactoside （IPTG） and identified by sodium dodecyl sul- fate polyacrylamide electrophoresis and LDH activity determination. After purified with Ni＇ -NTA agrose, the mLDHCA protein was probed with antisera from the pVAXI-mLDHCA 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 recombinam plasmids produced right fragment which was about 1000bp. Sequencing showed that the sequence of the cloned fragment was in agreement with sequence in Ge~ This recombinam vector was named as pET-28a （q-）-mLDHCA. 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 mLDHCA 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 mLDHCA protein could be expressed at a high level, the specificity of this protein was high and the activity was strong.
出处
《解放军医学杂志》
CAS
CSCD
北大核心
2008年第2期176-179,共4页
Medical Journal of Chinese People's Liberation Army
基金
福建省重点科技攻关项目(2002Y032)
全军医药卫生“十五”基金项目(01MA032)
关键词
乳酸脱氢酶类
基因表达
lactate dehydrogenase5
gene expression
